sign in sign up
<<
Home , Pekar, Spermatocytogenesis

HEALTH SCIENCES RESEARCH PAPERS

Kitap Adı : Health Sciences Research Papers İmtiyaz Sahibi : Gece Kitaplığı Genel Yayın Yönetmeni : Doç. Dr. Atilla ATİK Kapak&İç Tasarım : Didem Semra KORKUT Sosyal Medya : Arzu ÇUHACIOĞLU

Yayına Hazırlama : Gece Akademi Dizgi Birimi

Yayıncı Sertifika No : 42539 Matbaa Adı : GeceAkademi ISBN : 978-625-7958-05-9

Editors Assist. Prof. İbrahim ÇALTEKİN MD. Assist. Prof. Melike ÇALTEKİN MD.

The right to publish this book belongs to Gece Kitaplığı. Citation can not be shown without the source, reproduced in any way without permission. Gece Akademi is a subsidiary of Gece Kitaplığı.

Bu kitabın yayın hakkı Gece Kitaplığı’na aittir. Kaynak gösterilmeden alıntı yapılamaz, izin almadan hiçbir yolla çoğaltılamaz. Gece Akademi, Gece Kitaplığı’nın yan kuruluşudur. Birinci Basım/First Edition ©ARALIK 2019/Ankara/TURKEY ©copyright

Gece Publishing ABD Adres/ USA Address: 387 Park Avenue South, 5th Floor, New York, 10016, USA Telefon/Phone: +1 347 355 10 70

Gece Akademi Türkiye Adres/Turkey Address: Kızılay Mah. Fevzi Çakmak 1. Sokak Ümit Apt. No: 22/A Çankaya / Ankara / TR Telefon/Phone: +90 312 384 80 40 - +90 555 888 24 26

web: www.gecekitapligi.com –– e-mail: [email protected]

HEALTH SCIENCES RESEARCH PAPERS

CHAPTER 1 IN SITU HYBRIDIZATION METHOD Arzu GEZER...... 9 CHAPTER 2 IN LABORATORY ANIMALS Arzu GEZER...... 25 CHAPTER 3 APPROACH TO CARDIOMYOCYTES PROLIFERATION AND CARDIAC REGENERATION Aynur KARADAĞ GÜREL ...... 51 CHAPTER 4 ADULLT CARDIOMYOCYTE AND EXISTING CELL CYCLE Aynur KARADAĞ GÜREL ...... 63 CHAPTER 5 BIOECOLOGY OF INFECTIOUS DISEASES: AN ANTHROPOLOGICAL APPROACH Berna ERTUĞRUL ...... 75 CHAPTER 6 IMPACT OF IRON AND FOLIC ACID SUPPLEMENTATION DURING PREGNANCY ON NEWBORN BODY SYMMETRY Berna ERTUĞRUL ...... 91 CHAPTER 7 HOLISTIC CARE IN CARDIAC SURGERY PATIENTS Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ ...... 99

CHAPTER 8 PATIENT REPORTED OUTCOME MEASURES IN KNEE OSTEOARTHRITIS Fatih ÖZDEN, Özgür Nadiye KARAMAN ...... 113 CHAPTER 9 VıOLENCE PHENOMENON IN EARLY MARRIAGES Funda ÖZPULAT ...... 129 CHAPTER 10 THE DUTIES AND RESPONSıBıLıTıES OF THE NURSE IN SEXUAL ABUSE CASES TOWARDS CHıLDREN Funda ÖZPULAT ...... 147 CHAPTER 11 EVALUATION OF INFERTILITY ETIOLOGY IN WOMEN AND MEN AND TREATMENT OPTIONS Gizem KÖPRÜLÜLÜ ...... 165 CHAPTER 12 INVESTIGATION OF THE PERCEPTION / THE ATTITUDE LEVELS OF DIFFERENT GENERATION FREE / INDEPENDENT PHARMACISTS FOR PHARMACEUTICAL CARE: ADIYAMAN CASE Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR ...... 185 CHAPTER 13 TOOTH EXTRACTION REASONS AND TECHNIQUES IN PEDIATRIC PATIENTS Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN ...... 209

CHAPTER 14 CONSCIOUSNESS LEVEL ANALYSIS OF HEALTH PERSONNEL TOWARDS INTERNATIONAL PATIENT SAFETY GOALS: A Case Study Of Private Hospital In Turkey Nuray GİRGİNER, Mine İSKENDEROĞLU ...... 223 CHAPTER 15 REVIEW Monosodium Glutamate Nurhayat ATASOY ...... 243 CHAPTER 16 LASERS IN DENTISTRY Özge PARLAR ÖZ, Mahmut ERÇİL ...... 253 CHAPTER 17 SPECIAL USAGE AREAS OF LASERS IN DENTISTRY Özge PARLAR ÖZ, Mahmut ERÇİL ...... 267 CHAPTER 18 Herbal Therapy for Leishmaniasis: Role of Natural Products Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN ...... 281 CHAPTER 19 FOOD SUPPLEMENTS FOR MOTHERS-BABIES AND RELATED ANALYSIS Esra KIZAR, Serap SAĞLIK ASLAN ...... 299 CHAPTER 20 THE ROLE OF IMMUNE SYSTEM IN PHOTODYNAMİC THERAPY Sercin OZLEM CALISKAN, Rahsan ILIKCI SAGKAN ...... 319

CHAPTER 21 A HOLISTIC APPROACH TO CHRONIC LEG ULCERS Overview Of Diagnosis And Differential Diagnosis ...... 335 Sevgi AKARSU ...... 335 CHAPTER 22 WAR SURGERY AND NURSING CARE Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA ...... 357

IN SITU HYBRIDIZATION METHOD

CHAPTER

1

Arzu GEZER1

1 Atatürk Üniversitesi Sağlık Hizmetleri Meslek Yüksek Okulu Erzurum, [email protected]

Arzu GEZER • 11

1. INTRODUCTION The In Situ Hybridization (ISH) technique was described in 1969 and applied to RNA molecule in 1981. This technique is used to determine the location of a particular nucleic acid sequence in the cell or tissue using complementary and labeled RNA / DNA nucleic acid strands capable of binding to specific RNA or DNA sequences. This method allows nucleic acids to be visualized in their cellular environment(Kocks, Boltengagen, Piwecka, Rybak-Wolf, & Rajewsky, 2018). The In Situ Hybridization technique is called In Situ Hybridization when it is applied directly to cell and tissue sections, smears, and chromosomes scattered in mitosis. This method is ideal for determining the cells in which a specific gene has been read, or locating a gene on a specific chromosome, if a specific DNA sequence (a gene or part of a gene) is present in the cell. (Mukai, 2017) Under favorable conditions, this method is based on the formation of hybrid molecules by pairing with complementary sequences of single- stranded nucleic acid molecules to provide identification of sequences on DNA or RNA molecules. Hybridization of the target mRNA with the labeled nucleic acid probe is achieved in histological tissues, creating stable hybrids, thereby allowing the location of the probe to be made visible. In cytological preparations it allows direct localization of DNA sequences or genes on chromosomes (Beliveau, 2015). With this method, it is possible to make definite inferences in gene expression studies for cell types in different species. Nowadays, it is preferred as a genetic diagnosis method for toxicological studies, cancer research and chromosomal anomalies (Mukai, 2017).

2. IN SITU (NUTRITION) HYBRIDIZATION METHOD This is intended to identify specific sequences in RNA or DNA molecules by hybridization based on the principle of forming hybrid molecules by matching with complementary sequences of single-stranded nucleic acid molecules under appropriate conditions (Balajee & Hande, 2018). In situ hybridization procedures are used to localize specific nucleic acid sequences in cells of tissue samples. ISH is used to show various specific nucleic acid sequences, including genomic sequences on viral DNA and RNA, mRNAs and chromosomes in infected tissues and cells (Kocks et al., 2018).. 12 • Health Sciences Research Papers

If mRNA can be detected in a tissue, it can be understood which gene is read or active. In order to read the gene of interest, it is necessary to produce mRNA in the tissue. It is possible to produce protein by producing mRNA (Carter, Caron, Dogan, & Folpe, 2015; Evyapan, Ay, Cömertpay, & Lüleyap, 2019). 2.1. Preferred Inverts in In Situ Hybridization: DNA in the cell is denatured using heat or debris. The stranded DNA strands are separated from one another and are ready to be "hybridized" with the DNA or RNA fragment that corresponds to the desired sequence on the single strand. This series is called a probe (Tang et al., 2015). Several probes have been described. These probes include; DNA probes: These probes, which are highly active, should be subjected to denaturation prior to use. RNA probes: Unlike DNA probes, they have high stability. RNA-RNA hybrids are the most stable hybrids and can be easily destroyed by RNases (Beliveau, 2015). Oligonucleotide probes: Oligonucleotide probes consisting of 15-50 nucleotides are highly stable to the target sequence. Due to their small size, they can easily enter the target area. Very good penetration and single- stranded probes are very advantageous for ISH (Beliveau, 2015; ÖZDEMİR, MURANLI, & KANEV, 2015). When selecting probes to be used in ISH, the appropriate type of marking and the type of nucleic acid preferred as the probe should be considered. Marker probe: This method is called Fluorescent In Situ Hybridization (FISH) if it uses a fluorescent substance that can be detected by immunofluorescence microscopy. If the marker probe is not a fluorescent substance but only a dyestuff, the method is called Chromogenic In Situ Hybridization (CISH) and evaluation is made by light microscope. 2.2. Labels Preferred for In situ Hybridization: Labeling for probe marking is the realization of binding by means of antibodies. Probe labeling is done by chemical or enzymatic reactions. The most preferred label is digoxigenin. The biotin label is used in conjunction with avidin (Beliveau, 2015). For fluorescent labeling, the most sensitive imaging of the preferred labels is with alkaline phosphatase. Biotin is one of the most widely used Arzu GEZER • 13 non-radioactive nucleic acid labels. Antisense RNA (asRNA) can be used to stop protein synthesis of mRNAs (Choi, Kim, Baek, & Choi, 2015). Label (marker Test Explanation molecule) Digoxigenin Anti- No problem with endogenous digoxigenin marker molecules. Biotin Avidin Avidin is a glycoprotein. Causes high levels of unwanted binding.

Better than avidin, however it is a Streptavidin large molecule. Weak penetration of tissue. Enzyme (Peroxidase, Has large conjugates and weak Alkaline penetration. Phosphatase) Table 1. Most commonly used label materials

2.3. Stages of In Situ Hybridization It is possible to sort the stages of in situ hybridization as follows. Preparation of Biological Material and Marking of Probe Denaturation Hybridization Fixation and Washing Fastening and Permeability Preparation of the Biological Material and Marking of the Probe: Prior to hybridization, the preparations are subjected to certain procedures to reduce some of the interactions of the probe with structural elements and to prevent binding to molecules outside the target site. The probe can be cloned by multiplying the target sequence with polymerase chain reaction (PCR) or by synthesizing the desired sequence short. The probe should usually be labeled with a modified nucleotide marker, which can be determined by a radioactive isotope or immunohistochemistry. In general, it involves fixing a sample onto the microscope slide, hybridizing the target sequences with a labeled probe, and specifically detecting probe molecules that form target hybrids (Choi et al., 2015). In order to detect DNA sequences, probe hybridization of intracellular RNA molecules is 14 • Health Sciences Research Papers prevented. Removal of endogenous RNA reduces unwanted soil staining. The endogenous biotin is removed by the acetylation process, which is a suitable step for in situ hybridization(Zouari, Campuzano, Pingarrón, & Raouafi, 2017). Denaturation: Target sequences and probe denaturation should be performed for ISH. Denaturation is carried out by application of acid, alkali or heat. Because it is a more effective and simple application, the most preferred control method is heat. Proteases digest proteins that act as preservatives to target nucleic acids and enhance tissue permeability, allowing probes and labels to be easily introduced into the tissue. If the applied protease density is not sufficient, hybridization occurs under the standard. In this case it is necessary to increase the density of the protease used. If the samples are damaged, the density should be reduced again. Triton-X and Sodium Dodecyl Sulfate (SDS) are the most commonly used agents. The endogenous enzyme in the preparation should be denatured in the presence of the enzyme preferred as the probe (Singh, Verma, & Arora, 2015). It may be necessary to resuspend preparations following the administration of protease. Fixation prior to hybridization is performed to prevent the loss of nucleic acids in the preparation. For in situ hybridization between two DNAs, the probe and target DNA sequences should be denatured (Wang, Lim, & Son, 2014). Hybridization: Once the target nucleic acids and probe are single stranded, they are incubated at 50-55 °C for RNA and RNA hybrids, and overnight at 37 °C for DNA and DNA hybrids. There is a direct proportion between probe length and density. The labeled nucleic acid probes are placed in a hybridization mixture containing blocking DNA or tRNA, dextran sulfate, formamide, SDS, various salts and bovine serum albumin (BSA) (Tang et al., 2015). Dextran sulfate; increases the rate of hybridization. Formamide; is used in denaturation and hybridization solutions. It acts at the appropriate temperature for a reaction so as not to disturb the tissue morphology. SDS; increases the permeability of the tissue and allows the probe to enter the tissue. BSA; reduces the non-specific interactions of the probe with the tissue. Arzu GEZER • 15

Salts; stabilize the hybrids of the nucleic acids while adjusting the ionic strength of the denaturation and hybridization solutions (Carter et al., 2015). The hybridization application can be completed in about 3-5 hours. However, overnight incubation increases the accuracy of the procedure. Leme Washings after hybridization is only applied to release the correct matched ones and to clean the weakly connected probe. RNA probes are prone to adhesion resulting in an intense ground signal. For this reason, RNase administration can be performed as a posthybridization procedure (Jaouannet et al., 2017). Fixation and Washing: The fixation phase also includes permeabilization of the cells. The cells to be analyzed are fixed to maintain their original shape integrity, which makes them permeable to short oligonucleotides. Commonly preferred fixation solutions are alcohols and aldehydes. While alcohols precipitate the protein, the aldehydes cross cell membranes (Carev et al., 2017; Carter et al., 2015). The duration of tissue fixation is of no importance for hybridization activity, but optimal signal quality is achieved by fixing overnight (24 hours). Good adhesion of the tissue is ensured by keeping it in the oven overnight at 56 °C using positively charged signalized slides (Bogdanovska-Todorovska, Petrushevska, Janevska, Spasevska, & Kostadinova-Kunovska, 2018). Washing is the removal of excess and non-hybridized probes, and this step is performed sequentially. Washing steps affect the frequency conditions and overall hybridization. Washing temperature, salt concentration and incubation time in washing are important parameters for hybridization (Carev et al., 2017). Fixation and Permeability: At this stage, an appropriate amount of biological material or tissue sample (typically 1 ml) should be taken and centrifuged for 3 minutes. The sample is washed once with phosphate buffered saline (PBS) and 1 ml of PBS is added and mixed using a vortex. The solution is centrifuged at 13,000 g for 3 minutes and the supernatant removed. The pellet (sedimentation at the bottom after centrifugation) is resuspended in 0.25 ml of PBS and then mixed with 0.75 ml of paraformaldehyde (PFA). Subsequently, the sample is centrifuged at 13,000 g for 3 minutes and the supernatant removed. After fixation, the cells are washed, centrifuged at 13,000 g for 3 minutes and the supernatant 16 • Health Sciences Research Papers is removed and 1 ml of PBS is added to the mixture. Consequently, the sample is centrifuged at 13,000 g for 3 minutes, the supernatant is removed and PBS and absolute ethanol (1: 1, v / v) are added to the mixture. This suspension can be stored at -20 °C for a long period (about 1 year) (Kulikova, Franken, & Bisseling, 2018; Nakaya, Tanabe, Takamatsu, Hosokawa, & Mitani, 2017).

3. Fluorescence In Situ Hybridization (FISH) This technique combines molecular and cytogenetic techniques. Standard banding methods are insufficient to query the entire genome for a cell in only one experiment. However, FISH provides a good evaluation with banding methods as a method in which chromosomes and genes are used in dyeing fluorescent molecules (Pekar-Zlotin et al., 2015). It is a method that allows the desired DNA region of the genome to be observed in situ by staining with fluorescent RNA or DNA probes. Interphase allows evaluation of metaphase chromosomes and cell nucleus. However, fluorescence in situ hybridization is often used with fluorescently labeled oligonucleotide probes. It can be applied to morphologically conserved chromosome preparations, tissue sections or fixed cells (Williamson et al., 2014). Fluorescent marking is a process that forms a signal with the formation of a hybrid structure between the probe according to the principle of in situ hybridization. FISH performs the same function as the Southern blot technique. FISH is a technique using labelling with fluorescent signals, requiring complementary DNA, short sequences of single-chain DNA (probes) that undergo hybridization or hybridize in order to see the placement of these DNA sequences (ÖZDEMİR et al., 2015). This method is used for the identification of chromosome aberrations and gene mapping. While many of the preferred methods for studying chromosomes are active dividing cells (metaphase chromosomes), FISH can also study cleaved (interphase nucleus) cells (Liew, Rowe, Clement, Miles, & Salama, 2016). It is used to determine the number of metaphase deletions and chromosomes in the interphase phase and to rearrange chromosomes. Although FISH has been developed with well-defined applications, if the selection of the filter is not done correctly, and if the appropriate probe is not selected incorrect probes will arise. For the FISH method, preparations should be prepared using interphase nucleus or metaphase chromosomes. After dehydration in ethanol, DNA is denatured at 70 °C. For hybridization Arzu GEZER • 17 by denaturation of the labeled probe, incubation is performed at 37 °C for 4-16 hours (Hu et al., 2017). For the FISH technique, one example of the sample to be analyzed is sample fixation with ethanol and paraformaldehyde, hybridization of fluorescently labeled probes, washing of non-hybridized probes, and finally microscopic visualization by fluorescent or confocal scanning laser microscopes (Williamson et al., 2014). The FISH method has made a significant contribution to cytogenetics, including prenatal chromosome diagnosis, cell biology, tumor cytogenetics, biological dosimetry, chromosome evolution, and gene mapping (Wedi et al., 2017). It is also used for characterization of bacteria. It analyzes the structure composition of bacteria and determines the numerical aspects of bacteria. In general, the bacterial ribosomes comprise a copy of each of the 5S, 16S and 23S subunits. Bacterial phylogeny focuses on 16S rRNA (Machado, Castro, Cereija, Almeida, & Cerca, 2015) Hybridization in FISH is the binding of fluorescently labeled probes to ribosomal oligonucleotides in fixed cells. For hybridization, the presence of target nucleotides depends on the conditions of hybridization hardness with fluorescently labeled probes and target nucleotides. The hardness of the hybridization is optimal for the specific binding of labeled probes to target oligonucleotides. This can be counted as temperature, salt concentration and denaturation concentration (Pernthaler, Glöckner, Schönhuber, & Amann, 2001). Other types of FISH can be listed as follows: Fiber FISH Multicolor - FISH Two Color FISH

3.1. Fiber FISH Chromosome fibers are concentrated in the metaphase. However, the fluorescence obtained with the probes can be detected at a distance of 2-3 Megabases (Mb), while at close distances, the probe radiation is superimposed and the signal obtained is perceived as a group. If this hybridization is carried out in the interphase and the fibers can be 18 • Health Sciences Research Papers monitored clearly, the distances between the two probes is reduced to 50 kilobases (kb) in order to monitor the signal (Machado et al., 2015). High resolution chromosome mapping is possible by labeling DNA fragments with fluorescent dyes using chromatin yarns, which are shortened and start to thicken on the interphase. Labels of several hundred kilobase in the interphase phase can be determined by comparing with each other when they are marked with different colors. By determining the genes around the gene studied with the Fiber FISH method, information can be obtained about the presence of the gene in the field (Williamson et al., 2014).

3.2. Multicolor - FISH This is an application developed based on the FISH method in later years. 3.2.1. Spectral karyotyping (SCI): Fluorescent In situ hybridization is one of the main types of this technique; each of the 5 fluorochromes based on 24 chromosome staining probes prepared with different mixtures and simultaneous hybridization of 24 chromosomes and a single filter fluorescence microscope. With this method, 31 different targets can be distinguished and all chromosomes in the metaphase plate are displayed in different colors (Machado et al., 2015). Measurement of radiation emissions is effective in the evaluation of hybridization. Chromosomes are observed in their own wavelength or fluorescence. With spectral karyotyping, less than 1.5 Mb component changes can be detected. Genome changes that cause cancer can be more precisely understood than with clinical banding methods. Arrangements occurring in nucleic acid sequences or chromosomes can be easily determined (Leach & Jackson- Cook, 2001). 3.2.2. Multiplex-FISH (M-FISH): This is a method based on the understanding of hybridization with probes prepared with different combinations of 5 fluorochromes and different fluorescent signals. Only CM-multiplex FISH applied for centromeres and TM-Multiplex FISH techniques for telomeres are available (Huc, Sapierzynski, Rygier, & Pienkowska-Grela, 2015). 3.2.3. Cobra-FISH = Combined binary ratio: This is similar to other FISH methods. However, the difference with other methods is that 12 of 24 chromosomes can be divided into one group and the remaining 12 may Arzu GEZER • 19 be divided into other groups. While the first group is hybridized with 3 different fluorochromes, a fourth fluorochrome is used in addition to the second group. This technique can also be performed with the 5th fluorochrome as it provides specific staining for chromosome arms (Liehr, Othman, & Rittscher, 2017). Genomic In Situ Hybridization: A molecular cytogenetic method that analyzes the number of copies in the DNA sequence throughout the genome. With this method it is possible to determine the amount of chromosomal fragments between the different numbers of chromosomes, the number of chromosomal abnormalities such as amplification, duplication, insertion or deletion, or the number of copies of the chromosomes or the trisomy and monosomy regions on the DNA (Balajee & Hande, 2018; Kallioniemi et al., 1992). 3.3. RxFISH = (Cross-species color) As probes, gibbon chromosomes showing approximately 98% similarity with the homology of the human chromosome are used. With this method both chromosomal part changes and chromosomal changes can be determined. When used with G-banding technique, it gives 400 times more sensitive results (Williamson et al., 2014).

3.4. Comparative genomic hybridization (CGH) For the first time, Kaolinimi and colleagues used this method with the purpose of demonstrating the reduction or increase in DNA copy numbers. Thus, it was possible to identify chromosomal disorders more comprehensively and in detail (Kallioniemi et al., 1992). The CGH technique is a molecular cytogenetic technique which is based on the FISH technique, which shows fluorescent color differences obtained by binding different fluorescent dye stained reference and patient DNA samples to normal chromosomes. With this method, amplification or chromosomal loss of a certain region is shown in patient DNA (Zhang, Cerveira, Rens, Yang, & Lee, 2018). With the use of two-color imaging, it is more advantageous to determine chromosomal anomalies on a metaphase plate more reliably than normal karyotype analysis and to compare at least two genomes with the M-FISH method. Genomic DNA was obtained from the reference and test cells and the resulting DNA samples were obtained with different color fluorochromes [Cy3 (green) and Cy5 (red)], and the detection of amplified 20 • Health Sciences Research Papers regions or losses in chromosomes was noted according to color differences (Shalon, Smith, & Brown, 1996). Genomic DNA containing a large amount of IRS (interspersed repetitive sequence) is used as a probe for CGH. DNA probes are combined with Cot 1 DNA to suppress non-specific hybridization signals from IRSs. If the denaturation of chromosomal DNA in the preparation is not sufficient, good hybridization cannot be established (ÖZDEMİR et al., 2015). With conventional epifluorescence microscopy, fluorescent signals are monitored and displayed with a camera system. With this method, the aim is to hybridize test and control DNAs on the metaphase plate without anomaly. A more accurate and detailed analysis of the unstable material in the genome is used to identify unspecific deviations in a genome that is particularly stable. It is also used to screen the tumor genome for gene amplification and to determine the chromosomal location of amplification and has the advantage of analyzing all genomes in a single experiment (Liehr et al., 2017).

3.5. Array based genomic hybridization (A-CGH) Cytogenetics, FISH and molecular methods have recently been included in routine examinations for leukemia. In terms of diagnosis, classification, prognosis, follow-up and response to treatment criteria, cytogenetic, FISH and molecular genetic analyses are used. In cytogenetic analysis, chromosomal abnormalities> 5-10 Mb in DNA detect smaller submicroscopic changes (1-3 Mb) in FISH (Poorman et al., 2015).

3.6. Two Color FISH It is possible to make two-color or one-color FISH using one or two different probe DNAs. Two different probe DNAs can be labeled with two different modified nucleotides with a two-color FISH technique. These probes are co-hybridized on the same metaphase chromosomes. Thus, the location and distribution of two independent DNAs can be determined on the same chromosome set. The analysis to be made in this way allows determination of the relationship and organization which cannot be determined with any other method (Pekar-Zlotin et al., 2015). With ISH, viral, fungal and bacterial organisms can be detected morphologically. This method was reported as a more valid and ideal molecular method than the detection of EBV latent membrane protein by Arzu GEZER • 21

Epstein Bar Virus (EBV) expression or the immunohistochemical (IHC) method by PCR technique. With the advantage of rapid results, ISH is also very illuminating for understanding the relationship between DNA sequences and chromosome biology. This association, especially when combined with FISH and immunofluorescence, can combine properties of the chromatin structure with control of gene expression. Today, toxicological studies, cancer research and chromosomal anomalies are preferred as genetic diagnosis methods and molecular genetic analysis is much more sensitive with this technique, making it possible to determine the status of minimal residual disease (MRD).

22 • Health Sciences Research Papers

REFERANCES Balajee, A. S., & Hande, M. P. (2018). History and evolution of cytogenetic techniques: Current and future applications in basic and clinical research. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 836, 3-12. Beliveau, B. J. (2015). Oligopaints: Highly programmable oligonucleotide probes forvisualizing genomes in situ: Harvard University. Bogdanovska-Todorovska, M., Petrushevska, G., Janevska, V., Spasevska, L., & Kostadinova-Kunovska, S. (2018). Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience. Bosnian journal of basic medical sciences, 18(2), 132. Carev, I., Ruščić, M., Skočibušić, M., Maravić, A., Siljak‐Yakovlev, S., & Politeo, O. (2017). Phytochemical and cytogenetic characterization of Centaurea solstitialis L.(Asteraceae) from Croatia. Chemistry & biodiversity, 14(2), e1600213. Carter, J. M., Caron, B. L., Dogan, A., & Folpe, A. L. (2015). A novel chromogenic in situ hybridization assay for FGF23 mRNA in phosphaturic mesenchymal tumors. The American journal of surgical pathology, 39(1), 75-83. Choi, Y. S., Kim, Y. C., Baek, K. J., & Choi, Y. (2015). In situ detection of bacteria within paraffin-embedded tissues using a digoxin-labeled DNA probe targeting 16S rRNA. JoVE (Journal of Visualized Experiments)(99), e52836. Evyapan, G., Ay, G., Cömertpay, G., & Lüleyap, H. Ü. (2019). Tümörogenezisde endoplazmik retikulum stres cevabının rolü. Cukurova Medical Journal, 44(1), 241-248. Hu, L., Yin, X., Sun, J., Zetterberg, A., Miao, W., & Cheng, T. (2017). A molecular pathology method for sequential fluorescence in situ hybridization for multi- gene analysis at the single-cell level. Oncotarget, 8(31), 50534. Huc, T., Sapierzynski, R., Rygier, J., & Pienkowska-Grela, B. (2015). Diagnostyka cytogenetyczna-kliniczne zastosowanie w medycynie weterynaryjnej. Życie Weterynaryjne, 90(10). Jaouannet, M., Nguyen, C. N., Quentin, M., Jaubert-Possamai, S., Rosso, M.-N., & Favery, B. (2017). In situ hybridization (ISH) in preparasitic and parasitic stages of the plant-parasitic nematode Meloidogyne spp. Bio- protocol, 8(6), np. Kallioniemi, A., Kallioniemi, O.-P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F., & Pinkel, D. (1992). Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science, 258(5083), 818-821. Arzu GEZER • 23

Kocks, C., Boltengagen, A., Piwecka, M., Rybak-Wolf, A., & Rajewsky, N. (2018). Single-molecule fluorescence in situ hybridization (FISH) of circular RNA CDR1as. In Circular RNAs (pp. 77-96): Springer. Kulikova, O., Franken, C., & Bisseling, T. (2018). In Situ Hybridization Method for Localization of mRNA Molecules in Medicago Tissue Sections. In Functional Genomics in Medicago truncatula (pp. 145-159): Springer. Leach, N. T., & Jackson-Cook, C. (2001). The application of spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) technology to determine the chromosomal content (s) of micronuclei. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 495(1-2), 11-19. Liehr, T., Othman, M. A., & Rittscher, K. (2017). Multicolor karyotyping and fluorescence in situ hybridization-banding (MCB/mBAND). In Cancer Cytogenetics (pp. 181-187): Springer. Liew, M., Rowe, L., Clement, P. W., Miles, R. R., & Salama, M. E. (2016). Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system. Journal of pathology informatics, 7. Machado, A., Castro, J., Cereija, T., Almeida, C., & Cerca, N. (2015). Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method. PeerJ, 3, e780. Mukai, Y. (2017). In situ hybridization. In Plant Chromosomes (pp. 155-170): CRC press. Nakaya, M., Tanabe, H., Takamatsu, S., Hosokawa, M., & Mitani, T. (2017). Visualization of the spatial arrangement of nuclear organization using three-dimensional fluorescence in situ hybridization in early mouse embryos: A new “EASI-FISH chamber glass” for mammalian embryos. Journal of Reproduction and Development, 2016-2172. ÖZDEMİR, K., MURANLI, F. D. G., & KANEV, M. O. (2015). In situ Hibridizasyon Yöntemleri. Düzce Üniversitesi Bilim ve Teknoloji Dergisi, 3(2). Pekar-Zlotin, M., Hirsch, F. R., Soussan-Gutman, L., Ilouze, M., Dvir, A., Boyle, T., . . . Palmer, G. A. (2015). Fluorescence in situ hybridization, immunohistochemistry, and next-generation sequencing for detection of EML4-ALK rearrangement in lung cancer. The oncologist, 20(3), 316-322. Pernthaler, J., Glöckner, F.-O., Schönhuber, W., & Amann, R. (2001). Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. Methods in microbiology, 30, 207-226. 24 • Health Sciences Research Papers

Poorman, K., Borst, L., Moroff, S., Roy, S., Labelle, P., Motsinger-Reif, A., & Breen, M. (2015). Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization. Chromosome Research, 23(2), 171-186. Shalon, D., Smith, S. J., & Brown, P. O. (1996). A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome research, 6(7), 639-645. Singh, A., Verma, H., & Arora, K. (2015). Surface plasmon resonance based label- free detection of Salmonella using DNA self assembly. Applied biochemistry and biotechnology, 175(3), 1330-1343. Tang, K. F., Pantoja, C. R., Redman, R. M., Han, J. E., Tran, L. H., & Lightner, D. V. (2015). Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp. Journal of invertebrate pathology, 130, 37-41. Wang, X., Lim, H. J., & Son, A. (2014). Characterization of denaturation and renaturation of DNA for DNA hybridization. Environmental health and toxicology, 29. Wedi, E., Müller, S., Neusser, M., Vogt, P., Tkachenko, O., Zimmer, J., . . . Nayudu, P. (2017). Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes. Reproduction, Fertility and Development, 29(5), 913-920. Williamson, I., Berlivet, S., Eskeland, R., Boyle, S., Illingworth, R. S., Paquette, D., . . . Bickmore, W. A. (2014). Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization. Genes & development, 28(24), 2778-2791. Zhang, C., Cerveira, E., Rens, W., Yang, F., & Lee, C. (2018). Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome. Current protocols in human genetics, 99(1), e70. Zouari, M., Campuzano, S., Pingarrón, J., & Raouafi, N. (2017). Competitive RNA-RNA hybridization-based integrated nanostructured-disposable electrode for highly sensitive determination of miRNAs in cancer cells. Biosensors and Bioelectronics, 91, 40-45.

MALE REPRODUCTIVE SYSTEM IN LABORATORY ANIMALS

CHAPTER 2

Arzu GEZER1

1 Atatürk Üniversitesi Sağlık Hizmetleri Meslek Yüksek Okulu Erzurum, [email protected]

Arzu GEZER • 27

1. INTRODUCTION Animals used in research and biological studies designed according to scientific rules are called experimental animals or laboratory animals. The reasons why these animals are preferred as experimental animals are; ease of cultivation, ease of performing complex genetic applications, short pregnancy duration and sexual cycles, easy feeding and care, no need for large cultivation areas and sufficient knowledge gathered over many years. According to the International Laboratory Animals Committee; 40% to 80% of the preferred laboratory animals were reported to be mice (Akman, 2007). Laboratory animals can be listed as follows according to the frequency of experiments. 1. Mice 2. Rats 3. Rabbit 4. Guinea pigs The male reproductive system and production, nutrition and storage of haploid male sex cells is responsible for the production and secretion of male sex hormones. Male reproductive system organs include effluent channels that transmit these cells from testes that express gonad cells and secrete androgens, of the tubuli recti, , ductuli efferentes, ductus , ductus deferens, ductus ejaculatorius and ; and the gland, seminal vesicle and bulbourethral glands and external genital (Bernal, Aya, De Jesus-Ayson, & Garcia, 2015). 2. TESTIS The testes are a couple of organs that have an exocrine and endocrine function, which are responsible for the production of steroid hormones (steroidogenesis) and the production of gametes (gametogenesis), and affect sexual maturation and reproductive function. The exocrine function starts the production of spermatozoa in puberty and develops secondary sex characteristics, and the endocrine function ensures continuity of spermatozoa production in adults, protection of secondary sex characteristics and affects the function of the auxiliary glands (Prihatno et al., 2018; Schatten & Constantinescu, 2017). 28 • Health Sciences Research Papers

The testes are suspended in the so-called by the , which is called the funiculus spermaticus, including the ductus deferens, the cremator muscle, the spermatic artery and the pampiniform plexus veins. They are found in the abdomen until near birth, but shortly before birth they pass through the inguinal canal to the scrotum and have a temperature 2-3 C below body temperature (Ross, Pawlina, & Barnash, 2016). However, it was determined that the descend into the scrotum in the reproductive period and then withdraw into the abdominal cavity in rabbits and bats (Alves-Lopes & Stukenborg, 2017). In wild rabbits, it was reported that the testicles were in the scrotum in the month of July and that they were pulled towards the canal inguinalis in October and November (Kılınç, 2015). In humans and in most mammalian animals, the testes are in an ovoid-shaped double organ, while the rat has a single pouch and the shape is close to round. In the rat, it was observed that the testes are located anterior ventral with preputium and the anus (ANTEPLİ & Beyaz, 2017). In the rabbit, the testes have a flat oval shape and are located in the scrotum between the hind legs, which is slightly across the long axis of the body, slightly in the 2-month-old rabbit, and clearly visible by 4-6 months of age. Additionally, rabbit testes were found to be extremely advanced during adolescence. It was reported that the spermatic cord is longer in the rabbit than in guinea pigs and rats and the ductus deferens is located in the dorsal aspect of the corpus epididymis. The testicles are oval in shape, larger in comparison to rabbit and rat testes, and fuller than the rabbit's testicles, flush between the two hind legs and in a single pouch. Almost all values for the left testis (hormone and biochemical) in rabbits, guinea pigs and rats are stated to be higher than the right (Powers et al., 2019). The testes are wrapped in a three-layer capsule from the outside to the inside. -: A double-leaf serous membrane formed by mesothelial cells originating from the peritoneum. -: A large amount of irregularly settled collagen bundles, a smaller amount of elastic yarns and muscle fibers are found in the connective tissue structure. Tunica albuginea forms a which thickens at the back of the testis and provides blood and lymphatic vessels enter the organ and discharge channels from the organ. The fibrous connective tissue compartments inserted into the testis from Arzu GEZER • 29 the mediastinum testis separate the into 250-300 lopps (lobuli testis) (Rodriguez et al., 2017). -Tunica vasculosa: This blood vessel rich, loose connective tissue structure is the innermost layer (Bucci et al., 2017). 2.1. Seminiferous Tubules: Seminiferous tubules are tubules with a diameter of 150-300 vem in most animal species, and a highly curved event that begins at the blunt end of 30-70 cm and ends in the rete testis (Gümüşel, 2013). There are about 30 separate tubules in rats. While 80% of the testicles of adult animals are formed of seminiferous tubules, the remaining 20% of the testes comprise supporting connective tissue (Wahyuni et al., 2018). Seminiferous tubules are covered with multiple-layer germinal epithelium. These tubules include collagen and reticular fibers and loose interstitial connective tissue consisting of various connective tissue cells. This connective tissue contains a large amount of blood and lymphatic vessels and interstitial cells (Leydig cells) (X. Li et al., 2016). A from the outside to the lumen; Lamina propria, Basal lamina and Seminiferous epithelium (Gümüşel, 2013). Lamina propria: Contains type I collagen fibers, fibroblasts, actin filaments, and myoid cells. The myoid layer, which has rhythmic contractile activity and helps in the progression of non-motile sperm to the retic testis, has 3-5 rows in humans and mammals but has a single row in rodents and a flat sequence (Wahyuni et al., 2018). There is an increase in thickness of the lamina propria with aging. This thickening is accompanied by a reduction in the rate of sperm production and a general reduction in the sizes of seminiferous tubules. In the early development period, thickening of lamina propria and infertility are reported to be related (X. Li et al., 2016). Basal lamina: The seminiferous tubule has a continuous basal membrane. The electron microscope usually observes close myoid cells in the basal membranes (Şahin, 2016). 30 • Health Sciences Research Papers

Seminiferous epithelium: This is located on the basal lamina and contains germ cells in various stages along with Sertoli cells (Şahin, 2016).

2.2.1. In 1865, Italian scientist Enrico Sertoli discovered the surface cells of the testis, also known as support cells, extending from the basal laminate to the lumen of the seminiferous tubules, are prismatic and the only non- germinal cells in the seminiferous tubules (Dias et al., 2017). These cells, divided into 4 parts as basal part, main body, lateral cell extensions and apical cell surface, are morphologically divided into two different categories called type A and type B. Type A are mature that are ready to be discharged into the lumen in Sertoli cells, and they are located very close to hard spermatids and pachytene among the Sertoli cell crypts. In contrast, type B Sertoli cell spermatids are elevated and close to the lumen. Sertoli cells, seen during stage V of the seminiferous epithelium cycle, have more of type A, VI-VIII. Sertoli cells seen through the stages are evaluated as type B Sertoli cells. This situation is very important in the formation of epithelial cells along with germ cells (Dimitriadis et al., 2015). The Sertoli cell number is determined only in the prepubertal period, since only immature Sertoli cells are capable of proliferation. The main characteristics of Sertoli cells, such as proliferation period, varies between species. They multiply in the peripubertal period and fetal or neonatal period in humans (Franca, Hess, Dufour, Hofmann, & Griswold, 2016). Sertoli cell proliferation in rodents occurs in the fetal and neonatal period, whereas Sertoli cells in rhesus monkeys proliferate in the peripubertal period. The proliferation of adult Sertoli cells in rats begins on the 16th day of fetal life and reaches the maximum level on day 2 after birth. There are 1 million Sertoli cells in the rat testis at birth, and with increasing proliferative activity there are about 40 million on the 15th day after birth. From the 15th day onwards, the proliferation stops and differentiation and maturation begin; after that, it is assumed that the numbers remain stable throughout adult life (Hutson & Lopez- Marambio, 2017). Arzu GEZER • 31

Sertoli cells mature for various functions. A disorder in any of its functions may damage the production of spermatozoa (Cheng & Mruk, 2015). Functions of Sertoli cells: 1) To support, nurture and protect developing spermogenic cells, 2) To phagocyte the residual bodies discarded by spermatids at the end of , 3) To secrete androgen binding protein (ABP), 4) To facilitate the release of mature spermatids into the seminiferous tubule lumen, a process called sperming, 5) To produce and release a liquid rich in ions and proteins from the seminiferous tubule lumen. 6) To secrete inhibin and activin (inhibin, FSH secretion on negative, activin positive effect). 7) To provide differentiation of male genital release channels during the development of anti-Müllerian hormone secretion and thus Muller channels suppressing Wolff channels, 8) To establishing the blood-testicular barrier (Aktaş, 2017; Hutson & Lopez-Marambio, 2017). Sertoli cells in the testicles of domestic mammals contain varying amounts of lipid and glycogen inclusions. Microfilaments, microtubules and granulous endoplasmic reticulum are abundant in the structure, while few granular endoplasmic reticula are known. The nuclei of the Sertoli cells are corrugated. On average, 10 Sertoli cells are seen in the cross- sections of seminal tubules in an adult male (Şahin, 2016). These cells are closely linked to each other through the tubules external Zonula Occludens, adhesion connections sp Zonula Adherens and their communication links “Gap Junction” and thus form the blood-testicular barrier, which protects the developing spermatocytes and spermatids from autoimmune reactions and other harmful external factors. The blood-testicle barrier in seminiferous epithelium is divided into three areas; two permanent (basal and adluminal) and a temporary (intermediate) area (Cheng & Mruk, 2015). 32 • Health Sciences Research Papers

Basal area: and spermatocytes in the early phase of meiosis (preleptotene and leptotene spermatocytes) are found in this area. Adluminal area: This is the part close to the lumen. This area hosts all other germ cells. Intermediate area: Formed from the basal to the adluminal area during the passage of leptotene cells through the destruction and re-renewal of tight junctions. The presence of the intermediate area allows protection of the Sertoli cell integrity from the basal to the adluminal area during the passage of the cells (Kılınç, 2015). Cells associated with the basal membrane are only Sertoli cells and spermatogonium. More mature germ cells are located in the spaces between the Sertoli cells. These germ cells move towards the lumen as they mature (Fujitani, Otomo, Wada, Takamatsu, & Ito, 2016).

2.2.2. Spermatogenetic Cells 2.2.2.1. Spermatogonium: The basal membrane has 12-15 cells around the diameter. Since the age of sexual maturity, mitosis starts to multiply and create new cells. In the final stage of A pale (Ap) and A dark (Ad) types, Aβs are transformed into spermatogonium type B, after several mitoses these spermatogonium Bs undergo a meiotic division and form spermatocytes I (Fujitani et al., 2016). 2.2.2.2. Spermatocytes: Spermatocytes I, composed of mitotic division of type B spermatogonia, contain 46 chromosomes (diploid) (44 + XY) and 4N DNA. Immediately after mitosis, these cells enter the prophase stage of meiosis, which takes about 22 days. I is approximately 16 cells in size and is the largest of the spermatogenic cells. After the first meiotic division, smaller cells with 23 chromosomes (haploid) are formed, which are called spermatocytes II. The amount of DNA in these cells is halved. The division of these cells by spermatocyte I is very short, so they are rarely seen in the testicular sections. Spermatids occur as a result of division of spermatocytes II (Baysal, 2017; Cheng & Mruk, 2015). 2.2.2.3. Spermatids: Spermatids formed after the second meiosis of spermatocytes have a haploid chromosome and these cells are close to the lumen of the seminiferous tubule. Spermatids undergo a differentiation process called spermiogenesis. Spermatozoa take various names according to the stages they are in during the formation. Generally, they are small Arzu GEZER • 33 elongated cells and have a large nucleolus. Spermiogenesis is the last stage of spermatogenesis and spermatids take the form of spermatozoa that are specialized in transferring male DNA into the ovary at this stage (Ge et al., 2005; Zelený, 2017).

2.3. Seminiferous Epithelium Cycle Different steps are expressed as seminiferous epithelial cycles until the same cell group model seen anywhere in the seminiferous tubule appears a second time. In the cyclic functioning of spermatogenesis, each distinct cell cluster is defined as a step (Morais et al., 2013). For each type, the number of steps in the cycle is constant, while the duration and number of cycles of the seminiferous tubule cycle varies by type. For example, for rats this is 4 cycles of 4 days, 4 cycles of 6-8 days for mice and 6 cycles of 16 days for humans. In other words, a cycle of 6 cycles is seen in people and one cycle lasts 16 days. In rabbits this cycle was divided into eight phases by Swierstra and Foote (1963) (Swierstra & Foote, 1963). As human components appear as irregular patches in a mosaic pattern, these stages are not as clearly observed as in rodents (Baysal, 2017; Nyongesa, Patel, Wango, & Onyango, 2017). 2.4. Spermatogenesis: The sequence of events from spermatogonium to spermatozoa is called spermatogenesis and occurs in 3 stages: Spermatocytogenesis, Meiosis, and Spermiogenesis (Zelený, 2017). 2.4.1. Spermatocytogenesis: During spermatogenesis, spermatogonium proliferates with mitosis, and A-spermatogonium, I- spermatogonium (intermediate), B-spermatogonium, and eventually preleptotene primary spermatocytes are formed. Primary spermatocytes do not divide by mitosis after this stage, and they pass 2 meiosis divisions, which quadruple the number of germ cells (Tan, 2015). Cells that provide the widest contact with the basal lamina of the largest spermatogonium and seminiferous tubules are A-spermatogonium. This has a prominent nucleolus, pale and blurry-looking nuclei. Spermatogonium B has a round core containing a large amount of chromatin particles and are connected to each other by cytoplasmic extensions. As a result of splitting of spermatogonium, preleptotene primary spermatocytes are formed. Preleptotene primary spermatocytes gradually lose their contact with the basal laminate and tend to interfere with adipinal adenomas between the 34 • Health Sciences Research Papers cells of the Sertoli cells (Fouladi-Nashta, Raheem, Marei, Ghafari, & Hartshorne, 2017; Zahrina, 2015). 2.4.2. Spermiogenesis: The differentiation of spermatids to spermatozoa is called spermiogenesis. The most important morphological changes that occur during spermiogenesis are formation of acrosome, condensation of core chromatin, growth of a moving tail and loss of unnecessary parts for spermatozoa which will occur later in material. Spermiogenesis consists of Golgi, cap, acrosomal and maturation stages (Vernet et al., 2016). 2.4.3.1. Golgi stage: Proacrosomal granules appear in Golgi vesicles. The proacrosomal granules melt and fuse with each other to form an acrosomal granule in a single acrosomal vesicle. These two structures determine the anterior pole of the sperm head which will be formed by contacting the inner side of the nucleus (Aitken, Gibb, Baker, Drevet, & Gharagozloo, 2016). 2.4.3.2. Cap phase: The acrosomal vesicle grows and forms the head cap that covers the 2/3-inch front of the core. In the late cap phase, immobile spermatids are polarized and the core and head cap are displaced eccentrically. Two centrioles are collected at the caudal pole of the nucleus and the distal centriole leads to the growth of flagellum (Morrow et al., 2017). 2.4.3.3. Acrosome Stage: In the early acrosomal phase, the nucleus and cell body begin to grow and the nuclei of the spermatids are directed towards the periphery of the tubules and their tails towards the lumen. Spermatids previously separated from the lateral extension of Sertoli cells are then embedded in the apical recesses of these cells. Once the nucleus starts to grow, the core histones are replaced by essential proteins. After this concentration, the DNA gains resistance. Protamine must be replaced by histones provided by oocytes before the condensation of the chromatin during fertilization (Vernet et al., 2016). 2.4.3.4. Maturation stage: The core concentration is completed at this stage. In the middle part of the , most of the mitochondria are collected in helix style around the axoneme. The outer fibrillary system and fibrous sheath of the main part develop. The volume of spermatid in the advanced stages of the maturation stage is 20% -30% of the spermatid in the cap phase. Autolysis is often held responsible for this reduction. Arzu GEZER • 35

Before the spermisation, the cytoplasm bridges between the spermatids in the maturation phase are separated from each other and the residual cytoplasm is removed as a residual body. The residual bodies have different ends, they either become phagocytosed by the Sertoli cells or disappear in the tubular lumen or they are rapidly autolyzed by connecting to the apical edges of the tubule epithelium (Aitken et al., 2016). 2.5. Spermatozoa: spermatozoa with lengths of approximately 60-75 spm under a light microscope have two parts; a head and tail. These comprise parts such as neck, middle piece, main piece and end piece (Vernet et al., 2016). 2.5.1. Head: The spermatozoon head, which is species-specific and therefore differs between species in both shape and size, is full of nuclei with a large proportion of genetic material. The shape of the head determines the shape of the nucleus. The top 2/3 of the core is surrounded by the acrosomal cap, which contains a large number of hydrolytic and proteolytic enzymes. Acrosomal enzymes are necessary for perforation of the zona pellucida during fertilization. The core is wrapped with a postacrosomal sheath containing sulfur-rich proteins (Vernet et al., 2016). The core membrane forms a joint-like groove on the caudal surface of the sperm head (Kim et al., 2017). The shape of the head has been reported to be important for fertilization (Górski, Kondracki, Wysokińska, & Nazaruk, 2016). Human; the head is oval, the borders are smooth, the core is composed of acrosomal with a smooth appearance. The vacuole number should be less than 2% and the size of the vacuole should not exceed 20% of the area covered by the head (Mortimer, 2018). Rabbit; the head shape is elliptical, elongated, with rough and regular boundaries (Kolesar et al., 2018). Rats; spermatozoa heads look like hooks. Mice and guinea pigs; the head of the spermatozoa is sickle-shaped (Creasy & Chapin, 2013). 2.5.2. Tail: the tail comprises a neck, middle part, the main parts and the last piece as seen with electron microscope. 36 • Health Sciences Research Papers

2.5.2.1. Neck: A short and narrow formation between the head and the middle part. It consists of a centrally located centriole with 9 fibrils in longitudinal location and with the outer fibrils of the middle part (Stewart, Wang, & Montgomerie, 2016). 2.5.2.2. Middle Part: This has a flagellum structure formed by microtubule and axial filament. These formations are surrounded by outer fibrils, which are longitudinally aligned joined by fibrils in the neck. They are surrounded by energizing mitochondria. Ring-like thickening of the mid-part plasma membrane determines the boundary of the core and the middle part (Kim et al., 2017). 2.5.2.3. Essential Part: This is the longest part of the spermatozoa. The structure of the axial filament complex resembles that of the middle part and continues with the outer fibers of the middle part. Fibrillations are subject to occasional changes in size and shape and gradually become thinner towards the end of the main part (Stewart et al., 2016). 2.5.2.4. End Piece: The last part of the fibrous sheath containing only the axial filament complex determines the beginning of the last piece. In the proximal part of the last piece, this complex consists of 9 pairs of peripheral microtubules which are characteristic, but at the distal, these pairs reduce to one and end at different levels (Garcia-Vazquez, Gadea, Matás, & Holt, 2016).

2.6. Interstitial Tissue of the Testis Also known as interstitial tissue or peritubular tissue, Leydig cells are loose ligaments including blood and lymph vessels, plasma cells, dendritic cells, fibroblasts, macrophages and mast cells (Wheeler, Sharma, Kavoussi, Smith, & Costabile, 2018). In the interstitial tissue organization, some species-specific differences were determined and 3 different interstitial tissue structures were established. In the first group (guinea pigs, chinchilla, rat and mouse), Leydig cells form a small portion of the testicular structure and form piles packed close to the blood vessels. However, a large proportion of interstitium is composed of widespread lymphatic sinusoid (Ross et al., 2016). In the second group that includes many mammals (bull, ram, elephant, ape, and human), Leydig cells are not clearly associated with blood vessels and are in the form of cells of various sizes distributed within the edematous loose connective tissue drained by Arzu GEZER • 37 the central or eccentrically located lymph vessels in the interstitial areas. In the third group (pigs, zebra and blind rats), tightly packed Leydig cells are distributed in large interstitial areas and constitute 20-60% of the testicular structure. In these species, there is very little interstitial connective tissue and small lymphatic vessels are less common (Stévant, Papaioannou, & Nef, 2018). 2.6.1. Leydig Cells: In 1850, Franz Leydig gave the name or interstitial cells to the cells he defined in the interstitial area in mammalian testis (Lebelo & van der Horst, 2017). Like all cells secreting steroids, Leydig cells are also too big comprising lipid droplets, granular endoplasmic reticulum (sER), and tubular crystal mitochondria. There are two different types of polygonal shaped and round nucleated Leydig cells; These are Fetal Leydig cells (FLC) and Adult Leydig cells (ALC). During embryonic development, FLCs in male fetuses produce androgens, which are essential for the development of male characteristics. In the early postnatal period, FLCs are repressed and ALCs from undifferentiated stem cells replace FLCs. ALCs produce testosterone the most important androgen source that is indispensable for sexual differentiation and reproductive functions in men (C. Li et al., 2018). It forms less than 10% of the testicular mass in humans and some other mammals (Tremblay, 2015). The function of Leydig cells are to carry anterior pituitary prolactin, which induces LH and LH receptor expression, which stimulates testosterone production (Öztaş et al., 2019). The main functions of testosterone are the creation of the Wolf channel and the differentiation of this channel to other channels and epididymis, initiation and maintenance of the development of penis, male gland and secondary sexual functions, increasing libido, and control of spermatogenesis with the FSH hormone and it is known to have stimulation and general anabolic effects (Chen, Wang, Ge, & Zirkin, 2017).

3. TESTER'S TRANSMISSION CHANNELS

3.1. Testicular Internal Channels 3.1.1. Tubuli Recti: The last section of each seminiferous tubule with a straight course is called the tubuli rectum. There are only Sertoli cells in the wall. As the lumens of the flat tubules become closer to termination, they open to retic testis (Fietz & Bergmann, 2017). 38 • Health Sciences Research Papers

3.1.2. Rete Testis: This is an anatomized channel which is paved with cubic or low prismatic epithelium. Epithelial cells contain a single apical cilium and a few short microvilli.

3.2. Non-Testicular Channels 3.2.1. Ductuli Efferentes: Ductuli efferentes, whose numbers range from 12 to 25, originate from the rete testis and are organized in a similar manner to a cone with an end point toward the rete testis. Ductuli efferentes are covered with ciliated multi-layer prismatic epithelium. In all species, the ductuli efferentes have two main cell types; ciliated and non-silane. However, a number of intraepithelial lymphocytes and macrophages are encountered (BEYAZ, BAYRAM, & ALAN, 2008). 3.2.2. Ductus Epididymis: The ductus epididymis wall comprises long-branched stereocilia false multi-layer prismatic epithelium and muscle layer. Macroscopically, the epididymis is divided into three different regions: the hood, the corpus and the cauda. Regional differences in the histological structure of the epididymal epithelium were first identified in dogs, rabbits, rats, stallions and bulls. In many animal species, the epididymis is divided into 4 different sections, including the initial segment, hood, corpus and cauda epididymis based on histological and cytochemical characteristics. The first three sections form part of the epididymis related to sperm maturation, while the last part is responsible for storing sperm (Akmal, Masyitah, Hafizuddin, & Fitriani, 2015). Nicander (1979) divided the region into 8 subregions in rabbits according to regional differences, lumen content and width, epithelium height, structure and cytochemical characteristics. However, when similar criteria are taken into consideration in the epididymis of guinea pigs there are 7 subregions, in cats there are 6, in goats 5, and in bulls, rams and stallions there are 6 regions differentiated (Cooper, 2012; Nicander & Plöen, 1979). Epididymis epithelium stereocilia; There are four types of cells: main, basal, apical, and open cells (Castro, Boretto, Blanco, & Acosta, 2018). Main Cells: These cells, also referred to as principle cells, are predominant cell types in the epithelium of the ductus epididymis in mammals, accounting for about 80% of cells in the epithelium. They are prismatic shaped cells extending from the basal to the lumen. Apical faces Arzu GEZER • 39 are branched stereocilia. In the head section of the epididymis, the length of the stereocilia is long, while the tail section is short. The kernels are long and notched. In the upper part of the core, the Golgi complex is quite evident (Mahfud, Winarto, & Nisa, 2016). The cytoplasm contains lysosomes and vesicles. They are known to secrete lycoprotein, carnitine, sialic acid and glycerolphosphorylcholine. They are capable of endocytosis and pinocytosis. Approximately 90% of the testicular fluid is absorbed by the essential cells in the epidural channels and the epididymis (Kuzelova, Vasicek, & Chrenek, 2015). Basal Cells: These are pyramidal shaped cells that are in close contact with the primary cells, sitting on the basal laminate. They are considered to be the undifferentiated precursors of the primary cells (Mahfud et al., 2016). Apical Cells: Ductus epididymis is observed in the head of the epidermis and a rounded nucleus in the lumen is proton pumped by the lumen content of the cells involved in acidification. Cytoplasm is rich in mitochondria (Basu et al., 2018). Open Cells: Ductus epididymis is seen in the tail region. They allow the removal of cytoplasmic residues of spermatozoa. The inner and outer longitudinal muscle layers surround the epithelial and basal lamina, starting with an inner circular smooth muscle layer that increases in thickness from the beginning to the tail. Muscle layers perform peristaltic movement to facilitate the transport of spermatozoa along the canal (Kuzelova et al., 2015). In addition, intraepithelial lymphocytes (halo cells) are observed located within the epithelium along the entire epididymal canal. These cells are important components of the immunological response of the epididymis (Mahfud et al., 2016). 3.2.3. Ductus Deferens (): This is a wide lumen and thick-walled channel with a length of about 40-50 cm and a diameter of 2 mm. It starts from the tail of the epididymis, terminates in the vesicula seminalis secretory channel to form the ductus ejaculatorius. The function of the ductus deferens function is the transport of spermium. Its wall consists of three layers from inside to outside (Kashmiri, Dighade, & Gotmare, 2016). 40 • Health Sciences Research Papers

Tunica Mucosa: Mucosa forms longitudinal folds. Absorption is covered by a stereocilia false multilayered prismatic epithelium with phagocytosis and secretion functions. Epithelial cells are also responsible for the synthesis and secretion of many substances, including glycoproteins. The cell cytoplasm at the apex, which protrudes outward from the stereocilia is called the apical bubble and functions in apocrine secretion (Anisatuzzahro & Luthfi, 2017). Tunica Muscularis: The thickness of this well-developed muscle layer is 1-1.5 mm. Spermatozoa are pushed forward by contraction of the muscle layer, which shows transverse, internal and external lengths (Kashmiri et al., 2016). Tunica Adventitia: This is connective tissue rich in veins and nerves. The bulb with the enlarged portion of the ductus deferens links directly into the prostate gland. The epithelium in the ampulla thickens and folds and the lumen is wide. Here, spermatozoa accumulate (Kashmiri et al., 2016). 3.2.4. Ductus Ejaculatorius: The ductus deferens reaches the by entering the prostate and the ductus deferens is attached to the prostate and is called ductus ejaculatorius. This section consists only of the ductus deferens mucosa. There is no muscle layer except mucosa. It is a short, folded canal that passes through the prostate and opens into the prostatic part of the urethra (Schimming, Pinheiro, de Matteis, Machado, & Domeniconi, 2015; Weinbauer, Hoffmann, & Luetjens, 2016). Ejaculatory channels on the prostate posterior face of the segment of the longitudinal muscle layer, have thickened collagen in the middle and in the inner mucosa. Lumen diameter narrows towards the proximal (ANTEPLİ & Beyaz, 2017). A circular muscle layer is not found at any point. Each ejaculator channel is surrounded by circular tissue and both ejaculator channels are surrounded by a common muscle envelope. The ejaculatory ducts are composed of cuboidal epithelium and columnar epithelial cells containing yellow pigment (Naz & Kamal, 2017). 3.2.5. Urethra: this is a fibromuscular tube that transports semen, and urine, from the external urethral opening to the external environment in Arzu GEZER • 41 males. The length, structure and function of the urethra differ in men and women. The length in males is about 20 cm. It consists of three parts; Prostatic urethra: This passes through the base of the bladder along the prostate gland and is covered with transitional epithelium. : This is on the external urethral sphincter and is covered with either multilayer or false cylindrical epithelium. Penile urethra: This is the end part of the penis. The proximal part is covered with cylindrical epithelium and the distal portion is covered with squamous epithelium (Drobnis & Nangia, 2017).

5. AUXILIARY COMPONENTS The prostate gland, seminal vesicle and bulbourethral glands are the auxiliary and exocrine glands of the male reproductive system. The secretions from each of the glands are different and the activity of these secretions is very sensitive to androgen levels. The weight change in prostate and seminal vesicle and changing cellular activities can be used as a rapid and good predictor for changing androgen levels in circulation (Drobnis & Nangia, 2017). 4.1. Prostate Gland: The prostate consists of many lobes around the urethra. This is a colorless, combined tubuloalveolar gland that secretes serous secretions through the urethra with several channels. In rats, a pair of ventral lobes, a smaller dorsal lobe and lateral lobes are present and these lobes are located on the neck of the bladder. Prostatic secretion constitutes 15-30% of ejaculate and includes proteolytic enzymes, zinc, inositol, transferrin and citric acid (Dere et al., 2018; Prins, 2016). 4.2. Seminal Vesicle: The wall structure consists of a circular layer of muscle and the epithelium is single-layer cylindrical. The seminal vesicle, which is thought to have an important role in male reproduction, is a long, hollow, bilateral gland filled with a greenish white viscous fluid (Ge et al., 2005). This gland is located next to the vas deferens ampoule and is ejected into the urethra via the . The apex of the round structure is conical. The seminal vesicle secretion constitutes 50-80% of ejaculate, and the main component is citric acid, which contains fructose, proteins, vitamin C and lactoferrin, which provide energy for sperm motility (Evans, 2015). 42 • Health Sciences Research Papers

4.3. Bulbourethral Glands (Cowper's Glands): These are two united tubuloalveolar glands located on the urogenital diaphragm behind the membranous urethra. The channels are combined with the penile urethra. The outer face of the striped muscle containing connective tissue is surrounded by capsules. The septum separated from this capsule separates the organ into lobules (Fouladi-Nashta et al., 2017). The connective tissue of the septum is rich in elastic fibers, smooth muscle and striated muscle fibers. The secretory section is covered with a single-layer cubic or prismatic epithelium secreting mucus. Clear mucus, which acts as a secretion product lubricant, constitutes the major part of the preseminal fluid (Sulaiman & Coey, 2018).

5. PENIS The penis is an erectile organ composed of a spongious body and a double cavernous body. It was reported that the penis in the rabbit is found cranial of the pelvis and covered with feathers, and that the penis was more pronounced in the guinea pig than in the rabbit and rat (Schrader & Loreit, 2018).

Layers of the penis; Penis Skin: This has the flexibility to adapt to erection. The absence of subcutaneous adipose tissue and a thin skin cause the skin to become darker (Jefferson, 2018). Superficial Layer: The continuation of the membranous layer of the perineum and the groin with superficial penile vein and arteries. The arteries and the loose colles , starting from the bottom of the glans and surrounding the scrotum, extend to the urogenital diaphragm and continue as Scarpa's fascia on the anterior abdominal wall (Pinder, 2017). Tela Subfascialis: The cavernosal vein, artery, and nerve are thin tissue that is partially visible in the base of the penis under the surrounding Colles and fascia (Jefferson, 2018). Buck Fascia: Deep artery, veins and nerves pass under this fascia. It wraps the spongious and cavernous body, tightening rigidity of deep dorsal vein and circumflex veins by helping the fibrous sheath during erection (Levine, 2005). Arzu GEZER • 43

Tunica Albuginea: This is the deepest layer of the penis, which consists of two layers both circular and external in length, which provide stiffness, elasticity and tissue support and surround the spongious and cavernous bodies. This layer expands as much as the collagen fibers allow during erection and prevents venous return by compressing the emissary veins passing through it (Lerner, Melman, & Christ, 1993). In conclusion, the importance of both male and female reproductive systems in terms of both reproduction and quality of life cannot be overlooked. Attempts have been made to reveal the differences and similarities between the male reproductive system and the reproductive system in laboratory animals, but detailed studies were not enough. It is thought that detailed histological and molecular studies should be performed to resolve these deficiencies.

44 • Health Sciences Research Papers

REFERANCES Aitken, R. J., Gibb, Z., Baker, M. A., Drevet, J., & Gharagozloo, P. (2016). Causes and consequences of oxidative stress in spermatozoa. Reproduction, Fertility and Development, 28(2), 1-10. Akmal, M., Masyitah, D., Hafizuddin, H., & Fitriani, F. (2015). Epididimis Dan Perannya Pada Pematangan Spermatozoa. Jurnal Edukasi dan Sains Biologi, 4(2). Akman, O. (2007). Swiss albino ırkı farelerde sperma kalitesinin belirlenmesi amacıyla hos testi sonuçları ve diğer spermatolojik parametreler arasındaki ilişkinin araştırılması. Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü, Aktaş, S. (2017). Farelerde borik asidin testis dokusunda oksidatif stres ve spermatozoon dna hasarına olan etkisinin saptanması. Alves-Lopes, J. P., & Stukenborg, J.-B. (2017). Testicular organoids: a new model to study the testicular microenvironment in vitro? Human reproduction update, 24(2), 176-191. Anisatuzzahro, A., & Luthfi, M. J. (2017). Anatomical Study of Male Reproductive Organs of the Indonesian Short-Nosed Fruit Bat (Cynopterus titthaecheilus Temminck, 1825). Biology, Medicine, & Natural Product Chemistry, 6(1), 13-17. ANTEPLİ, N., & Beyaz, F. (2017). The immunohistochemical localization of the vascular endothelial growth factor and its receptors in the testes and epididymis of the rabbit. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 64(4), 289-299. Basu, S., Arya, S. P., Usmani, A., Pradhan, B. S., Sarkar, R. K., Ganguli, N., . . . Sarda, K. (2018). Defective Wnt3 expression by testicular Sertoli cells compromise male fertility. Cell and tissue research, 371(2), 351-363. Baysal, F. (2017). Anabolik androjenik steroid ile oluşan testis hasarında karnozin ve vitamin E’nin koruyucu etkileri. ESOGÜ, Sağlık Bilimleri Enstitüsü, Bernal, R. A. D., Aya, F. A., De Jesus-Ayson, E. G. T., & Garcia, L. M. B. (2015). Seasonal gonad cycle of the climbing perch Anabas testudineus (Teleostei: Anabantidae) in a tropical wetland. Ichthyological research, 62(4), 389- 395. BEYAZ, F., BAYRAM, G. K., & ALAN, E. (2008). Pre-pubertal ve Pubertal Dönemlerde Ankara Tavşanı Testis ve Epididimislerinde S100 Proteininin İmmunohistokimyasal Lokalizasyonu. Erciyes Üniversitesi Veteriner Fakültesi Dergisi, 5(2), 79-85. Arzu GEZER • 45

Bucci, S., Rizzo, M., Liguori, G., Umari, P., Chiriacò, G., & Bertolotto, M. (2017). The Testicles: Trauma, Inflammation and Testicular Torsion. In Atlas of Ultrasonography in Urology, Andrology, and Nephrology (pp. 493-509): Springer. Castro, S. A., Boretto, J. M., Blanco, G., & Acosta, J. C. (2018). Adjustment of the reproductive activity of vulnerable lizard Phymaturus williamsi at high altitudes. Herpetol Conserv Biol, 13, 283-293. Chen, H., Wang, Y., Ge, R., & Zirkin, B. R. (2017). Leydig cell stem cells: identification, proliferation and differentiation. Molecular and cellular endocrinology, 445, 65-73. Cheng, C. Y., & Mruk, D. D. (2015). Biochemistry of Sertoli cell/germ cell junctions, germ cell transport, and spermiation in the seminiferous epithelium. In Sertoli cell biology (pp. 333-383): Elsevier. Cooper, T. G. (2012). The epididymis, sperm maturation and fertilisation: Springer Science & Business Media. Creasy, D. M., & Chapin, R. E. (2013). Male reproductive system. In Haschek and Rousseaux's Handbook of Toxicologic Pathology (pp. 2493-2598): Elsevier. Dere, E., Anderson, L. M., Huse, S. M., Spade, D. J., McDonnell-Clark, E., Madnick, S. J., . . . Vanlandingham, M. M. (2018). Effects of continuous bisphenol A exposure from early gestation on 90 day old rat testes function and sperm molecular profiles: a CLARITY-BPA consortium study. Toxicology and applied pharmacology, 347, 1-9. Dias, T. R., Alves, M. G., Silva, J., Barros, A., Sousa, M., Casal, S., . . . Oliveira, P. F. (2017). Implications of epigallocatechin-3-gallate in cultured human Sertoli cells glycolytic and oxidative profile. Toxicology in Vitro, 41, 214- 222. Dimitriadis, F., Tsiampali, C., Chaliasos, N., Tsounapi, P., Takenaka, A., & Sofikitis, N. (2015). The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone. Hormones, 14(4), 479-503. Drobnis, E. Z., & Nangia, A. K. (2017). Male reproductive functions disrupted by pharmacological agents. In Impacts of Medications on Male Fertility (pp. 13-24): Springer. Evans, T. J. (2015). Reproductive toxicity and endocrine disruption of potential chemical warfare agents. In Handbook of Toxicology of Chemical Warfare Agents (pp. 599-613): Elsevier. 46 • Health Sciences Research Papers

Fietz, D., & Bergmann, M. (2017). Functional Anatomy and Histology of the Testis. Endocrinology of the Testis and Male Reproduction, 1-29. Fouladi-Nashta, A. A., Raheem, K. A., Marei, W. F., Ghafari, F., & Hartshorne, G. M. (2017). Regulation and roles of the hyaluronan system in mammalian reproduction. Reproduction, 153(2), R43-R58. Franca, L., Hess, R. A., Dufour, J., Hofmann, M.-C., & Griswold, M. (2016). The Sertoli cell: one hundred fifty years of beauty and plasticity. Andrology, 4(2), 189-212. Fujitani, K., Otomo, A., Wada, M., Takamatsu, N., & Ito, M. (2016). Sexually dimorphic expression of Dmrt1 and γH2 AX in germ stem cells during gonadal development in Xenopus laevis. FEBS open bio, 6(4), 276-284. Garcia-Vazquez, F. A., Gadea, J., Matás, C., & Holt, W. V. (2016). Importance of sperm morphology during sperm transport and fertilization in mammals. Asian journal of andrology, 18(6), 844. Ge, R.-S., Dong, Q., Sottas, C. M., Chen, H., Zirkin, B. R., & Hardy, M. P. (2005). Gene expression in rat Leydig cells during development from the progenitor to adult stage: a cluster analysis. Biology of Reproduction, 72(6), 1405-1415. Górski, K., Kondracki, S., Wysokińska, A., & Nazaruk, A. (2016). The importance of ejaculate volume for the physical parameters of ejaculates and sperm morphology of Hypor boars. Kafkas Univ Vet Fak Derg, 22, 493-501. Gümüşel, A. (2013). Adolesan çağdaki sıçanların epididiminden alınan spermlere ve testis leydig hücrelerine sigara dumanının ve kurkumin antioksidanının etkilerinin mikroskobik incelenmesi (Doktora Tezi), İstanbul Üniversitesi, İstanbul. Hutson, J. M., & Lopez-Marambio, F. A. (2017). The possible role of AMH in shortening the gubernacular cord in testicular descent: A reappraisal of the evidence. Journal of pediatric surgery, 52(10), 1656-1660. Jefferson, M. (2018). On Michael Jackson: Granta Books. Kashmiri, Z., Dighade, S., & Gotmare, V. (2016). Histopathological Effect of Chemotherapeutic Agent Cyclophosphamide on The Cauda Epididymis of Male Albino Rat (Rattus norvegicus). RESEARCH JOURNEY, 62. Kılınç, L. (2015). Metotreksatın neden olduğu testis hasarına karşı curcuminin koruycu etkisinin incelenmesi. Kim, K.-W., Bennison, C., Hemmings, N., Brookes, L., Hurley, L. L., Griffith, S. C., . . . Slate, J. (2017). A sex-linked supergene controls sperm morphology Arzu GEZER • 47

and swimming speed in a songbird. Nature ecology & evolution, 1(8), 1168. Kolesar, E., Tvrda, E., Halenar, M., Schneidgenova, M., Chrastinova, L., Ondruska, L., . . . Massanyi, P. (2018). Assessment of rabbit spermatozoa characteristics after amygdalin and apricot seeds exposure in vivo. Toxicology reports, 5, 679-686. Kuzelova, L., Vasicek, J., & Chrenek, P. (2015). Influence of macrophages on the rooster spermatozoa quality. Reproduction in domestic animals, 50(4), 580-586. Lebelo, S. L., & van der Horst, G. (2017). Histological and ultrastructural features of the Leydig cells and their association with other testicular cells of the vervet monkey, Chlorocebus aethiops. Pakistan Journal of Zoology, 49(5). Lerner, S. E., Melman, A., & Christ, G. J. (1993). A review of erectile dysfunction: new insights and more questions. The Journal of urology, 149(5 Part 2), 1246-1255. Levine, K. L. (2005). No penis, no problem. Fordham Urb. LJ, 33, 357. Li, C., Gao, S., Chen, S., Chen, L., Zhao, Y., Jiang, Y., . . . Zhou, X. (2018). Differential expression of micro RNA s in luteinising hormone‐treated mouse TM 3 Leydig cells. Andrologia, 50(1), e12824. Li, X., Wang, Z., Jiang, Z., Guo, J., Zhang, Y., Li, C., . . . Lian, Q. (2016). Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes. Proceedings of the National Academy of Sciences, 113(10), 2666- 2671. Mahfud, M., Winarto, A., & Nisa, C. (2016). MIKROMORFOLOGI ALAT KELAMIN PRIMER BIAWAK AIR (Varanus salvator bivittatus) JANTAN (Micromorphological Structure of Primary Reproductive Organ of Male Water Monitor Lizard (Varanus salvator bivittatus)). Jurnal Kedokteran Hewan-Indonesian Journal of Veterinary Sciences, 10(1), 72- 76. Morais, D. B., Cupertino, M. C., Goulart, L. S., Freitas, K. M., Freitas, M. B., Paula, T. A., & Matta, S. L. (2013). Histomorphometric evaluation of the Molossus molossus (Chiroptera, Molossidae) testis: The tubular compartment and indices of sperm production. Animal reproduction science, 140(3-4), 268-278. Morrow, S., Gosálvez, J., Lopez-Fernandez, C., Arroyo, F., Holt, W., & Guille, M. J. (2017). Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa. Reproduction, Fertility and Development, 29(8), 1556-1566. 48 • Health Sciences Research Papers

Mortimer, D. (2018). The functional anatomy of the human spermatozoon: relating ultrastructure and function. MHR: Basic science of reproductive medicine, 24(12), 567-592. Naz, M., & Kamal, M. (2017). Classification, causes, diagnosis and treatment of male infertility: A review. Oriental pharmacy and experimental medicine, 17(2), 89-109. Nicander, L., & Plöen, L. (1979). Studies on regional fine structure and function in the rabbit epididymis. International Journal of Andrology, 2(1‐6), 463- 481. Nyongesa, A., Patel, N., Wango, E., & Onyango, D. (2017). High khat dose and long-term exposure impairs spermatogenesis: experimental study using rabbit model. Journal of Morphological Sciences, 34(03), 156-167. Öztaş, E., Yılmaz, T. E., Güzel, E., Sezer, Z., Okyar, A., & Özhan, G. (2019). Gliclazide alone or in combination with atorvastatin ameliorated reproductive damage in streptozotocin-induced type 2 diabetic male rats. Saudi Pharmaceutical Journal. Pinder, S. O. (2017). Michael Jackson and the Quandary of Black Identity. In Michael Jackson (pp. 51-63): Routledge. Powers, S. A., Odom, M. R., Pak, E. S., Moomaw, M. A., Ashcraft, K. A., Koontz, B. F., & Hannan, J. L. (2019). Prostate-Confined Radiation Decreased Pelvic Ganglia Neuronal Survival and Outgrowth. The journal of sexual medicine, 16(1), 27-41. Prihatno, S. A., Padeta, I., Larasati, A. D., Sundari, B., Hidayati, A., Fibrianto, Y. H., & Budipitojo, T. (2018). Effects of secretome on cisplatin-induced testicular dysfunction in rats. Veterinary world, 11(9), 1349. Prins, G. S. (2016). The Endocrine Society Centennial: Hormones and Apoptosis in the Prostate Gland… Live and Let Die. In: Oxford University Press. Rodriguez, I. M., Cuevas, M., Silvero, A., Cañete-Portillo, S., Sanchez, D. F., Barreto, J., & Cubilla, A. L. (2017). Novel Histologic Finding: Adipose Tissue Is Prevalent Within Penile Tunica Albuginea and Corpora Cavernosa. The American journal of surgical pathology, 41(11), 1542- 1546. Ross, M. H., Pawlina, W., & Barnash, T. A. (2016). Atlas de histologia descritiva: Artmed Editora. Schatten, H., & Constantinescu, G. M. (2017). Animal models and human reproduction: John Wiley & Sons. Arzu GEZER • 49

Schimming, B., Pinheiro, P., de Matteis, R., Machado, C., & Domeniconi, R. (2015). Immunolocalization of aquaporins 1 and 9 in the ram and epididymis. Reproduction in domestic animals, 50(4), 617-624. Schrader, J., & Loreit, F. (2018). Professionalisierung bei Lehrkräften der Erwachsenen-und Weiterbildung: Individuelle und kollektive Perspektiven. In Das Personal in der Weiterbildung (pp. 283-308): Springer. Stévant, I., Papaioannou, M. D., & Nef, S. (2018). A brief history of sex determination. Molecular and cellular endocrinology, 468, 3-10. Stewart, K. A., Wang, R., & Montgomerie, R. (2016). Extensive variation in sperm morphology in a frog with no sperm competition. BMC evolutionary biology, 16(1), 29. Sulaiman, S., & Coey, J. (2018). Clinical Reproductive Science. In M. Carroll (Ed.), Male and Female Reproductive Anatomy (pp. 35-44). Manchester, UK: Wiley Blacwell. Swierstra, E., & Foote, R. (1963). Cytology and kinetics of spermatogenesis in the rabbit. Reproduction, 5(3), 309-NP. Şahin, T. C. (2016). Testiste oluşturulan iskemi reperfüzyon modelinde montelukast’ın koruyucu etkisi. ESOGÜ, Sağlık Bilimleri Enstitüsü, Tan, S. (2015). Varikoselli Sıçanlarda Spermatojenik Kök Hücrelerin Durumunun İmmünohistokimya Teknikleri İle Belirlenmesi. Pamukkale Üniversitesi Sağlık Bilimleri Enstitüsü, Tremblay, J. J. (2015). Molecular regulation of steroidogenesis in endocrine Leydig cells. Steroids, 103, 3-10. Vernet, N., Mahadevaiah, S. K., Decarpentrie, F., Longepied, G., de Rooij, D. G., Burgoyne, P. S., & Mitchell, M. J. (2016). Mouse Y-encoded transcription factor Zfy2 is essential for sperm head remodelling and sperm tail development. PLoS One, 11(1), e0145398. Wahyuni, S., Gholib, G., Adnyane, I., Agil, M., Hamny, H., Agungpriyono, S., & Yusuf, T. L. (2018). Characterization of Seminiferous Epithelium Stages in the Wild Javan Muntjac (Muntiacus muntjak muntjak) Using the Tubular Morphology Method. Veterinary medicine international, 2018. Weinbauer, G., Hoffmann, G., & Luetjens, M. (2016). Options for enhanced analysis of spermatogenesis in the nonhuman primate model. Paper presented at the 37th Annual Meeting of the ACT, Abstract. 50 • Health Sciences Research Papers

Wheeler, K. M., Sharma, D., Kavoussi, P. K., Smith, R. P., & Costabile, R. (2018). Clomiphene Citrate for the Treatment of Hypogonadism. Sexual medicine reviews. Zahrina, A. D. (2015). Uji Aktivitas Antifertilitas Ekstrak Etanol 96% Daun Sambiloto (Andrographis paniculata Nees.) Pada Tikus Jantan Galur Sprague-Dawley Secara In Vivo. Zelený, T. (2017). Minoritní členové tubulinové superrodiny.

APPROACH TO CARDIOMYOCYTES PROLIFERATION AND CARDIAC REGENERATION

CHAPTER 3

Aynur KARADAĞ GÜREL1

1 Uşak University, Faculty of Medicine, Department of Medical Biology

Aynur KARADAĞ GÜREL • 53

INTRODUCTION Cardiovascular diseases play an increasingly important role in becoming a major cause of mortality and morbidity worldwide. World health organization data report that cardiovascular diseases are the number one cause of death in the world and 17.9 million people die each year from CVDs and account for 31% of all deaths in the World (Akar et al., 2009). Nearly half of deaths in developed countries and one third in developing countries are due to cardiovascular diseases. Of these deaths, 7.6 million were due to heart attacks and 5.7 million to stroke. By 2030, 23.4 million people are expected to die from cardiovascular diseases (WHO http://www.who.int/cardiovascular_diseases/en/, Schafer and Northoff, 2008). Heart failure syndrome caused by coronary artery diseases, cardiomyopathies, pericardial and cardiac heart anomalies leads to left ventricular (LV) myocardial damage, systolic and diastolic dysfunction. Coronary artery disease and dilated cardiomyopathies are the most common cause of heart failure in 60-70% of patients. (Cowie et al., 1999). Left ventricular systolic dysfunction and chronic heart failure is a disease progressing with complex cellular mechanisms. Left ventricular dysfunction begins with trauma and cardiac stress caused by the onset of modifications in cellular metabolism. As a result of these changes, the cardiac remodeling process begins and thus cardiac remodeling causes long term cardiac dysfunction, cardiomyocyte death and survival myocytes and extracellular matrix changes (Delgado et al., 1999). Heart transplantation remains the gold standard for the treatment of terminal heart failure. However, approximately 20-40% of transplant candidates are lost during waiting and 20% are lost in the first year of transplantation, with a mortality of 5% per year after this first year (Hosenpud et al., 2001). In addition, the high cost, immunosuppression complications and the considerable morbidity of other treatments should be noted. In this status, in addition to increasing organ transplant waiting time of the patients as well as their orientation has led to the need for alternative treatment methods. The most common etiologies are ischemic coronary artery disease, systemic hypertension, structural heart valve disease and dilated cardiomyopathy. Despite improvements in treatment strategies, hospitalization rates and mortality remain high, especially in patients with 54 • Health Sciences Research Papers heart failure who have reduced ejection fraction. Implantation of left ventricular assist devices (LVAD), which was originally developed as a bridge to transplantation, has become a target treatment for most patients. In Germany, the number of LVAD implantations per year rose to 915, while heart transplantation remained below 300 in 2015 (Beckmannet al.,2016). The results of LVAD treatment have improved significantly in terms of quality of life and survival, since 1 and 2-year survival rates have been reported to be 80% and 70%, respectively. However, bleeding, thromboembolism and its complications (stroke and neurological deficiencies), pump failure and infections remain major problems that strongly influence long-term survival rates (Kirklin et al., 2014; Teuteberg et al., 2015). Therefore, new regenerative approaches are needed to improve cardiomyocyte loss and left ventricular function.

The Organization of Myocytes in the Heart The heart is the first organ in the vertebrate embryo and is the main organ of the circulatory system adjacent to the sternum and some cartilage ribs on the sides of the right and left lungs in the anterior mediastinum in the thoracic cavity. The heart is a muscular organ, somewhat pyramid-like in shape and located in the pericardium in the mediastinum. Pericardium is free inside with the outside of his connections to the major blood vessels at the base. Although the weight varies according to the weight of the human body, it is 250-300 g for women and 300-350 g for men. The free wall thickness is 3-5 mm in the right ventricle and 13-15 mm in the left ventricle (Snell, 2004). The heart is divided into four chambers: a vertical septum and right-left atria and ventricles. The right atrium is located in front of the left atrium and the right ventricle is in front of the left ventricle. The heart is composed of endocardium composed of endothelial cells, the inner part of which covers the heart cavities, the muscle layer called myocardium in the middle and the outer part of the pericardial layer consisting of fibrous, inner serous membrane (mucus structure more slippery) (Snell, 2004). The heart muscle cell or myocyte is the main structural component of the myocardium, which normally covers 70% of the ventricular wall volume. Cardiac myocytes are 10 to 20 µm in size and 80 to 100 µm in length, similar to the major axis ellipsoid cylinders. Myocytes settle end- to-end and each merges with the others to form a three-dimensional (3D) cell network. The heart is highly organized and consists of ventricular or Aynur KARADAĞ GÜREL • 55 atrial cardiomyocytes, Pace maker cells, Purkinje cells, blood vessels and connective tissue. There are five basic components of cardiac myocytes. These are 1. cell membrane (sarcolemma) 2. Sarcoplasmic reticulum 3. Contractile elements 4. Mitochondria (cardiac myocytes have more mitochondria than striated skeletal muscle, mitochodria are 23% in cardiac myocytes due to aerobic respiration, and 2% in skeletal muscle due to aerobic respiration, 5. Nucleus (Snell, 2004; Nakano et al., 2012). Myocardial cells, which enable the heart to function as a pump with its contraction feature, have different characteristics in different parts of the heart. For example, atrial, ventricular and stimulating conduction cells have different characteristics, but they also have different features in themselves. Cardiomyocytes account for about 90% of myocardial volume. Ventricular cardiomyocytes are columnar cells with a diameter of 20 μm and a length of 60-140 μm. Ventricular cardiomyocytes account for about 50% of heart weight and 2-4 billion cardiomyocytes are found in the human left ventricle. Atrial cardiomyocytes are elliptical cells with a diameter of 5 μm and a length of 10-20 μm. In a single contracted cardiomyocyte, myofibrils account for about 50% of the cell volume and mitochondria account for 25% of the cell volume. The remaining portion of the core, creates sarcoplasmic reticulum and the cytosol. Cardiomyocytes have myofibril aggregation. myofibrillary rod-shaped structures are contraction elements of cardiomyocytes. One of the special structures of cardiomyocytes is a smooth sarcolemma associated with the plasma membrane and the basement membrane. This conformational states allows sarcolemma to regulate the interaction with the intracellular and extracellular environment (Nassar, 2005). The other cells in the heart are interstitial cells, endothelial cells that form a rich capillary network, and connective tissue elements. Atrial myocytes are usually smaller in diameter than ventricular myocytes because their functional requirements are different. Structural features are also less. Some atrial cells contain electron-dense granules in their cytoplasms called specific atrial granules. These are atrial natri uretic peptide stores. It is synthesized in the atrial muscle and is given when the atrial pressure increases and distension occurs. Atrial natriuretic peptide is produced in various physiological and pathological conditions (Nakano et al., 2012). 56 • Health Sciences Research Papers

Proliferation and Developmental Stages of Cardiomyocytes The growth and proliferation of cardiomyocytes consists of two different stages in time. During embryogenesis, heart mass increases with the proliferation of cardiomyocytes in this process known as hyperplasia in which karyokinesis and cytokinesis occur. Cardiomyocytes, exiting the cell cycle shortly after birth, lose their division characteristics (Clubb and Bishop, 1984). In mammalian hearts, cardiomyocytes proliferate prenatally and are withdrawn from the cell cycle 7 days after birth. 3 days after birth, cardiomyocytes maintain a constant cell volume and are essentially single nuclei. 1-2 weeks later, multi-nucleated cardiomyocytes begin to form with DNA replication without cytokinesis (Li et al. 1996, Ahuja et al., 2007, Porrello et al. 2011). When cell division in the mammalian heart stops, cell growth occurs with increasing cell size. This condition is called hypertrophy (Oparil et al., 1984). In the early neonatal period, the transition from hyperplasia to hypertrophy is defined by the phenomenon of binucleation (nuclear division without cytoplasm division) in some species. Before exiting the cell cycle, cardiomyocytes cannot perform cytokinesis, only karyokinesis (nuclear division) occurs. Thus, they cannot complete the final cell division (Li et al., 1996). In the mouse heart,> 90% of cardiomyocytes are binuclear. Unlike mammals, lower vertebrates do not lose their ability to postnatal division. Zebrafish and salamander are also renewed after damage to the heart tissue. Endogenous repair mechanisms in neonates and amphibians after heart injury allow complete restoration of myocardium. In adult mammalian heart, the stimulation of pre-existing cardiomyocytes with the same endogenous repair mechanism can be considered as the most appropriate method for reconstructing the heart in a clinical setting. Molecular mechanisms under which cell proliferation and cardiomyocytes exit from the cell cycle in adults are not fully understood (Bettencourt et al., 2003; Poss et al., 2002; Puente et al., 2014). Cardiomyocytes show three developmental forms of cell cycle control and expansion. These include proliferation binucleation and hypertrophy. There is little evidence of postnatal cardiomyocyte proliferation and regeneration. Many forms of heart disease are associated with the absence or loss of functional myocardium. Postpartum damage such as ischemia / reperfusion caused by myocardial infarction or interventions leads to loss of cardiomyocyte and reduced cardiac function. Cardiomyocyte Aynur KARADAĞ GÜREL • 57 proliferation in adult mammalian hearts under basal conditions occurs at a rate of 0.5-1% per year and replaces the cardiomyocytes that die due to apoptosis. Although cardiomyocyte proliferation rate increases after heart injury, heart regeneration is insufficient (Bergmann et al., 2009; D'Uva.et al., 2015). In mammalian postnatal life, the mechanisms regulating myocyte proliferation, withdrawal from the cell cycle, and myocyte hyperplasia have not been fully understood and are largely unknown (Ahuja et al., 2007). Regardless of its efficacy, the human heart has been shown to be capable of producing new cardiomyocytes after birth (Beltrami et al., 2001). However, the source of these new cardiomyocytes is still under discussion. Do these new cardiomyocytes come from the differentiation of targeted / resident stem cells in the heart or the division of pre-existing cardiomyocytes? Embryonic cardiomyocytes; Increased cardiac mass during the embryonic period is predominantly by proliferation of mononuclear contractile cardiomyocytes. In embryonic cardiomyocytes G1, S, G2 and M-phase cyclin (Cyclin D1, Cyclin D2, Cyclin A, B1 and E) and cyclin- dependent kinases (CDC2, CDK2, CDK4 and CDK6) were overexpressed, whereas cyclin inhibitors were expressed low they become (Sherr, 1994). Neonatal period and binucleation; Cardiomyocytes proliferate rapidly during fetal development, but most mammals lose this ability in the neonatal period shortly after birth. Immediately before exiting the cell cycle, the myocytes undergo a final DNA synthesis, but the absence of cytokinesis results in binuclear cardiomyocytes. Cytokinesis is the final stage of cell division and is responsible for equal division between sister cells to complete mitosis. DNA synthesis in the neonatal period usually results in binuclear. The process of nuclear division before cellular division is complete is known as acidokinetic mitosis. Cytokinesis is a complex process involving regulatory and cell skeletal elements. Cytokinesis is not only progression through the G1/S phase by DNA synthesis, but also progression through the G2/M phase. Distinct complexes between transitions at each stage are regulated by cyclin and cyclin-dependent kinases (Fededa and Gerlich, 2012). Existing of cardiomyocytes from the cell cycle leads to reduced expression of some of the cell cycle regulators (Zebrowski et al., 2016). To complete cell division, fully differentiated embryonic cardiomyocytes need to separate 58 • Health Sciences Research Papers their myofibril complex, which takes place in two steps. First, the M band is separated, then the Z-disk and the thin actin proteins are separated respectively. Increasing complexity of myofibrils on cardiomyocytes is observed as terminal differentiation after postnatal period (Hyams, 2005; Lebart and Benyamin, 2006;. Rodent heart begins to accumulate binuclear cardiomyocytes within 3-4 days in the postnatal period, and more than 90% after 3 weeks are binuclear cardiomyocytes. In humans, this rate is 25% postpartum and remains stable throughout life (Olivetti et al., 1996).

Conclusion Although the heart is believed to have regenerative capacity after injury, such capacity is too small to provide adequate repair. The main objective of scientists was the possibility that medical advances could provide new therapeutic advantages to find ways to increase cardiomyocytes and division rates for heart disease. The main strategy in stimulating the heart for regeneration is the elimination of factors that prevent the proliferation of terminally differentiated cardiomyocytes and the use of factors or cells to facilitate proliferation. Cardiomyocyte proliferation is the most important point in cardiac regeneration. The regulation of cardiomyocyte divisions has a complex molecular mechanism. Numerous factors are involved in maintaining strict control of this important event in heart development. Cell cycle and cytokinesis are key processes associated with cellular division. In particular, stimulants and inhibitors at any time regulate the general cardiomyocyte population. While finding methods to facilitate the proliferation of cardiomyocytes in the developing heart or damaged heart, it is very important to know the mechanisms that prevent excessive hyperplasia. The occurrence of nuclear division without cytokinesis causes a decrease in the proliferative factors of multinucleated cells, while a simultaneous increase in inhibitors occurs. This comprehensive regulation demonstrates the importance of maintaining optimal cardiomyocyte count. Many studies have focused on the manipulation of the cell cycle to promote proliferation of cardiomyocytes, while many studies have focused on cellular-based studies. The function of the heart muscle functions in the damaged or incapacitated heart, the pre-predetermined state, the new regeneration, serves as the basis of modern regenerative cardiovascular medicine. Aynur KARADAĞ GÜREL • 59

REFERENCES Ahuja P, Sdek P, MacLellan WR. Cardiac myocyte cell cycle control in development, disease, and regeneration. Physiol Rev 2007;87:521-44. Ahuja P, Sdek P, MacLellan WR. Cardiac myocyte cell cycle control in development, disease, and regeneration. Physiol Rev 2007;87:521-44. Akar, A.R., Durdu, S., Arat, M., Kılıçkap, M., Küçük, N. O., Arslan, O., Kuzu, I., Ozyurda, U. 2009. Five-year follow-up after transepicardial implantation of autologous bone marrow mononuclear cells to ungraftable coronary territories for patients with ischaemic cardiomyopathy. Eur J Cardiothorac Surg.;36(4):644-650. Beckmann A, Funkat AK, Lewandowski J, Frie M, Ernst M, Hekmat K, Schiller W, Gummert JF, Welz A (2016) German heart surgery report 2015: the annual updated registry of the German society for thoracic and cardiovascular surgery. Thorac Cardiovasc Surg 64:462–474 Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science 2009;324:98-102. Bettencourt-Dıas, M., Mıttnacht, S., Brockes, J.P. (2003). Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes. J Cell Sci.;116(Pt 19):4001-4009. Clubb FJ, Jr and Bishop SP (1984) Formation of binucleated myocardial cells in the neonatal rat. An index for growth hypertrophy. Lab Invest 50:571–577. Cowie MR, Wood DA, Coats AJ, Thompson SG, Poole-Wilson PA, Suresh V, Sutton GC. Incidence and aetiology of heart failure; a population-based study. Eur Heart J. 1999 Mar;20(6):421-8. Delgado, C., A. Artiles, A. M. Gomez, and G. Vassort. 1999. Frequency- dependent increase in cardiac Ca2+ current is due to reduced Ca2+ release by the sarcoplasmic reticulum. J. Mol. Cell Cardiol. 31:1783–1793. D'Uva G, Aharonov A, Lauriola M, et al. ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nat Cell Biol 2015;17:627-38.

Fededa JP, Gerlich DW. Molecular control of animal cell cytokinesis. Nat Cell Biol (2012) 14:440–7. Hosenpud JD, Bennett LE. Mycophenolate mofetil versus azathioprine in patients surviving the initial cardiac transplant hospitalization: an analysis of the Joint UNOS/ISHLT Thoracic Registry. Transplantation. 2001; 72: 1662-5. http://www.who.int/cardiovascular_diseases/en/ 60 • Health Sciences Research Papers

Hyams, J.S. (2005) Cytokinesis: the great divide. Trends Cell Biol.;15(1):1. Kirklin JK, Naftel DC, Pagani FD, Kormos RL, Stevenson LW, Blume ED, Miller MA, Baldwin JT, Young JB (2014) Sixth INTERMACS annual report: a 10,000-patient database. J Heart Lung Transplant 33:555–564. Lebart, M.C., Benyamın, Y. (2006). Calpain involvement in the remodeling of cytoskeletal anchorage complexes. FEBS J.;273(15):3415-3426. Li F, Wang X, Capasso JM, et al. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737-46. Nakano, S., Muramatsu, T., Nishimuraand S., Senbonmatsu T., (2012). "Current Basic and Pathological Approaches to the Function of Muscle Cells and Tissues - From Molecules to Humans", book edited by Haruo Sugi, Chapter 8 Cardiomyocyte and Heart Failure 161-182. Olivetti, G., Cıgola, E., Maestri, R., Corradı, D., Lagrasta, C., Gambert, S.R., Anversa, P. (1996) Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart. J Mol Cell Cardiol.;28(7):1463-1477. Oparil S, Bishop SP, Clubb FJ, Jr. (1984) Myocardial cell hypertrophy or hyperplasia. Hypertension 6: III38-43. Porrello ER, Mahmoud AI, Simpson E, et al. Transient regenerative potential of the neonatal mouse heart. Science 2011;331:1078-80. Poss K.D., Wilson L.G., Keating M.T. (2002) Heart regeneration in zebrafish. Science.;298 (5601):2188-2190. Puente B N, W. Kimura, Shalini A. Muralidhar, J. Moon, James F. Amatruda, Kate L. Phelps, Hesham A. Sadek. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response Cell, 157 (2014), pp. 565-579. Schafer R., Northoff H. (2008). Cardioprotection and cardiac regeneration by mesenchymal stem cells. Panminerva Med 50:31–39. Sherr C.J. (1994) G1 phase progression: cycling on cue. Cell.;79(4):551-555. Snell, R.S. Toraks II Göğüs Boşluğu. İçinde: Klinik Anatomi. Yıldırım M (çev. ed.), Klinik Anatomi. 6. Baskı, İstanbul: Nobel Tıp Kitabevleri, 2004: s. 77-136. Teuteberg JJ, Slaughter MS, Rogers JG, McGee EC, Pagani FD, Gordon R, Rame E, Acker M, Kormos RL, Salerno C, Schleeter TP, Goldstein DJ, Shin J, Aynur KARADAĞ GÜREL • 61

Starling RC, Wozniak T, Malik AS, Silvestry S, Ewald GA, Jorde UP, Naka Y, Birks E, Najarian KB, Hathaway DR, Aaronson KD (2015) The HVAD left ventricular assist device: risk factors for neurological events and risk mitigation strategies. JACC Heart Fail 3:818–828. Zebrowski DC, Becker R, Engel FB. Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation. Am J Physiol Heart Circ Physiol. 2016 May 1; 310(9):H1045-54.

ADULLT CARDIOMYOCYTE AND EXISTING CELL CYCLE

CHAPTER 4

Aynur KARADAĞ GÜREL1

1 Uşak University, Faculty of Medicine, Department of Medical Biology

Aynur KARADAĞ GÜREL • 65

INTRODUCTION In chronic cardiovascular patients who develop heart failure, new treatment methods are needed to recover the contractile properties of the myocardium. Although attempts to minimize damage to the heart tissue have been successful, new approaches and treatments are still required. Cardiac regenerative medicine offers potential inspiring strategies, including the use of progenitor cells, cardiac stem cells, or small molecules to replace the lost cell population. The future objectives of these approaches are to further explore the potential of the identified molecules and to reduce the costs and toxic effects of therapeutics and accelerate the improvement. With cellular reprogramming, the concept of terminal differentiation is re-discussed and ways of increasing the division capacity of cardiomyocytes are sought. Thus, the conventional view known as having completed terminal differentiation during development has been modified to include new levels of cellular plasticity. The ability of the cardiac tissue to regenerate after injury depends on the proliferation capacity of cardiomyocytes. Traditionally, since cardiomyocytes forming the mammalian heart are terminally differentiated cells, they were considered to respond to physiological and pathological stresses with hypertrophy (Linzbach, 1976). To complete their differentiation, cardiomyocytes exit the cell cycle and stop their division (Naqvi et al., 2009). Therefore, the heart is considered as a postmitotic organ without regeneration capacity after injury (Akar et al., 2006). It was considered that the heart itself failed to repair tissue damage by traumas such as ischemic conditions leading to cardiac infarction. Capacity of cardiomyocyte regeneration have been identified in studies with raydoactive C in humans. It is estimated that the left ventricle decreases 3% per year at 75 years and 1% turnover at 20 years (Bergmann et al., 2009). Shortly after birth, cardiomyocytes complete terminal differentiation, and later, expression of tissue-specific genes, including structural proteins such as cardiac actin and α-myosin heavy chain (α-MHC), begins to increase. In both embryonic and neonatal periods, the highest β-myosin heavy chain (β-MHC) and skeletal actin are expressed. Another indicator of terminal differentiation in myocytes is that although most are stimulated by 66 • Health Sciences Research Papers mitogens or physiological stress, most of them do not enter the cell cycle. However, there is evidence that cardiomyocytes re-enter the cell cycle in response to injury in the human heart (Anversa eyt al.,2002). DNA labeling studies have shown that DNA synthesis in adult mouse ventricular cardiomyocytes is normally 0.0005% per minute, but 0.008% in case of damage. In contrast to the mouse heart, the human heart re-enters the cell cycle in response to cardiomyocyte damage at a rate of 1-4% after injury. The fate of these proliferating cells and how they proliferate is still unresolved. Species-specific changing cardiomyocyte proliferation is not yet known (Soonpaa and Field, 1997).

Studies to Determine Cardiomyocyte Proliferation Many studies have demonstrated the presence of cardiomyocyte regeneration by examining cardiomyocyte cell cycle activity by comparing damaged adult hearts to normal hearts, and studies in zebrafish and neonatal mouse hearts have supported this. Genetic manipulations in mice have shown that differentiation of adult cardiomyocyte proliferation can be prevented based on knowledge of normal cardiomyocyte development. Unlike adult mammals, amphibians, reptiles and zebra fish continue proliferation of cardiomyocytes after myocardial injury (Jopling et al., 2010; Oberpriller and Oberpriller, 1974). Research has focused on the use of stem and progenitor cells in the repair of damaged myocardium and the development of endogenous regenerative mechanisms (Garbern and Lee, 2013). Mitosis of cardiomyocyte nuclei has been shown in studies over the last 100 years, but evidence for cardiomyocyte division remains unclear. The first systematic investigations of cellular mechanisms of human heart development were demonstrated in the 1950s. When the cardiomyocyte cross-sectional area increase in the left ventricular papillary muscle in humans was examined, it was shown that cardiomyocyte growth could fully explain the physiological myocardial growth between birth and adulthood (Linzbach, 1950; Linzbach, 1960). Nevertheless, historically, the mammalian heart has been seen as a post-mitotic organ where primary parenchymal cells and cardiomyocytes do not increase after birth (Zak, 1973; Linzbach, 1950). Even though mitotic shapes are shown under the microscope as an indicator of cardiomyocyte proliferation, these Aynur KARADAĞ GÜREL • 67 approaches probably did not have the precision required for precise visualization of cardiomyocyte cytokinesis. All these results are uncertain, but have led to two paradigms: First, unlike skeletal muscle, the adult heart does not have progenitors that support the formation of new cardiomyocytes. The second paradigm argued that there was a single cellular mechanism of postnatal developmental and pathological heart growth: cardiomyocyte expansion (Borisov and Claycomb, 1995). In the second century, technical advances have provided new data, including the use of confocal microscopy to visualize cell cycle events, automated analysis of large cell populations, and genetic and metabolic labeling for cellular fate mapping. While combining the important role that cardiomyocyte expansion plays in physiological and pathological heart growth, it has developed a new cellular paradigm involving adult cardiomyocyte regeneration. Recognition of endogenous cardiomyocytes as a new source of cardiomyocytes increases the likelihood of this process being stimulated for myocardial repair. By understanding the mechanisms that limit the regenerative response of mammalian myocardium, therapeutic strategies can be developed. To realize this therapeutic potential, it needs to address questions that include the precise cellular source, regulatory signaling pathways, underlying cellular mechanisms, and the rate and dynamics of production for new cardiomyocytes. Many studies have examined cardiomyocyte cell cycle activity in normal and injured adult hearts, and the presence of ongoing cardiomyocyte regeneration has long been recognized. For example, in uninjured adult rat hearts, ventricular cardiomyocyte cell cycle activity (usually used as a carrier marker for cardiomyocyte regeneration) values ranged from 0 to 3.15% [9]. The duration of the S-phase is much longer than the M-phase, and monitoring the synthesis of cardiomyocyte DNA (tritium-thymidine or bromodeoxyuridine labeling) will result in a higher rate of regeneration than the presence of mitotic figures. Other markers (such as Proliferating Cell Nuclear Antigen or Ki67 immune reactivity) expressed throughout the majority of the cell cycle will lead to even higher regeneration rates.

Investigation of cardiomyocyte regeneration mechanisms is a challenge Cardiomyocyte formation in adult mammalian hearts is a slow process compared to blood, skin and digestive system. At cellular resolution, in situ imaging of the beating mammalian heart is technically challenging and a 68 • Health Sciences Research Papers small amount of tissue from myocardial biopsies from patients makes it difficult to characterize (Hashimoto et al., 2014; Hesse et al., 2012). The heterogeneity of the cells in the myocardium and their proximity to neighboring cells prevent the identification of cardiomyocyte nuclei (Soonpaa and Field, 1997; Soonpaa et al., 2012). Perhaps the most difficult aspect is that cardiomyocytes exhibit non-proliferative cell cycles that increase the DNA content without cell division. Despite these biological features that make regeneration difficult, several methods have advanced our understanding of cardiomyocyte proliferation. Classical and novel methods have advanced cardiac regeneration research. The labeled thymidine and thymidine analogs are stably incorporated into the genome during DNA synthesis and are used to track cell cycle events. Cell proliferation markers, BrdU, PCNA and Ki-67 tissue distributions can be examined immunohistochemically. 5-Bromo-2-Deoxyuridine (BrdU), Proliferated cell nuclear antigen (PCNA) and Ki-67 are tests to demonstrate cell proliferation. BrdU is a group of halogenated pyrimidines and can replace the thymidine molecule during DNA synthesis and enter the structure of the DNA chain. Therefore, these substances are called ‘thymidine analogues. This method is based on BrdU labeling of only S phase cells and monitoring the progression of this labeled cell group in the cell cycle. Ki-67 is the non-histone protein found in all active phases of the cell cycle except G0 (Iatropoulos et al., 1996; Assy et al.,1997). The use of tritium thymidine is limited by potential radioactive toxicity due to prolonged administration compared to confocal microscopy (Soonpaa and Field, 1997). Thymidine analogs such as bromo- deoxyuridine (BrdU) have also been administered for up to several weeks to determine cardiomyocyte cell cycle activity. Although high BrdU concentrations may affect cell proliferation and differentiation, no side effects have been reported in long-term labeling (Wilson et al., 2008; Malliaras et al., 2013). Using tritium labeling and a single injection of BrdU, progressive cardiomyocyte-DNA synthesis was observed in rats within ten days of birth (Li et al., 1996; Soonpaa et al., 1996). However, cell cycle activity was determined in cardiomyocyte nuclei in mice up to 21 days using Ki67 as a marker (Walsh et al. 2010). Naqvi et al. 2014 detected BrdU uptake in 13-15 days old mice and markers of mitosis and cytokinesis in cardiomyocytes. In the adult heart, when the mice were examined 2 hours after the administration of tritium-thymidine, the incidence of 0.0005% labeled cardiomyocytes expressing a transgenic Aynur KARADAĞ GÜREL • 69 marker from the alpha-MHC promoter was determined (Soonpaa and Field, 1997). More recently, the use of long-term BrdU labeling has shown low basal activity of cardiomyocyte proliferation (Li et al., 1996; Malliaras et al., 2013). Antibodies used against markers of the cell cycle can be used for visualization by microscopy (eg Ki67 for G1, S, G2 and M-phase, phospho-histone3 for M-phase, Aurora B kinase for cytokinesis) (Senyo et al., 2013). ; Malliaras et al., 2013; Ellison et al., 2013; Mollova et al., 2013). Automated systems offer an unbiased approach to a large number of cardiomyocyte samples, but they require separated cells. Flow cytometry (FACS) and laser scanning cytometry (LSC) are highly efficient methods for detecting and measuring isolated cardiomyocytes or nuclei by antibody (Malliaras et al., 2013; Walsh et al., 2010; Mollova et al., 2013). Recently, humans have demonstrated a reduction in cardiomyocyte proliferation between birth and 20 years post-mortem when measured by LSC using the M phase marker phospho-histone3 in cardiomyocyte cell cycle. In humans, LSC and FACS in mice demonstrated a common consensus that the frequency of cardiomyocyte cell cycle activity decreased after birth (Walsh et al., 2010; Mollova et al., 2013). Carbon-14 (C-14) is an approach designed by Frisen et al to monitor cellular increase in tissue (Bergmann et al. 2009). C-14 was taken by diet by humans and was used to calculate the average birth date of a stable cell population (Bergmann et al., 2009). The carbon-14 content in the cardiomyocyte nuclear DNA was measured using accelerator mass spectrometry. The mean age of cardiomyocytes was compared with the age of the source individual and ~ 1% new cardiomyocyte formation was observed after 25 years of age. The accuracy of this approach depends on the quality of cardiomyocyte nuclei isolated from post-mortem myocardium. The analysis is based on additional assumptions for mathematical modeling of data (Bergmann et al., 2009). Potential changes in cardiomyocyte count after birth include assumptions about cardiomyocyte cell death rates and the prevalence of non-proliferative cell cycles (multinucleation and ploidy) with age (Elser and Margulies, 2012). The source of cardiomyocytes produced during the turnover remains an active research area. The data supports both progenitor differentiation and cardiomyocyte proliferation. Previous studies have shown that new cardiomyocytes can be produced in the heart of adult mammals, but the origin of these new cardiomyocytes was uncertain. Zeng et al. Reported that existing adult cardiomyocytes 70 • Health Sciences Research Papers may re-enter the cell cycle and are able to form new cardiomyocytes through a three-step process involving differentiation, proliferation and re-differentiation. "Myocyte proliferation is usually rare and therefore difficult to work with," he explains. Senior researcher Chunyu Zeng emphasized that they have developed an experimental system to induce new cardiomyocyte formation from adult cardiomyocytes and will use this system to investigate the underlying mechanisms of new cardiomyocyte formation. Genetically labeled cardiomyocytes from adult mouse hearts were cultured with neonatal rat ventricular myocytes and evaluated by time-lapse imaging and immuno-labeling to observe cytokinesis of proliferating adult cardiomyocytes. Adult cardiomyocytes in the co- culture were positive stained for many proliferation markers and showed that these cells re-enter the cell cycle by differentiation. These differentiated adult cardiomyocytes developed an organized contraction apparatus on day 3 and were reported to reach the highest number of beating cardiomyocytes on day 7. This in vitro system is involved in separation from neighboring cardiomyocytes, separation and proliferation of undivided myocytes, differentiation of these cells into functional cardiomyocytes, and formation of new cardiomyocytes from pre-existing adult cardiomyocytes. Both single-core and dual-core adult cardiomyocytes have been shown to proliferate with complete cytokinesis. In vivo studies in mouse models of myocardial infarction have shown that increasing electrical binding between adult cardiomyocytes in the infarct border region may increase the number of newly formed cardiomyocytes and improve cardiac function (Wang et al., 2017).

Aynur KARADAĞ GÜREL • 71

REFERENCES Akar, A.R, Durdu, S., Corapcioglu, T., Ozyurda, U. ( 2006) Regenerative medicine for cardiovascular disorders-new milestones: adult stem cells. Artif Organs;30:213-232. Anversa P, Leri A, Kajstura J & Nadal-Ginard B (2002). Myocyte growth and cardiac repair. J Mol Cell Cardiol 34, 91–105. Beltrami, A.P, Urbanek, K., Kajstura, J., Yan, S. M., Fınato, N., Bussanı, R., Nadal-Gınard B, Sılvestrı F, Lerı A, Beltramı Ca, Anversa P. (2001). Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med; 344:1750-1757. Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science 2009;324:98-102. Borisov, A.B., Claycomb, W.C., 1995. Proliferative potential and differentiated characteristics of cultured cardiac muscle cells expressing the SV40 T oncogene. Ann. N. Y. Acad. Sci. 752, 80 Ellison G.M., et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair Cell, 154 (2013), p. 827. Elser J.A., Margulies K.B. Hybrid mathematical model of cardiomyocyte turnover in the adult human heart PLoS One, 7 (2012), p. e51683. Garbern, J.C., Lee, R.T., 2013. Cardiac stem cell therapy and the promise of heart regeneration. Cell Stem Cell 12, 689 (Jun 6). Hashimoto, H., et al., 2014. Time-lapse imaging of cell cycle dynamics during development in living cardiomyocyte. J. Mol. Cell. Cardiol. 72C, 241 (Apr 3). Hesse, M., et al., 2012. Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle. Nat. Commun. 3, 1076. Jopling, C., et al., 2010. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464, 606 (Mar 25). Li F, Wang X, Capasso JM, et al. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737-46. Linzbach, A.J. (1976).Hypertrophy, hyperplasia and structural dilatation of the human heart. Adv Cardiol;18:1-14. Linzbach, A.J., 1950. The muscle fiber constant and the law of growth of the human ventricles. Virchows Arch. 318, 575. 72 • Health Sciences Research Papers

Linzbach, A.J., 1960. Heart failure from the point of view of quantitative anatomy. Am. J. Cardiol. 5, 370 (Mar). Malliaras K., et al. Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart EMBO Mol. Med., 5 (2) (2013), pp. 191-209. Mollova M., et al. Cardiomyocyte proliferation contributes to heart growth in young humans Proc. Natl. Acad. Sci. U. S. A., 110 (4) (2013), pp. 1446- 1451. Naqvi, N., Lı, M, Yahıro, E., Graham, R.M., Husaın, A. (2009). Insights into the characteristics of mammalian cardiomyocyte terminal differentiation shown through the study of mice with a dysfunctional c-kit. Pediatr Cardiol. Jul;30(5):651-8. Jan 23. Oberpriller, J.O., Oberpriller, J.C., 1974. Response of the adult newt ventricle to injury. J. Exp. Zool. 187, 249 (Feb). Rumyantsev, P.P. B.M. Carlson Growth and hyperplasia of cardiac muscle cells. Soviet medical reviews. Supplement series. Cardiology. Harwood Academic Publishers, London, U.K. ; New York, N.Y., U.S.A (1991), p. 376. Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, Wu TD, Guerquin-Kern JL, Lechene CP, Lee RT. Mammalian heart renewal by pre- existing cardiomyocytes. Nature. 2013;493:433–436. doi: 10.1038/ nature11682. Soonpaa M.H., Field L.J. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am. J. Physiol., 272 (1997), pp. Soonpaa, M.H., et al., 1997. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J. Clin. Invest. 99, 2644 (Jun 1). Soonpaa, M.H., Field, L.J., 1997. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am. J. Physiol. Heart Circ. Physiol. 272, H220. Soonpaa, M.H., Kim, K.K., Pajak, L., Franklin, M., Field, L.J., 1996. Cardiomyocyte DNA synthesis and binucleation during murine development. Am. J. Physiol. 271, H2183 (Nov). Soonpaa, M.H., Rubart, M., Field, L.J., 2012. Challenges measuring cardiomyocyte renewal. Biochim. Biophys. Acta 1833, 799 (Apr). Aynur KARADAĞ GÜREL • 73

Walsh S., A. Ponten, B.K. Fleischmann, S. Jovinge. Cardiomyocyte cell cycle control and growth estimation in vivo—an analysis based on cardiomyocyte nuclei Cardiovasc. Res., 86 (2010), p. 365. Wang WE, Li L, Xia X, Fu W, Liao Q, Lan C, Yang D, Chen H, Yue R, Zeng C, Zhou L, Zhou B, Duan DD, Chen X, Houser SR, Zeng C. Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury. Circulation. 2017 Aug 29;136(9):834-848. Zak, R., 1973. Cell proliferation during cardiac growth. Am. J. Cardiol. 31, 211 (Feb).

BIOECOLOGY OF INFECTIOUS DISEASES: AN ANTHROPOLOGICAL APPROACH

CHAPTER 5

Berna ERTUĞRUL1

1 Sivas Cumhuriyet University, Department of Anthropology

Berna ERTUĞRUL • 77

“Many common diseases are ineffective in primitive societies and probably have not played a role in human evolution.” Francis L. Black

INTRODUCTION In addition to adapting the environment to itself, human is also under the influence of significant adverse environmental factors. Although it is capable of changing living conditions in order to bring them to optimal living standards, one has never been able to fully protect themselves from the harmful effects of these self-shaping conditions. It already owes its existence to the biological and cultural adaptations it has developed against these threats. In other words, if he/she had stayed away from these threats, she/he would not have been so different and competent as he/she would not have had to develop new adaptive abilities in the rugged paths of evolution. Negative environmental conditions affecting human beings manifested in different ways in almost every step of the adventure of evolution. For example, the threat of wild carnivores to the human species is a type of environmental pressure from the hunter-gatherer era thousands of years ago. Our ancestors did not suffer from the pressures such as illnesses that manifested in severe epidemics and environmental pollution seen in societies that have adopted settled life in these periods. Of course, these rotational differences shown by environmental pressures on human beings are closely related both to the change of environmental conditions and to the change of one's own way of life. The drastic changes in human life have brought with it many different environmental pressures and gained new adaptive abilities to resist these new pressures. For example, the “Neolithic Revolution”, which was described as a revolution in human life in the history of evolution, brought along many useful facts as well as harmful facts. Negative changes in human health can be considered among these harmful cases. In particular, the transition to settled life and the introduction of agro-animal husbandry has caused people to be exposed to some infectious diseases that they have never encountered before (Kottak 2001: 257). Before moving on to these infectious diseases, it is useful to reveal what has changed in human life with the transition to established life. 78 • Health Sciences Research Papers

Origin of Infectious Diseases Up to 10 thousand years ago, people adopted the hunter and gatherer lifestyle. After this period, it is known that agriculture and livestock started in certain parts of the earth. The transition to this different mode of production took place at different times in different parts of the earth. In previous periods, people lived in hunting and gathering in small groups with a population not exceeding 100 (Table 1). These pre-agricultural groups hunted some large animals and, therefore, moved frequently in parallel with changes in animal geography. In other words, these communities were permanently nomadic (Black 1975, Kelley 1989: 192). Table 1: Relationship between population density and cultural change (Muscie-Taylor 1993:7)

Before Generation Cultural Situation Population Size Today 1.000.000 50.000 Hunter and Scattered groups not gatherer exceeding 100 10.000 500 Discovery of Villages with population agriculture less than 300 5.500 220 Discovery of Cities with population irrigated around 10,000 and villages agriculture with population around 300 250 10 Discovery of steam Cities with a population of power 100,000-500,000, villages with a population of around 1000. 130 6 Initiation of health Cities with a population of reforms 100,000-500,000, villages with a population of around 1000. 0 ------Cities with a population of 500.000-5.000.000, villages with a population of at least 1000.

Continuous relocation requires constant adaptation to different environments. Therefore, these people should not be exposed to infectious diseases which can be considered as environmental pressures. Because many diseases are unique to different environmental conditions, frequent relocation has prevented parasitic organisms that form these diseases from sheltering human groups for a long time. In particular, epidemics spread Berna ERTUĞRUL • 79 rapidly in non-dynamic groups, where larger groups of people live. The fact that the hunter and gatherer groups consisted of small groups and adopted a dynamic life style did not allow many diseases to find a host for long time (Black 1975, Kelley 1989: 192). But this does not mean that pre- agricultural societies do not suffer from any disease. Probably a number of viral and bacterial diseases existed in hunter-gatherer groups, but the course of these diseases never played an important role in the evolutionary adventures of our ancestors who lived during this period (Black 1975). The period in which infectious diseases started to influence human groups emerged with settled life that began approximately 8-10,000 years ago. During this period, small village groups with a population of approximately 300 emerged. The population living together increased and dry farming started in the Middle East about 7,500-10,000 years ago and the first animals were domesticated (Kottak 2001: 235). The increase in the population has brought some problems. As a first step in history, the problem of human waste has emerged as a result of the transition to settled life and the increase in population, which foreshadows a period when some infectious diseases have started to threaten people for the first time in history. Indeed, there is a significant relationship between population growth and resettlement and infectious diseases. In this period, the first animals were domesticated in some regions, especially in the Middle East, and the milk of these animals began to be utilized. Tuberculosis (Mycobacterium bovine), which is known to be transmitted to human beef cattle during these periods (about 7,000 years ago), came into being by domesticating cattle and feeding many of them in their own homes. This disease lies next to the cattle, meat and milk, the first agriculture and animal husbandry people who benefited and after a while adapting to humans has become a disease specific to it (Nikforuk 1991: 170). It should be pointed out that many of the diseases that originate from animals adapt to the resident living person and become a disease specific to it. In addition to domesticated diseases, resident life has caused undesirable animals to cause problems for humans in places where people live. For example, mosquitoes and rats are parasites bearing important health problems in humans (Cockborn 1977: 108). Such animals are pests living as settlers in settled villages. For example, settled villages are communities far from effective fighting against mosquitoes. The endemic malaria is a common disease in non-dynamic tropical communities. According to Zulueta, even though people lived in tropical areas during the 80 • Health Sciences Research Papers hunter-gatherer turnover, they were not much affected by malaria. Because these groups could easily displace the effects of these parasites (Muscie- Taylor 1993: 8). Therefore, tropical parasites such as malaria and schistosomiasis have made itself felt more in agriculture and animal husbandry. According to Poulin, the settled lifestyle of the Neolithic man, who lives on agriculture and animal husbandry, is still maintained in many different contemporary traditional communities today (Black 1975). Therefore, it is useful to look at today's traditional communities to establish the life profile of the settled Neolithic communities. The relationship between lifestyle and infectious diseases in today's traditional communities is the subject of the next chapter.

Infectious Diseases in Today's Traditional Societies There are many infectious diseases known to be effective on humans. The number of such diseases known to be caused by many bacteria and viruses exceeds 250. Some of these diseases are observed in many regions of the world as mentioned before; some of them are endemic and specific to some regions. If we classify infectious diseases exceeding 250, we will encounter six basic groups of diseases. These are viral diseases, rickettsia diseases, bacterial diseases, fungal diseases, protozoon diseases and helminth diseases (Çetin 1979). While some of these diseases are lethal, some of them may cause damages that cannot be considered as important in the daily life of the infected individuals. The increase in the population due to collective life, which is seen in many parts of the world and with the transition to settled life, has accelerated the spread of infectious diseases along with the maintenance of traditional livelihoods and unhygienic environmental conditions (Cockborn 1977: 108). Today, there are many groups of people who maintain the traditional way of life. They are especially concentrated in regions such as Africa, South America and South Asia. There are many infectious diseases that occur in these societies that maintain their traditional lifestyles, namely agriculture and animal husbandry. These diseases include typhoid fever, tuberculosis, dysentery, pinta, malaria, schistosoma and trachoma. The faults known to pass from animals to humans are called zoonos. Some living things that live as parasites in animals can pass on to humans Berna ERTUĞRUL • 81 and cause some important diseases. These diseases are generally common in societies that have adopted the traditional way of life. In these societies, the widespread tradition of feeding various animals and making use of them is an important factor in the spread of these diseases. In particular, the use of faeces of some animals as fertilizer increases the frequency of faults through faeces. For example, this practice is quite common in many villages in South Korea where traditional agriculture is practiced. Some hookworms entering through the skin to the village people working barefoot in the fields cause important parasitic diseases. In addition, diseases such as typhoid, which are transmitted by the ingestion of plants grown on farmland fertilized with animal faeces, also threaten these societies. According to a study in rural areas of South Korea, intestinal parasites are reported in 90% of the population. Research in contemporary traditional societies is a risky situation, as it is possible that the infections that are unfamiliar to these societies are transmitted by the researchers to these societies and that some diseases seen in these societies are transmitted to the researchers. Therefore, such research on traditional societies is very limited (Black 1975). However, the World Health Organization and some international organizations are conducting various studies about the distribution of some tropical diseases known to be endemic on the earth. For example, malaria, known to be endemic and one of the most common diseases in the world, appears to have influenced many societies that have adopted a traditional way of life around the world. There are four types of parasites that cause malaria. They are transmitted by mosquitoes in areas where malaria is very common. This disease, especially prevalent in tropical regions, is common in tropical regions where irrigated agriculture is common in communities living on the water's edge. According to the World Health Organization, 40% of the world's population, especially Africa, Latin America and South Asia, are at risk. According to experts, 270 thousand people develop this disease annually and 2 million people die every year (Popkin 1982). In particular, half of the cases reported in one year occur in groups living in sub-Saharan Africa. The abundance of mosquitoes carrying malaria germs in this region and abundant irrigation channels are factors that increase the risk of malaria (Sladen and Bang 1969, Frideman 1994). 82 • Health Sciences Research Papers

Another most common disease after malaria is shistosomiasis. Like malaria, this disease is unique to tropical regions. Shistosomiasis worm is particularly common in African countries. In addition to this region, the maggots that cause this disease are abundant in the Southeast American and Caribbean islands. It is reported that this disease is also encountered in villages engaged in agriculture in South East Anatolia in our country (Çetin, 1979). The widespread use of agriculture-related lifestyles worldwide has caused this disease to spread. Shistosomiasis affects about 200 million people in at least 74 countries, and 400 million people live under the threat of this disease (WHO, 1986; Muscie-Taylor, 1993: 23). These worms, which are found in some snails living in irrigation channels opened for irrigation of agricultural lands, can easily be transmitted to humans in case of contact with these waters. The increase in ponds for irrigation in Africa in recent years has led to the rapid spread of schistosomiasis after malaria and tuberculosis (Sladen and Bang, 1969). This disease is also very common in the Philippines. Large-scale studies initiated by the WHO in the 1960s have helped to control the disease in this region. But the problem remains particularly important in Africa. In 1962, Hairston's Inland village of Leyte (Philippines), 469 out of 768 individuals were found to have Schistosoma japonicum eggs in their liver. Accordingly, approximately 65% of this group examined have this disease. This disease is widespread in this village, the most important reason, especially in the irrigation channel of the crustaceans carrying the disease is consumed as a food source (Sladen and Bang, 1969). In addition, the fact that the swamp areas are abundant and rice cultivation is widespread in this region increases the risk of transmission of the disease. Therefore, this disease is also seen in Japan and China, where rice cultivation is very common. Walking around barefoot in rice cultivated land causes these maggots to enter the body from the soles of the foot easily. (Guo et al. 2001). The most important reason for the prevalence of schistosomiasis is the rapid expansion of irrigation canals created for irrigation of agricultural lands in traditional regions. For example, agricultural innovations initiated in Egypt in the 1930s led to the spread of irrigation canals and small dams in many regions. Following these reforms, schistosomiasis disease was reported to increase from 2-11% to 44-75% in four different regions studied in Aswan between 1934 and 1937 (WHO, 1986). In Sudan, another African country where agricultural reforms were initiated, the course of Berna ERTUĞRUL • 83 this disease increased in parallel with these reforms. After the construction of the Sennar dam in the Gezira region of Sudan in 1924, the incidence of the disease was 5.1% between 1924 and 1944, while in 1952 it was 21% for adults and 45% for children. Likewise, in Ghana, the prevalence of this disease was 5-10% before the construction of irrigation canals, while the frequency of the disease increased significantly after the construction of irrigation canals in the 1950s (WHO 1986). S. Haematobium and S. Japonicum are particularly common in the north of S. Africa and west of Central Africa and west of Madagascar. In addition, it is remarkable that these two parasites are also present in South Eastern Anatolia in our country. The other two species, S. Mansoni and S. Intercalatum, are found in similar regions of Africa, but also occur in the east of South America. On the other hand, on the island of Madagascar, these two diseases are seen in the western regions (WHO 1986). West and North Africa are areas where traditional agriculture and animal husbandry are common. For this reason, animal-to-human diseases depending on the way of life are quite common here. For example, Archer et al. Conducted a health survey in 1981 in three villages in the Karamoja region of Uganda, where 10 types of parasitic diseases were found to be prevalent in this region (Archer et al. 1982). Table 3 shows the distribution of diseases according to the number of individuals. Table 2: Distribution of parasitic diseases in the Karamoja region of Uganda (Archer et al. 1982) Parasite type N (%) Malaria (n=148) 58 39,7 Helminth diseases (n = 138) Hookworm 54 37,7 Tapeworm 32 22,5 Enterobius 7 5,1 Intestinal helminthiasis 5 3,6 Schistosoma 5 3,6 Strongyloidiasis 2 1,5 Subulara 6 4,3 Protozoa (n=138) Amoebiosis 42 30,4 Giardiasis 27 19,6

84 • Health Sciences Research Papers

According to Table 2, 157 individuals were examined and it was seen that 95% of the individuals examined had parasitic diseases. In particular, the incidence of malaria in 58 out of 148 individuals examined shows that this disease is quite common in this region. After Malaria, the two most common diseases in Karamoja are hookworm and tapeworm. These two diseases caused by maggots living in the small intestine of a human are passed from cattle to human beings. The tapeworm, 5-10 microns in length, settles in the striated muscles of the animals. In Karamoja, tapeworm cattle are eaten without good cooking, resulting in 22.5% of the disease. In addition to these three diseases mentioned in the population of Karamoja engaged in agriculture and animal husbandry, the other seven diseases can be seen in various proportions, the lack of clean drinking water in this region, and close contact with the fed cattle are among the important reasons. According to Biellik and Henderson, especially after the 1970s, the Karamoja people gave more weight to animal husbandry and irrigated agriculture, causing hookworm, schistosomiasis and malaria to increase rapidly in this region (Biellik and Henderson 1981). If we look at Gambia, another traditional community of West Africa from the Karamojo region of Uganda, it will be seen that there are many diseases caused by the traditional way of life in this country. In a study initiated by Ian Mc Gregor in the Kenaba region in 1949, a long-term study was initiated on many individuals living in an area of 40 x 20 km2. In this region, which consists of a large savannah area and where approximately 9 thousand people live, the people continue to live with agriculture and animal husbandry to a significant extent (Lewis 1993: 75). It is known that drinking water shortage is an important problem in this region when the season is dry. For example, during the dry season, many parasitic diseases arise from water resources shared with other animals. In dry seasons, pathogenic organisms increased by up to 100% in 1979 by Barell and Rowland's research (Lewis, 1993.87). Another disease in which important studies have been carried out among African infectious diseases is African Sleep Disease, which is transmitted from flies (Cheche fly). This disease is common in 36 countries, especially in Central and West Africa. Especially Sleep Disease has been an important problem in Uganda and Sudan recently. Following these diseases that are transmitted from animals to humans in African countries that have adopted a traditional way of life, it is Berna ERTUĞRUL • 85 necessary to look at the diseases specific to the societies that have traditionally lived in the south of the New World. For example, the most common disease of this region is the American Lechemniasis, which is transmitted by tsar flies. This disease is especially prevalent in the northern regions of S. America. In these societies, which have traditional lifestyles, carcinoma is transmitted to human beings in wetlands, especially by flies that lay eggs around irrigation canals. Tsetse flies only this disease moving to human by the female can not only be seen in this region, for example, one of three species N. cutaneus and mucocutaneous leşemniyaz in West Africa, around the Mediterranean, in Turkey, is common around the Arabian Peninsula and the Caspian Sea (WHO 1984) . Visceral leishemniasis, another species of the disease, is also abundant in the east of S. America, East and North Africa, around the Mediterranean Sea and in rural areas of India. Leshemchinia has been recognized as a public health problem in some countries. Visceral leishemniasis, especially after the Second World War, became widespread in D. Africa. In addition, experts reported that several thousand cases of cutenous leukemniasis were reported in 1976 in Africa in Magre, Africa (WHO 1986). Chagas disease is another common disease seen in the poor people living in the region after the leukemniasis in America. This disease is very common in rural areas of the United States and poorer areas of cities. It is estimated that this disease, carried by insects such as triatomide, threatens around 65 million people in S. America. In the 1980s, it was revealed by the research conducted by the Pan American Health Organization that the number of people with this disease is 15-18 million. Chagas disease is very common in countries in the southernmost tip of S. America and in the central-northern regions of Brazil (WHO 1986). Experts report that the people of the Amazon of America live a traditional life that can be considered quite primitive. These groups consist of tribes consisting of several hundred people. In 1975, Black brought together two surveys of 9 tribes in the Amazon region. If we look at the infectious diseases which are observed in these tribes which have traditional life style and are transmitted from animals, it will be seen that there are many different diseases in these 9 tribes. Diseases caused by herepes simplex and cytomegaloviruses, which are common after leechemniasis in this region, are present in these communities as well as in many primitive tribes (Table 3). 86 • Health Sciences Research Papers

Table 3 shows that many zoonotic diseases exist in these communities. For example, tuberculosis and malaria that have recently moved to this continent are present in 4 out of 8 tribes (Black 1975). However, in other studies such as Suia and Txukharamia, these diseases have never been found. This indicates that two diseases have recently entered this region. In addition to these two diseases, the high incidence of yellow fever, arbovirus A and toxoplasmic diseases in these populations can be attributed to close contact with animals. Table 3: Distribution of Infectious Diseases Observed in Some Tribes in South Africa by Number of Individuals * (Black 1975)

Researched tribes

Diseases Tiro Ewarho Xikrin Gorotire Kuben Mekra Xavante yona Kran noti Suıia Kegn Herpes 91 75 90 93 Yellow Fever 14 37 4 5 5 3 8 Arbovirus A 47 46 49 30 18 Toxoplasma 43 46 52 52 100 Tuberculosis 27 39 4 1 Malaria 41 6 84 62 * Zoonotic diseases were selected from the original table. Another study conducted in Peru in S. America shows that parasitic diseases are common in this region. According to this study conducted in 4 villages in different altitudes of Peru, it can be said that parasitic diseases especially from domestic pigs are abundant in these villages, as in other American countries, mosquitoes and black flies are frequently encountered in these villages. Another remarkable point in this study is that as the altitude increases, the incidence of diseases decreases (Harrison et al. 1989: 512). It is useful to examine another common disease in Europe after African and S. African specific diseases. Brucellosis is a disease that passes from animals to humans and is found in many geographies throughout the Mediterranean. In particular, this disease is transmitted from farm animals to milk. This disease, also known as Mediterranean Fever, is particularly common in the Mediterranean Sea. This disease, which is widespread in countries such as Spain and Italy, where livestock farming is widespread, is endemic in Lebanon. Tohme et al. can be considered as one (Tohme et al. 2001). Arribas et al. (1989) reported that there were 246 cases of burucellosis between 1981-1987 in Miguel Servet Hospital in Spain. It is Berna ERTUĞRUL • 87 reported that 25,165 cases of burucellosis were encountered in the rural areas of Italy where the disease was widespread between 1987 and 1986 (Ortona et al. 1988). As it is known, most of the people in the eastern and southeastern regions of our country continue their livelihood with agriculture and animal husbandry. Therefore, depending on the way of life, people of Anatolia also suffer from many parasitic diseases. Many of the diseases mentioned above in traditional societies are also common in these regions. Diseases seen in rural areas of Anatolia include tuberculosis, brucella, malaria, typhoid and typhus. Tuberculosis disease is very common in the eastern regions of our country because of the widespread animal husbandry. In a study conducted by Türkdoğan et al. In Van province, the prevalence of this disease was found to be 3/1000 in rural areas (Türkdoğan et al. 1996). Similarly, malaria and brucella are diseases specific to rural areas in Anatolia. Especially in hot regions in the southeast, malaria is endemic. In provinces with warm climates such as Adana and Diyarbakır, malara is quite common. The construction of irrigation canals initiated within the framework of the GAP project causes malaria to become widespread in this region (Canda 1991). According to the experts in our country until 1970, malaria was a disease controlled to a significant extent. After this date, however, there was a significant increase in the frequency of malaria, especially in the southeastern regions in 1977. According to experts, the socioeconomic status of people living in regions known to be endemic to malaria, the way of living, livelihoods related to irrigated agriculture and migration should be held responsible (Barut et al. 1992). Another common infectious disease in the eastern and southeastern regions is brucella. This disease is quite common in rural people who consume fresh cheese and drink without boiling milk (Aslan et al. 1995). Diseases in different traditional populations are not limited to those listed above. Many diseases are transmitted from animals to humans and are endemic. These diseases include Asian Colera, Histoplasmosis, Northern Queensland Tick Typhus, North American Blastomycosis, South African Fever, Venezuelan Horse Encephalitis, Scotland Encephalitis Colorado Tick Fever, Far East Encephalitis, West Nile Fever, Crimean Hemorrhage Fever, North Asian Tick Fever. Diseases have been named according to the regions where they are seen (Çetin 1979).

88 • Health Sciences Research Papers

Conclusion With the transition to settled life, the human species has been under the grip of many negative environmental conditions. Living permanently in a particular region has led to a rapid increase in the population here, as well as some of the parasites that are specific to this region to adapt to these people rapidly. The increase in the number of farmland and livestock to feed the growing population has also increased the number of diseases caused by this lifestyle. In parallel with this increase, the problem of eliminating human wastes has created another problem that cannot be overcome. The industrial revolution and the rapid advances in health at the beginning of the last century have led to an increase in medical facilities, enabling us to better recognize diseases that threaten our health. This has eradicated many diseases in developed societies in traditional societies. Today, however, almost half of the world's population still maintains its traditional way of life, such as agriculture and animal husbandry. In societies that have adopted this way of life, the number of those who still maintain these sources of livelihood under primitive conditions is considerable. In particular, it can be said that the main traditional diseases mentioned above are mostly seen in the regions suitable for the spread of these diseases and that the primitive agriculture and animal husbandry techniques cause these diseases to become widespread. It is important to note that such diseases are not only caused by changes in human life but also by natural conditions. For example, infectious diseases are more common in African and South American societies interested in agriculture and animal husbandry, not only because of this lifestyle, but also because of the climatic characteristics of these regions (Harrison et al. 1989: 512).For example, parasitic diseases such as malaria and schostomiasis are common in these regions and the tropical structure of these regions, while the traditional lifestyles of people living in this region are among the reasons that increase the effectiveness of these parasites. For example, ponds and canals opened to irrigate agricultural land - as mentioned in the previous chapters - increase the incidence of infectious diseases by causing many parasites to breed there. In addition, the fact that these societies are quite backward in terms of hygiene and health accelerates the spread of such diseases.

Berna ERTUĞRUL • 89

REFERENCES Archer, T.J., Mc Donald, P.J., Klıdjian, A.M., Karran, S.J. (1982) Parasitic diseases in Karamoja, Uganda. Lancet. 17: 912-913 Arribas, L.J.L., Navarro, G.J.F., Hernandez, N.M.J., Muniesa, C.M.P., Garcia, M.J.R., Rosas, N.A., Sarda, J.N., Barrasa, V.I., Amorin, C.M.J. (1989) Epidemiology of brucellosis. Retrospective study of 246 hospital cases. Rev. Clin. Esp. 185: 60-64. Aslan, T., Genç, M., Güneş, G., Pehlivan, E., Günal, S. (1995) Malatya ilinde seçilmiş bazı risk gruplarında Wright Tekniği ile brusella taraması. İnönü Üniversitesi Turgut Özal Tıp Merkezi Dergisi, 2: 354-358. Barut, A., İnal, A., Özgüven, V., Hacıbektaşoğlu, A., Özer, F.T. (1992) Türkiye’de sıtma epidemiyolojisi ve eradikasyon çalışmalarının başarısı. Türk Hijyen ve Deneysel Biyoloji Dergisi, 49: 181-190. Biellik, R.J., Henderson, P.L. (1981) Mortalitiy, nutrition status and diet during the famine in Karamoja, Uganda, 1980. Lancet. ii: 1330-1333. Black, F.L. (1975) Infectious disease in primitive societies. Science, 187: 515-518. Canda, M.Ş. (1991) Sıtmanın ekopatolojisi ve ülkemiz açısından önemi. Enfeksiyon Hastalıkları, 15: 1-12. Çetin, E.T. (1979) İnfeksiyon Hastalıkları. İstanbul Tıp Fakültesi Klinik Ders Kitapları, İstanbul: Çeliker Matbaacılık Koll. Şti. Cockburn, T.A. (1977) Where did our infectious disease come from? The evolution of infectious disease. In: Healt and Disease in tribal Societies pp. 103-113, Ciba Foundation Symposium 49 (new series), Frideman, G.D. (1994) Primer of Epidemiology. 4 ed. Mc. Grow Hill İnc. Health Professions Divisions. Guo, J.G., Ross, A.G., Lin, D.D,. Williams, G.M., Chen, H.G., Li, Y., Davis, G.M., Feng, Z., Mc Manus, D.P,. Sleigh, A.C. (2001) A baseline study on the importance of bovines for human Schistosoma japonicum infection around Poyang Lake, China. Am. J. Trop. Med. Hyg. 65: 272-278. Harrison, G.A., Tanner, J.M., Pilbeam, D.R, Baker, P.T. (1989) Human Biology. Oxford University Press. Kelley, M.A. (1989) Infectious Disease. In: Reconstruction of Life from the Skeleton Ed: İşcan, M.Y., Kennedy, pp. 191-199, K.A.R., New York: Alan R. Liss, Inc. Kottak, F. (2001) Antropoloji: İnsan Çeşitliliğine Bir Bakış. Ankara: Ütopya Yayınevi. 90 • Health Sciences Research Papers

Lewis, G. (1993) Some Studies of Social Causes of and Cultural Response to Disease. In: The Anthropology of Disease. Ed. Mucsie – Taylor, C.G.N, Oxford University Press. Mucsie – Taylor, C.G.N. (1993) The Biologic Anthropology of Disease. In: The Anthropology of Disease. Ed. Mucsie – Taylor, ss:1-65, C.G.N, Oxford University Press. Nikforuk, A. (1991) Mahşerin Dördüncü Atlısı. Çev. Selahattin Erkanlı, İstanbul: İletişim Yayınları. Ortona, L., Cauda, R., Ventura, G., Tumbarello, M., Ballada, D., Branca, G. (1988) Ten years of brucellosis in Italy (1977-1986). Eur. J. Epidemiol., 4: 503-505. Popkin, B.M. (1982) A household framework for examining the social and economic consequences of tropical diseases. Soc. Sci. Med. 16: 533-543. Sladen, B.K., Bang, F.B. (1969) Biology of the Populations. New York: American Elsevier Publishing Com. Inc. Tohme, A., Hammoud, A., el Rassi, B., Germanos-Haddad, M., Ghayad, E. (2001) Human brucellosis. Retrospective stıdies of 63 cases in Lubnan. Presse Med. 30: 1339-43. Türkdoğan, M.K., Tuncer, İ., Algün, E., Alıcı, S. (1996) Van yöresinde abdominal tübeklülozlar. Gastroentroloji, 7:121. WHO (1984) Technical Report Series, No: 701. WHO (1986) Major parasitic infectious: A global review. Wld. Hlth. Statist. Quart., 39: 145-160.

IMPACT OF IRON AND FOLIC ACID SUPPLEMENTATION DURING PREGNANCY ON NEWBORN BODY SYMMETRY

CHAPTER 6

Berna ERTUĞRUL1

1 Sivas Cumhuriyet University, Department of Anthropology

Berna ERTUĞRUL • 93

INTRODUCTION Although our species has a bilateral symmetrical structure, it is seen that this symmetrical structure is not perfect when examined in detail. Different stressors in humans may reduce developmental stability and cause growth to deviate from its normal course. Studies have shown that environmental factors are largely responsible for deviations from bilateral symmetry (Palmer and Strobeck 1986, Zakharov 1992). Numerous studies have shown that body symmetry is a good indicator of the individual's developmental stability or homeostasis (see Palmer and Strobeck 2012, Moller and Swaddle 1997). The type of asymmetry commonly seen in bilateral traits is fluctuating asymmetry (FA). Fluctuating asymmetry defines small land random asymmetrical deviations in bilateral organs. (Palmer and Strobeck, 1986; Palmer and Strobeck, 2003; Van Valen, 1962; Zakharov, 1992). FA is used as an important indicator in developmental stability studies since it reflects the phenotypic and genotypic quality of a population. Long-term studies of various species have shown that many factors are responsible for the deviation of bilateral characters. These factors include high temperature, environmental pollution, malnutrition, inbreeding, infections, parasites, and some genetic diseases leading to increased homozygosity (Moller and Swaddle 1997, Thornhill and Moller 1997, Mazzi et al., 2002). Studies have shown that negative maternal factors decrease the developmental quality of the fetus as well as the developmental stability and increase both the dermatoglyphic and anthropometric asymmetry levels during pregnancy. During pregnancy, iron and folic acid supplementation is important to reduce the risk of maternal anemia and low birth weight. The World Health Organization (WHO) states that the use of 60 mg iron + 0.4 mg folic acid as a standard dose is important for all pregnant women (WHO 2012). The aim of this study was to determine whether iron and folic acid use had any effect on the body symmetry of newborns.

Subject and Method This study was carried out in Sivas Province of the Central Anatolian Region of Turkey. This province, with a population of approximately 300,000, is ranked 49th of 81 provinces according to the Survey of Socio- Economic Development Rankings of the Provinces and Regions of Turkey 94 • Health Sciences Research Papers

(issued in 2012 by the Ministry of Development). The study group consisted of full-term NB with gestational ages between 37 and 41 weeks. After deliveries in the NB clinic, babies were monitored for 2-3 days. Newborns were examined in two groups as using iron and folic acid combination and non-use during pregnancy. In order to determine the fluctuating asymmetry levels of the newborns, bilateral ear lengths, widths and second and fourth digit lengths were measured with 0.01 mm accuracy by blind measurement technique. Of the 200 infants in this study, 60 (30 males, 30 females) were measured twice to estimate measurement error. A mixed-model ANOVA was used to estimate repeatability of the asymmetry. Two-way mixes model analysis of variance was used to determine whether there was a difference between the composite fluctuating asymmetry levels of the newborns used with and without vitamin combination.

Results Analysis revealed that the infants of mothers who did not use iron and folic acid combinations had higher levels of composite asymmetry and this effect was more pronounced in male newborns (Figure 1) (F1,224 = 9.47, P = 0.002). The interaction of sex and vitamin use on composite asymmetry was not significant (Table 1). Table 1: Two-way analysis of variance results (Dependent variable = composite FA, Fix factors = sex * drug use) Type III df Mean F P Sum of Square Squares Corrected 4.187 3 1.396 4.666 0.004 Model Intercept 141.769 1 141.769 474.002 0.000 Sex 1.437 1 1.437 4.805 0.029 Drug use 2.833 1 2.833 9.473 0.002 Sex*drug use 0.669 1 0.669 2.236 0.136 Error 65.800 220 0.299 Total 209805 224 Corrected Total 69.986 223

Berna ERTUĞRUL • 95

1,2 1,09 1,1 1 0,9 0,8 Use 0,8 0,74 0,68 Not use 0,7 0,6 0,5 Male Female

Fig 1: Composite FA scores due to iron and folic acid use

Discussion According to the results of this study conducted on the newborns of predominantly lower socioeconomic families, it is observed that the babies of mothers who use iron and folic acid supplements during pregnancy have a more symmetrical body structure. In pregnancy, iron consumption increases due to the needs of the fetus and the increase in blood volume. It is reported that a total of 1000 mg of iron is required during the entire gestation period, while iron requirement reaches a high level, especially in the third trimester (WHO 2012). Iron requirement was 0.8 mg / day in the first trimester and 6.3 mg / day in the second and third trimesters. Although iron absorption is increased in pregnant women, dietary iron is not sufficient to meet daily requirements; therefore additional iron supplementation is required depending on the state of the iron stores in the body. Most women in developing countries have very low levels of iron stores due to malnutrition, infections and frequent and numerous pregnancies. It is seen that iron deficiency during pregnancy causes deterioration in the motor development and coordination of the fetus, growth retardation, impaired speech ability, decreased physical activity, and decreased resistance to attention deficit and infections (WHO 2012). The World Health Organization (WHO) recommends the daily folic acid intake of non-pregnant women and adults to be 170 mcg, especially considering its effect on neural tube defects and increasing this amount to 370-470 in pregnancy and 270 mcg in lactation (WHO 1998). Folic acid 96 • Health Sciences Research Papers has a very important function in terms of protein synthesis in the fetus, cell proliferation and bone marrow function. Studies have shown that adequate folic acid intake during pregnancy significantly reduces the likelihood of central nervous system abnormalities in the baby (WHO 2012). Few studies have been conducted on neonates with regard to fluctuating asymmetry, which is a good indicator of phenotypic and genotypic stresses. Livshits and Kobyliansky (1989) found that anthropometric asymmetry was increased in babies born to mothers who had lung diseases during pregnancy. In the same study, it was observed that the factors leading to preterm birth had an effect on increasing anthropometric asymmetry. In a different study conducted by 216 newborns by the same researchers, it was observed that asymmetry increased in infants with low values in terms of eight anthropometric traits. In this study, it was also found that cardiovascular and infectious diseases observed in the mother cause anthropometric asymmetry in the infant, but asymmetry increased in newborns in parallel with increasing maternal age (Livshits et al. 1988). Kieser and Groeneveld (1994) conducted a study on the babies of mothers who smoked during pregnancy, and it was found that the babies of mothers who smoked had a higher amount of dental asymmetry than non-smokers. Similar results are observed in a study conducted by Kieser et al. (1997). This study also shows that obese mothers tend to give birth to babies with more asymmetric upper jaw teeth. In a study by Sing and Rosen (2001) on 277 prospective mothers, there is a positive relationship between pregnancy obesity and developmental stability of the baby, as in the study mentioned above. In this study, it is also shown that maternal pregnancy diabetes and high blood pressure cause an increased tendency in fluctuating asymmetry in the newbons. As we have seen, worsening maternal factors reduce the quality of physical development of the fetus (as in this study). Another point that is emphasized in asymmetry studies is the level of developmental stability differences of the sexes. Some researchers who work on tooth asymmetry claim that females tend to be more symmetrical than men. According to these researchers, the presence of two X chromosomes in females provides a more stable structure during the development process. At the same time, females should be more resistant to environmental factors when they are in the womb, suggesting that they should be more symmetrical (Kowner, 2001; Özener, 2010). The findings obtained in this study show that babies of mothers who do not receive iron and folic acid supplements are more Berna ERTUĞRUL • 97 asymmetric. However, it can be said that the effect is more pronounced in male newborns. Female newborns with and without support are more symmetrical than boys. On the other hand, it is seen that the male infants of mothers who do not receive iron - folic acid support tend to be asymmetric. In conclusion, the results of this study suggest that iron and folic acid use during pregnancy has positive effects on the developmental stability of newborns -especially for male newborns- as expected.

98 • Health Sciences Research Papers

REFERENCES Kieser, J.A, Groenveld, H.T. (1988) Fluctuating odontometric asymmetry in an urban South African Black population. Journal of Dental Research, 67: 1200-1205. Kieser, J.A., H.T. Groenveld., Da Silva, P.C.F. (1997) Dental asymmetry, maternal obesity and smoking. American Journal of Physical Antrhropology, 102:133-139. Kowner, R. (2001) Psychological perspective on human developmental stability and fluctuating asymmetry: Sources, applications and implications. British Journal of Psychology, 3:447-469. Livshits, G., Davidi, L., Kobyliansky, E., Ben-Amıtai, D., Levi, Y., Merlob, P. (1988) Decreased developmental stability as assessed by fluctuating asymmetry of morphometric traits in preterm infants. American Journal of Medical Genetics, 29:793-805. Livshits, G., Kobyliansky, E. (1989) Study of genetic variance in the fluctuating asymmetry of anthropometrical traits. Annals of Human Biology, 16:121-129. Møller, A.P., Swaddle, J.P. (1997) Asymmetry, developmental stability, and evolution, Oxford: Oxford University Press. Özener B. (2010) Facial fluctuating asymmetry as a marker of sex differences of the response to phenotypic stresses. American Journal of Physical Anthropology, 143:321–324. Palmer AR, Strobeck C. (1986) Fluctuating asymmetry: measurement, analysis, patterns. Annual Review of Ecology, Evolution and Systematics 17:391-421. Palmer AR, Strobeck C. (2003) Fluctuating asymmetry analysis revisited. Polak M (ed): Developmental Instability: Causes and Consequences. Oxford University Press, 279-319. Singh, D., Rosen, V.C. (2001) Effects of maternal body morphology, morning sickness, gestational diabets and hypertension on fluctuating asymmetry in young women. Evolution and Human Behavior, 22:373-384. Van Valen L. (1962) A study of fluctuating asymmetry. Evolution 16:125-142. WHO (1998) Healty Eating During Pregnancy and Lactation. Training Course and Workshop Curriculum for Health Professionals Revısed Draft. WHO (2012) Guideline: Daily iron and folic acid supplementation in pregnant women. Geneva, World Health Organization. Zakharov VM. (1992) Population phenogenetics: analysis of developmental stability in natural populations. Acta Zoologica Fennica 191:7-30.

HOLISTIC CARE IN CARDIAC SURGERY PATIENTS

CHAPTER 7

Elif GEZGİNCİ1, Sonay GÖKTAŞ2, Kübra GÜNEŞ

1 Asst. Prof. 2 Assoc. Prof.

Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 101

INTRODUCTION Earlier, instead of the interaction between human body and mind, the organ where the pain was felt was concentrated on and the effects of the disease on mind were not given importance. Hippocrates argued that psychosocial changes in individual caused physical problems due to the interaction between body, mind and environment. This idea of Hippocrates revealed the holistic approach. Holistic concept pioneered by Jan Christian Smuts in 1926 pointed out that holistic approach in healthcare has individual, social and environmental aspects. The holistic approach enables the individual to receive the best care by arguing that it is not possible to distinguish between the physical, psychological, spiritual, sociocultural, intellectual and environmental dimensions of the individual (Papathanasiou, Sklavou & Kourkouta, 2013; Çalışkan & Gündüz, 2019). The holistic approach focuses on two important issues. First, each individual is distinctive and unique. Secondly, the unique individual is more complex than the concept of health and disease (Papathanasiou et al., 2013). In 1971, Joyce Travelbee spoke of the importance of holistic approach by saying ‟a nurse provides services not only for relieving the physical pain, but also to give holistic (comprehensive) care to the individual”. For example, ignoring the individual's psychological state after surgery and addressing the physiological needs only, indicates that holistic care is not provided. The mental and physical responses of the individual in case of health and illness should be evaluated as a whole. The American Holistic Nurses Association founded by nurses who adopted holistic concept in 1981, defined health as the existence of harmony between body, mind and soul, and focused on the relationship between the individual's bio-psycho-social-spiritual dimensions and argued that they are considered as a whole (Ergül & Bayık, 2004; Erişen & Karaca Sivrikaya, 2017; Çalışkan et al., 2019).

HOLISTIC APPROACH AND NURSING CARE It is the foundation of nursing profession to provide health care with holistic understanding in order to meet the existence and various needs of the individual. Care, which is one of the reasons for the emergence of nursing profession, is related to humanitarian values. Holistic care, derived from the concepts of humanity and holism, is a philosophy which is called the heart of nursing that directs the care of individuals. During a disease, 102 • Health Sciences Research Papers psychological, social and cultural needs adversely affect the daily life activities by disrupting the individual's balance. Holistic care addresses the physical and mental needs of individuals, restores their balance and improves their lives (Ergül et al., 2004; Jasemi et al., 2017). Florence Nightingale is the first nurse to highlight the importance of holistic care, revealing many modern nursing values with a holistic perspective. With her statement: ‟For health, spiritual needs are as important as the physical organs that make up the body, the physical state that we all observe can affect our minds and souls”, Nightingale emphasized the importance of holistic care. Holistic care is based on the humanistic view of unity and individual (Erişen et al., 2017). The concept of holistic care was introduced into the nursing literature by theorists such as Rogers, Parse, and Newman in 1980, and nurses at that time provided holistic care for the individuals traditionally. There is a holistic paradigm in the health systems of all cultures that exist in the world. In the meantime, each individual has the right to get holistic care regardless of race or religion, but there are too many individuals who cannot obtain this right. There is evidence that most nurses had a science- oriented training do not have a good knowledge of the concept of holistic care (Papathanasiou et al., 2013; Jasemi et al., 2017). This situation makes it impossible to focus on the concept of healing, which is the important point of the holistic approach, and results in taking into consideration only the physical needs of the patients. The lack of improvement in nurses' treatment-oriented care is not only insufficient for health recovery but also puts individuals under serious threat. The patients’ length of stay in the hospital is prolonged and treatment costs are increased. In many countries, such as the UK, Australia and Iran, healthcare requirements are not appropriate. Many aspects of health and illness status of individuals cannot be evaluated, and their needs cannot be met. When the reasons of neglect of holistic approach were examined, many factors such as inappropriate professional relations, incomplete nursing reports, insufficient time, clinical supervision, management, concepts like knowledge, unavailability of resources, motivational factors and misunderstanding in nurses were identified (Henderson, 2002; Olive, 2003; Bahrami, 2010). Although the holistic care approach is described as a work-oriented, time-consuming concept in the studies of McEvoy and Duffy (2008) and Bullington, and Fagerberg (2013), Bahrami (2010) emphasizes the Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 103 importance of holistic approach in nursing care. Similarly, the results obtained from the study of Jasemi et al. (2017) showed that holistic care approach did not only produce positive results for patients but also positively affected the treatment process. The findings of the literature suggest that the concept of holistic care should be understood, learned and applied correctly (Jasemi et al., 2017).

HOLISTIC APPROACH AFTER CARDIOVASCULAR SURGERY The holistic approach is important in the care process of individuals after cardiovascular surgery. Holistic approach is composed of physical, psychological, spiritual, sociocultural, intellectual and environmental dimensions (Çalışkan et al., 2019).

PHYSICAL DIMENSION Physical care in patients after cardiovascular surgery includes the provision of circulation and hemodynamic, oxygenation, bleeding prevention, pain control, early mobilization, nutrition and excretion intervention. Patients admitted to the Cardiovascular Intensive Care Unit after surgery are at high risk. Cardiovascular surgery can cause physical complications such as bleeding, cardiac tamponade, heart failure, dysrhythmias, angina, renal failure and respiratory failure (Morton & Fontaine, 2013; Leeper & Kaplow, 2019). Therefore, the physical dimension of the care required in the postoperative period is vital for the patient. The nurse tracks and evaluates blood pressure, heart rate and rhythm, central venous pressure and pulmonary hypertension, cardiac output, SpO2 value, acid-base balance and fluid-electrolyte balance in order to maintain the patient's hemodynamic. The observation of bleeding, which is a problem that disrupts hemodynamic, is also very important in nurse monitoring. The amount of drainage in the chest tubes of the patient should be checked every hour for bleeding and if the drainage is too high, it needs to be checked every 15-30 minutes and the surgical incision site should be observed (Morton et al., 2013; Ball, Costantino, & Pelosi, 2016). In the early period after cardiovascular surgery, the patient's breathing is provided by mechanical ventilation. During this period, patient's breathing sounds, chest movements and airway patency should be carefully monitored. If the patient needs oxygen after extubation, the respiration of the patient should be supported with a mask. In order to prevent pulmonary 104 • Health Sciences Research Papers complications, deep breathing and coughing exercises are performed and spirometer usage is advised (Çatal & Dicle, 2011; Morton et al., 2013; Kankaya & Bilik, 2018). The causes of postoperative pain are mostly due to trauma, intubation, catheter and chest tube. Pain has a negative effect on heart rate, blood pressure and respiratory function. The nature, location and duration of pain should be evaluated. A calm environment should be created for the patient and the patient should be rested with a suitable position and a healthy sleep pattern (Morton et al., 2013; Koyuncu, Eti Aslan, Karabacak & Demirkılıç, 2015). The patient's pain is controlled, and deep breathing and coughing exercises prevent the development of pulmonary complications Kankaya et al., 2018). In the study of McTier et al. (2016), it was found that pulmonary complications were reduced with the patient taking responsibility by participating in self-care and with the nurse providing support after the cardiac surgery. Individuals with limited mobility due to sedation and mechanical ventilation after cardiovascular surgery should be mobilized as soon as possible after extubation. During mobilization, vital signs and respiratory movements of the individual should be monitored. Prolonged immobilization leads to complications such as acidosis, atelectasis, pneumonia, deep vein thrombosis, pressure injury, organ failure, sepsis and increases the length of stay in the hospital as well as the cost of hospital stay in the intensive care unit (Zomorodi, Topley & McAnaw, 2012; Morton et al., 2013; Castelino et al., 2016). In the systematic review prepared by Santos et al. (2017), it is reported that with mobilization in the first 72 hours after surgery, the length of hospital stay of the patients is reduced and early mobilization is effective in preventing complications. Sedation and mechanical ventilation affecting mobilization also affect nutrition and excretion. In the study of Braga et al. (2009), it is determined that the rate of malnutrition observed in individuals after cardiac surgery was found to be 10-25% and it prolonged the length of stay in the intensive care unit by increasing the mortality and morbidity rates. It has been stated that early oral feeding will significantly reduce mortality rates. As a result of blood and fluid loss, immobility and inadequate feeding due to surgery, problems may occur in the excretory system. Nurses should monitor the patient's weight, hourly urine output, intestinal sounds and diarrhea by considering the hypovolemia and hypervolemia that may develop (Çatal et al., 2011; Morton et al., 2013). Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 105

Beyond vital needs, physical status also includes genetic, age, gender, race and development level. Gender is an important factor in the case of bleeding, which is one of the complications after cardiovascular surgery. In healthy women, fibrin formation and clotting power were found to be faster and stronger than men. Although the incidence of acute coronary syndrome (ACS), which may result in cardiovascular surgery, is equal in women and men until the age of 80, it is observed that the incidence of women is higher after age 80 (Lopes et al., 2015; Karaaslan & Demir, 2017).

PSYCHOLOGICAL DIMENSION Patients may have many problems that affect their emotional and mental state after cardiovascular surgery. Stress, commonly seen under the name of mental problems, worsens cardiovascular health (Stoll, Csaszar, Szoke, & Bagdi, 2014). Roohafza et al. (2010) stated that stress may be effective in the development of ACS. Depression and anxiety that may occur after stress affect the individual's body and mental health. In the study conducted by Kelleci et al. (2009), it was found that 35.3% of heart patients had the possibility of depression. Blumenthal et al. (2003) suggested that depression may trigger death after coronary artery bypass grafting (CABG). It has been observed that the rate of anxiety is high in individuals with acute coronary syndrome, although not as high as depression. Patients with a high rate of anxiety before cardiovascular surgery struggle in returning their daily life after the operation and recovery is delayed. Therefore, stress, depression and anxiety cause physiological impairments in individuals' lives and reduce the quality of life. Patients are concerned about their autonomy and control, dependence on other individuals may develop and as a result loss of self-confidence may occur (Çatal et al., 2011; Kocaman Yıldırım & Öztürk, 2016). After cardiovascular surgery, stress management techniques and cognitive behavioural therapies should be applied together with therapeutic touch to reduce anxiety and depression within the scope of holistic care. The individual who regains self-esteem and social abilities after the attempts can feel sufficient to establish a personal relationship. In this way, the individual’s physical health improves along with the well-being of mental health (Albus, 2010; Tan & Morgan, 2015).

106 • Health Sciences Research Papers

SPIRITUAL DIMENSION Emerged with the existence, spirituality is one of the essential elements of holistic care. Been mentioned with words such as inwardness, religion and belief, it has been associated with very different meanings. Today, beyond the meaning of religious belief, it is considered as “breathing”, “being alive”. Describing spirituality as the essence of the individual, Oldnall reflected this as a force that adds meaning to the individual's life and helps the individual to integrate his beliefs and values (Çınar & Eti Aslan, 2017; Erişen et al., 2017). Spirituality, accepted as the basic concept of holistic care, occurs when an individual begins to seek answers about the meaning, hope and eternity of life in a moment of excitement, stress, fear, crisis due to illness or trauma (Yılmaz, 2011; Çınar et al., 2017). After cardiovascular surgery, the individual being treated in the intensive care unit cannot see the relatives in an environment never experienced before and feels lonely. Feelings of abandonment can cause problems such as anxiety, sleep deprivation and even delirium and depression. Because of the psychological problems experienced, the individual avoids communicating with the health personnel, does not participate in self-care and is withdrawn. On the other hand, these behaviours interfere with the treatment of the individual and lead to regression rather than progression in recovery. This situation reveals the spiritual necessity of the holistic approach (Boztilki & Ardıç, 2017; Çınar et al., 2017; Erişen et al., 2017). In cases of crisis between mind and body, nurses should develop positive coping strategies within the scope of spiritual interventions, listen to patients' fears, reduce anxiety using calming techniques, provide empathy and emotional support, and increase communication with healthcare staff and family (Yılmaz, 2011). Despite the attempts, nurses seem to be inadequate about the spiritual dimension of care. When literature is examined, Chung et al. (2007) reported that nurses know the effects of the spiritual concept on patients, but they cannot adapt it to nursing culture due to the interventions and care carried out along with technological advances.

SOCIOCULTURAL DIMENSION A human being is a social entity that contains the sociocultural concept and cannot be separated from the environment, culture, family and beliefs. Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 107

The effects of sociocultural and socioeconomic conditions on human health have been investigated for a long time and it is stated that necessary treatment and care should be applied within the scope of sociocultural concept (Boztilki et al., 2017). It is argued that there is strong evidence that the lack of social support, insufficient socioeconomic status, lack of interpersonal relationships, living alone and low education level, which are accepted as sociocultural risk factors, lead to the development of coronary heart disease in individuals. Sociocultural factors that affect the development of the disease also affect the health of individuals after cardiovascular surgery (Çatal et al., 2011; Kocaman Yıldırım & Öztürk, 2016). In the study conducted by Barry et al. (2006), it was found that the quality of life of the patients who went through the CABG operation with the support of their families increased. Similarly, Alcan Okgun et al. (2017) emphasized that patients with high educational and socio-economic status gave more importance to a healthy lifestyle after CABG surgery. Following cardiovascular surgery, individuals may experience an imbalance between dependence and independence within the context of sociocultural status, loss of confidence and lack of support for healthcare professionals providing treatment and care, feeling of alienation, and lack of communication. Post-operative treatment and care by nurses and other health professionals should be carried out with sensitivity and through building trust. The health team and family should support the individual together and keep the individual in personal and social relationships (Çatal et al., 2011; Karabacak & Acaroğlu, 2011).

INTELLECTUAL DIMENSION In the case of cardiovascular disease and after surgery, the daily activities of individuals are affected. As a result, ineffective coping situations, lack of self-confidence and deterioration in quality of life can be seen in individuals. In order to provide a holistic care, the intellectual state of the patient should also be considered along with the physiological state (Özdemir & Taşçı, 2013; Karacaoğlu Vicdan & Birgili, 2018). Intellectual status includes past experiences, educational level, ability to develop and evaluate, cognitive ability, learning and memory. The individual who has a complaint after the operation first thinks that the problems will be eliminated by trying personal methods rather than sharing 108 • Health Sciences Research Papers this situation with the nurse. This situation is affected by many concepts such as age, education, past experiences, orientation and perception of the individual (Sucaklı, 2014; Çalışkan et al., 2019). For example; a patient with a blood pressure above the normal limits after cardiovascular surgery may refuse to use the drug necessary by considering the side effects. A diabetic patient may not want to eat the diet given at the hospital. Toxin substances, trauma, infection, mental problems that an individual is exposed to can cause intellectual trauma. When faced with such a situation, the nurse should communicate effectively with the individual to help for the understanding and learning of the situation (Karacaoğlu Vicdan et al., 2018; Çalışkan et al., 2019).

ENVIRONMENTAL DIMENSION Individuals interact with their environment. Environmental conditions significantly affect health and disease status. When the environmental conditions that affect patients physically and psychologically are examined within the scope of Roy's Adaptation Model, it can be seen that they are divided into three groups as focal, contextual and residual stimuli. The fact that cardiovascular surgery has been applied is perceived as a focal stimulus by the patients and this reduces their adaptability (Çatal et al., 2011; Boz, 2018). Considering the patients, intensive care units can be defined as an unknown environment due to the sounds of foreign people, where the physical examination is performed frequently by physicians and nurses, which is crowded and noisy, in which the lights are on for 24 hours, odours and cold are felt and there are too many foreign devices (So & Chan, 2004). In the studies conducted by So and Chan (2004) and Yaman Aktaş et al. (2015), it was found that environmental stimuli in the intensive care unit caused significant sleep deprivation. In addition, although catheters, drain and thorax tubes, dressing changes, position changes and mobilization are considered within the physical dimension required by surgery, they are considered as environmental stimuli and are claimed to cause pain in patients. Physically and psychologically affected patient, feeling alone in a foreign environment never experienced before, not being able to distinguish day and night due to sleep deprivation, and feeling pain due to care given may develop psychological problems (Badır & Eti Aslan, 2003; Hintistan, Nural, & Öztürk, 2009). Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 109

Florence Nightingale emphasizes that the environmental theory is based on the healing environment and that nurses have important responsibilities in providing this environment. Nightingale advocated supporting nursing care with a healing environment and stated that there is improvement in the healing process of individuals (Boz, 2018). Patients treated in the intensive care unit may experience various problems due to environmental stressors. These environmental factors that cause psychological stress in patients should be taken into consideration by nurses and minimized by appropriate interventions. In order to eliminate patients' sleep deprivation, noise from the devices should be reduced, physicians and nurses should be informed not to speak loudly, lighting should be reduced at night so as not to interfere with the work flow, and only necessary interventions should be performed when patients are asleep (Çınar & Özbayır, 2014; Yaman Aktaş et al., 2015).

CONCLUSION This review is written to emphasize the importance of holistic approach for patients as a multidimensional concept after cardiovascular surgery. With the increase in scientific knowledge about the human body, the physical, psychological, spiritual, sociocultural, intellectual and environmental dimensions of human structure have emerged. The physical and mental integrity of the individual is one of the important points to be considered in the treatment of the disease. Because of CABG surgery, the individual has the potential to develop ineffective adaptation behaviours in physical, mental and spiritual dimensions. In this period, the effort to survive, which is the basis of the harmony of physiological and psychological dimensions, is threatened with many stimuli. In order to maintain an effective postoperative life, the individual should perform adaptive behaviour in the early period. For this reason, the vital needs of the individual within the scope of the physical dimension should be met, anxiety and depression should be reduced and compliance with the treatment should be ensured. In order to eliminate the spiritual distress caused by questioning the meaning and hope of life, coping methods should be taught and social support should be provided as the individual feels lonely in the given environment. The understanding of a comprehensive holistic care will emphasize the professionalism of the nurses and help the needs of the individual to be managed with a more scientific and systematic approach. As a result, it is important to evaluate the individual's bio-psycho-social-spiritual dimensions as well as the physical needs during care procedures performed after cardiovascular surgery. 110 • Health Sciences Research Papers

REFERENCES Albus, C. (2010). Psychological and Social Factors in Coronary Heart Disease. Annals of Medicine, 42(7), 487-494. Alcan Okgün, A., Korkmaz, D. ve Çakmakçı, H. (2017). Koroner Arter Baypas Greft Cerrahisi Sonrası Sağlıklı Yaşam Biçimi Davranışlarının Değerlendirilmesi. Türk Göğüs Kalp Damar Cerrahisi Dergisi, 25(1), 45- 51. Badır, A. ve Eti Aslan, F. (2003). Yoğun Bakım Ünitelerinde Çok Konuşulan Az Sorgulanan Bir Sorun: Ağrı. Yoğun Bakım Hemşireliği Dergisi, 7(1), 100- 108. Bahrami, M. (2010). Do Nurses Provide Holistic Care To Cancer Patients? Iranian Journal of Nursing and Midwifery Research, 15(4), 245-251. Ball, L., Costantino, F. & Pelosi, P. (2016). Postoperative Complications of Patients Undergoing Cardiac Surgery. Current Opinion in Critical Care, 22(4), 386-392. Barry, L. C., Kasl, S. V., Lichtman, J., Vaccarino, V. & Krumholz, H. M. (2006). Social Support And Change İn Health-Related Quality of Life 6 Months After Coronary Artery Bypass Grafting. Journal of Psychosomatic Research, 60(2), 185-193. Blumenthal, J. A., Lett, H. S., Babyak, M. A., White, W., Smith, P. K., Mark, D. B., Jones, R., Mathew, J.P., Newman, M.F. & Investigators, N. (2003). Depression As a Risk Factor For Mortality After Coronary Artery Bypass Surgery. The Lancet, 362(9384), 604-609. Boz, İ. (2018). İyileştirici Bakım Çevresi. Balıkesir Sağlık Bilimleri Dergisi, 7(1), 56-61. Boztilki, M. ve Ardıç, E. (2017). Maneviyat ve Sağlık. Journal of Academic Research in Nursing, 3(1), 39-45. Braga, M., Ljungqvist, O., Soeters, P., Fearon, K., Weimann, A. & Bozzetti, F. (2009). ESPEN Guidelines on Parenteral Nutrition: Surgery. Clinical Nutrition, 28(4), 378-386. Bullington, J. & Fagerberg, I. (2013). The Fuzzy Concept of ‛Holistic Care’: A Critical Examination. Scandinavian Journal of Caring Sciences, 27(3), 493-494. Castelino, T., Fiore, J. F., Niculiseanu, P., Landry, T., Augustin, B. & Feldman, L. S. (2016). The Effect of Early Mobilization Protocols on Postoperative Outcomes Following Abdominal and Thoracic Surgery: A Systematic Review. Surgery (United States), 159(4), 991-1003. Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 111

Chung, L. Y. F., Wong, F. K. Y. & Chan, M. F. (2007). Relationship of Nurses’ Spirituality to Their Understanding And Practice of Spiritual Care. Journal of Advanced Nursing, 58(2), 158-170. Çalışkan, N. ve Gündüz, C. S. (2019). Cerrahi Hastasının Bütüncül Bakımı. Mevlüde Karadağ ve Hülya Bulut (Ed.), Cerrahi Hemşireliği Kavram Haritası ve Akış Şemalı (s. 487-504). Ankara: Vize Yayıncılık. Çatal, E. ve Dicle, A. (2011). Koroner Arter Bypass Greftli Hastalarda Erken Dönem Hemşirelik Bakımının Roy'un Uyum Modeline Göre Analizi. Hemşirelikte Araştırma Geliştirme Dergisi, 2, 68-80. Çınar, F. ve Eti Aslan, F. (2017). Spiritüalizm ve Hemşirelik: Yoğun Bakım Hastalarında Spiritüel Bakımın Önemi. Journal of Academic Research in Nursing, 3(1), 37-42. Çınar, S. ve Özbayır, T. (2014). Mide ve Doedenum Hastalıkları. Ayfer Karadakovan ve Fatma Eti Aslan (Ed.), Dahili ve Cerrahi Hastalıklarda Bakım (Geliştirilmiş 3. Baskı) (s. 657-680). Ankara: Akademisyen Tıp Kitabevi. Ergül, Ş. ve Bayık, A. (2004). Hemşirelik ve Manevi Bakım. Cumhuriyet Üniversitesi Hemşirelik Yüksekokulu Dergisi, 8(1), 37-45. Erişen, M. ve Karaca Sivrikaya, S. (2017). Manevi Bakım ve Hemşirelik. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, 6(3), 184-190. Gülel, O. (2012). Kardiyovasküler Risk Faktörleri. Journal of Experimental and Clinical Medicine, 29(3s), 107-116. Henderson, S. (2002). Factors İmpacting on Nurses’ Transference of Theoretical Knowledge of Holistic Care into Clinical Practice. Nurse Education in Practice, 2(4), 244-250. Hintistan, S., Nural, N. ve Öztürk, H. (2009). Yoğun Bakım Ünitesinde Yatan Hastaların Deneyimleri. Yoğun Bakım Hemşireliği Dergisi, 13(1), 40-46. Jasemi, M., Valizadeh, L., Zamanzadeh, V. & Keogh, B. (2017). A Concept Analysis of Holistic Care By Hybrid Model. Indian Journal of Palliative Care, 23(1), 71-80. Kankaya, E. A. ve Bilik, Ö. (2018). Kalp Kapak Ameliyatı Sonrası Güncel Hemşirelik Yaklaşımları: Bakım Neden Önemli? Balıkesir Sağlık Bilimleri Dergisi, 7(2), 101-111. Karaaslan, M. B. ve Demir, M. (2017). Yaşlı Kardiyovasküler Hastalarla İlgili Önemli Çalışmalar ve Sonuçları. Türk Kardiyoloji Derneği Arşivi, 45(5), 138-142. 112 • Health Sciences Research Papers

Karabacak, Ü. ve Acaroğlu, R. (2011). Konfor Kuramı. Maltepe Üniversitesi Bilim ve Sanat Dergisi, 4(1), 197-202. Karacaoğlu Vicdan, A. ve Birgili, F. (2018). Kronik Hastalıklara Uyumu Değerlendirme Ölçeğinin Geliştirilmesi, Geçerlik ve Güvenirlik Çalışması. Journal of Current Researches on Health Sector, 8(2), 135-144. Kelleci, M., Aydın, D., Sabancıoğulları, S. ve Doğan, S. (2009). Hastanede Yatan Hastaların Bazı Tanı Gruplarına Göre Anksiyete ve Depresyon Düzeyleri. Klinik Psikiyatri, 12, 90-98. Kocaman Yıldırım, N. ve Öztürk, S. (2016). Kardiyovasküler Hastalıklarda Güncel Psikososyal Yaklaşımlar. Kardiyovasküler Hemşirelik Dergisi, 7(2), 60-68. Koyuncu, A., Aslan, F. E., Karabacak, Ü. ve Demirkılıç, U. (2015). Nursing and Practices That Speed up Healing in Heart Surgery. GKDA Dergisi, 21(1), 42-50. Leeper, B. & Kaplow, R. (2019). Postoperative Complications of Cardiac Surgery and Nursing Interventions. Sonya R. Hardin & Roberta Kaplow (Ed.), Cardiac Surgery Essentials for Critical Care Nursing (s. 251-284): Jones & Bartlett Publishers. Lopes, C. T., Dos Santos, T. R., Brunori, E. H. F. R., Moorhead, S. A., Lopes, L. L. & Barros, A. L. B. L. (2015). Excessive Bleeding Predictors After Cardiac Surgery in Adults: Integrative Review. Journal of Clinical Nursing, 24, 3046-3062. McEvoy, L. & Duffy, A. (2008). Holistic Practice–A Concept Analysis. Nurse Education in Practice, 8(6), 412-419. McTier, L., Botti, M. & Duke, M. (2016). Patient Participation in Pulmonary İnterventions to Reduce Postoperative Pulmonary Complications Following Cardiac Surgery. Australian Critical Care, 29(1), 35-40. Morton, P. G. & Fontaine, D. K. (2013). Patient Management: Cardiovascular System. Patricia Gonce Morton & Dorrie K. Fontaine (Ed.), Essentials of Critical Care Nursing a Holistic Approach (s. 157-178): Wolters Kluwer Health| Lippincott Williams & Wilkins. Olive, P. (2003). The Holistic Nursing Care of Patients with Minor İnjuries Attending The A&E Department. Accident and Emergency Nursing, 11(1), 27-32. Özdemir, Ü. ve Taşçı, S. (2013). Kronik Hastalıklarda Psikososyal Sorunlar ve Bakım. ERÜ Sağlık Bilimleri Fakültesi Dergisi, 1(1), 57-72. Elif GEZGİNCİ, Sonay GÖKTAŞ, Kübra GÜNEŞ • 113

Papathanasiou, I., Sklavou, M., & Kourkouta, L. (2013). Holistic Nursing Care: Theories and Perspectives. American Journal of Nursing Science, 2(1), 1- 5. Roohafza, H., Talaei, M., Sadeghi, M., Mackie, M. & Sarafzadegan, N. (2010). Association Between Acute and Chronic Life Events on Acute Coronary Syndrome A Case-Control Study. Journal of Cardiovascular Nursing, 25(5), 1-7. Santos, P. M. R., Ricci, N. A., Suster, E. A. B., Paisani, D. M. & Chiavegato, L. D. (2017). Effects of Early Mobilisation in Patients After Cardiac Surgery: A Systematic Review. Physiotherapy, 103(1), 1-12. Selimen, D. & Andsoy, I. I. (2011). The İmportance of A Holistic Approach During The Perioperative Period. AORN Journal, 93(4), 482-490. So, H. M. & Chan, D. S. K. (2004). Perception of Stressors by Patients And Nurses of Critical Care Units in Hong Kong. International Journal of Nursing Studies, 41(1), 77-84. Stoll, D. P., Csaszar, N., Szoke, H. & Bagdi, P. (2014). The İmportance of Psychological Assessment and Support in Patients Suffering From Cardiovascular Disease or Undergoing Cardiac Treatment. Journal of Cardiovascular Diseases and Diagnosis, 2(4), 1-5. Sucaklı, M. H. (2014). Akut Hastalık Yönetiminin Psikososyokültürel ve Varoluşsal Boyutu. Turkish Journal of Family Medicine and Primary Care, 8(4), 117-123. Tan, M. P. & Morgan, K. (2015). Psychological Interventions in Cardiovascular Disease: An Update. Current Opinion, 28(5), 371-377. Yaman Aktaş, Y., Karabulut, N., Yılmaz, D. ve Özkan, A. S. (2015). Kalp Damar Cerrahisi Yoğun Bakım Ünitesinde Tedavi Gören Hastaların Algıladıkları Çevresel Stresörler. Kafkas Tıp Bilimleri Dergisi, 5(3), 81-86. Yılmaz, M. (2011). Holistik Bakımın Bir Boyutu: Spiritualite, Doğası ve Hemşirelikle İlişkisi. Anadolu Hemşirelik ve Sağlık Bilimleri Dergisi, 14(2), 61-70. Zomorodi, M., Topley, D. & McAnaw, M. (2012). Developing Mobility Protocol For Early Mobilization of Patients in A Surgical/Trauma ICU. Critical Care Research and Practice.

PATIENT REPORTED OUTCOME MEASURES IN KNEE OSTEOARTHRITIS

CHAPTER 8

Fatih ÖZDEN1, Özgür Nadiye KARAMAN2

1 Öğretim Görevlisi, Muğla Sıtkı Koçman Üniversitesi, [email protected] 2 Uzman Fizyoterapist, Muğla Sıtkı Koçman Üniversitesi Eğitim ve Araştırma Hastanesi, [email protected]

Fatih ÖZDEN, Özgür Nadiye KARAMAN • 117

INTRODUCTION Osteoarthritis (OA) is a degenerative joint disease. It is mostly observed in the knee joint and its prevalence increases with age. The main symptoms of knee OA are pain, disability and a decrease in quality of life (Martel- Pelletier & Pelletier, 2010; Paker, Buğdaycı, Sabırlı, Özel, & Ersoy, 2007). The main involvement in knee OA is observed in cartilage and subchondral bone, but it has effects on almost all periarticular tissues. Consequently, the range of motion and mobility of the patient are adversely affected (Brandt, Dieppe, & Radin, 2008; Felson & Neogi, 2004; Milne, Evans, & Stanish, 2000). Knee OA should not be considered only as a joint disease. Along with joint cartilage, subchondral bone, joint capsule, synovial membrane, ligaments and muscles around the joint are also affected (Brandt et al., 2008; Milne et al., 2000). Fibrillation, ulceration and consequently degenerative changes in articular cartilage occur in the cartilage region of the joint. Cysts and osteophytic formations are seen in bone tissue. In recent studies, it has been emphasized that OA does not start only in cartilage tissue, but is an organ disorder of synovial joint. Pathology can begin in any part of the organ and progress in different ways (Brandt et al., 2008; Kennedy, Stratford, Wessel, Gollish, & Penney, 2005). Symptoms such as pain, joint movement limitation, stiffness, proprioceptive disorders in the knee joint, limitation in functional activities are common. In addition, varus or valgus deformities seen in the knee. Also, instability in the joint, loss of strength in the periarticular muscles, especially the quadriceps muscle are common (Uysal & Basaran, 2009). Knee OA patients have pain and stiffness especially while walking, sitting- up, stair descending and squatting in daily life (Bijlsma, Berenbaum, & Lafeber, 2011; Michael, Schlüter-Brust, & Eysel, 2010; Naal et al., 2009; Oliveria, Felson, Reed, Cirillo, & Walker, 1995). Defects in functional activities due to loss of joint movement, stiffness and pain bring psychological and social disorders (Gonçalves, Tomás, & Martins, 2012; Ranawat, Ranawat, & Mehta, 2003). Recently, evaluations made by the patient's own statements have gained importance. Considering the shortcomings of evaluations made only by clinicians and containing only objective parameters, it has become important to perform objective and subjective evaluations together in knee osteoarthritis and knee arthroplasty patients (Theodoulou, Bramwell, Spiteri, Kim, & Krishnan, 2016). Orthopedic assessment measures have generally focused on objective parameters such as radiographic analysis or clinical tests. In recent years, 118 • Health Sciences Research Papers another dimension has been added to the clinical outcome evaluations by validating patient-oriented measurement questionnaires (Van Der Straeten, Witvrouw, Willems, Bellemans, & Victor, 2013). It is well known that to the patients with Total Knee Arthroplasty (TKA), an appropriate assessment should be made, especially their expectations and satisfaction levels should be determined (Hamamoto et al., 2015). Because the success of knee treatment depends not only on the positive results of the evaluation of the patient such as pain and function, but also on the improvement of the individual's psychosocial health (Mancuso et al., 2001). It is also known that patients with knee OA wish to have excellent knee function after TKA (Mota, Tarricone, Ciani, Bridges, & Drummond, 2012; Papakostidou et al., 2012). Therefore, it is recommended that the evaluations should be both clinical and subjective (Noble et al., 2012). In this book chapter, the most commonly used (Patient Reported Outcome Measurements) PROMs used in the evaluation of knee osteoarthritis and subsequent operation called “Total Knee Arthroplasty” are discussed.

Literature review The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC): WOMAC is a frequently used questionnaire to assess physical disability and symptoms in individuals with hip and knee OA. This assessment tool records the status of post-treatment responses and changes related to the patient. Approximately 28 years ago, Bellamy et al. conducted the test-retest reliability and validity study of this questionnaire in patients with OA who underwent TKA and Total Hip Arthroplasty operation or underwent conservative treatment with non-steroid anti- inflammatory drugs (Nicholas Bellamy, Buchanan, Goldsmith, Campbell, & Stitt, 1988). WOMAC conducts a 3-dimensional evaluation: pain, stiffness and physical function. Likert version of WOMAC is often made from 0 to 4. A lower score indicates a lower level of symptom and physical disability. Similarly, each sub-score can have a maximum score of 20, 8 and 68, respectively. In addition, the overall total score of this index is generally the score obtained by summing up 3 sub-scores (N. Bellamy, 1995). The WOMAC questionnaire was frequently used to evaluate knee or hip OA, TKA and total hip arthroplasty (Beswick, Wylde, Gooberman- Hill, Blom, & Dieppe, 2012; Hirschmann, Testa, Amsler, & Friederich, 2013; Katz et al., 2013). The Turkish validity and reliability study of the Fatih ÖZDEN, Özgür Nadiye KARAMAN • 119 questionnaire was conducted in patients diagnosed with knee OA (Tüzün, Eker, Aytar, Daşkapan, & Bayramoğlu, 2005). Knee Stroke and Osteoarthritis Result Score (KOOS): KOOS was developed as a continuation of the WOMAC questionnaire and was designed to measure short-term and long-term symptoms and functions in patients with knee injury or OA (Roos, Roos, Lohmander, Ekdahl, & Beynnon, 1998). KOOS consists of a total of 42 items and 5 separate subscores: Pain (9 items), Other Symptoms (7 items), Function of Daily Life Activities (17 items), Function in Sports and Recreational Activities (5 items) and Knee Quality of Life (4 items) (Roos & Toksvig-Larsen, 2003). The validity and reliability of this questionnaire was made with various orthopedic interventions such as TKA, meniscectomy and anterior cruciate ligament reconstruction as well as patient groups with knee OA (Englund, Roos, Roos, & Lohmander, 2001; Ornetti et al., 2008; Roos & Toksvig-Larsen, 2003). The answers to the questions are structured with a 5-point Likert system and 0 means that there are no problems and 4 states that there are extreme problems. After the calculation with these scores, the score is converted to a score between 0-100. In this scoring 0 represents extreme knee problems, while 100 points indicate that there are no knee problems (Ornetti et al., 2008). Turkish version, validity and reliability of KOOS were performed by Paker et al. patients diagnosed with knee OA (Paker et al., 2007). Oxford Knee Score (ODS): The Oxford Knee Score (ODS), developed by Dawson et al., is a short questionnaire that assesses pain and functional status in patients with TKA (Dawson, Fitzpatrick, Murray, & Carr, 1998). ODS is considered to be one of the most valid and reliable assessment tools among the specially developed questionnaires (Garratt, Brealey, & Gillespie, 2004). ODS has also been used in OA and osteotomy surgery patient groups (Khurana, Sankhala, Malik, Shekhawat, & Rathore, 2015; Tugay et al., 2016). The ODS, which consists of 12 items, is a valid, reliable and reliable questionnaire that is sensitive to practical, clinically important changes (Dawson et al., 1998; Garratt et al., 2004). Each question has an answer system consisting of 5 categories that are rated with 0 to 4 points. The total score is determined as 0 worst and 48 best, and there is also a revised version of the questionnaire (Murray et al., 2007). The Turkish version, cultural adaptation and validity study of the Oxford Knee Score was performed in a patient group diagnosed with knee OA (Tugay et al., 2016). 120 • Health Sciences Research Papers

Index of Severity for Knee Osteoarthritis - Severity Index for Knee Osteoarthritis (ISK): ISK is an assessment tool that measures the severity of OA in the knee. Consists of three parts; Pain or discomfort (5 questions), walking distance (2 questions) and daily living activities (4 questions). The total score ranges from 0 to 24. The lower score indicates better functional status. A score of 14 and below indicates an extremely severe, 11 to 13 very severe, 8 to 10 severe, 5 to 7 moderate and 1 to 4 low level of disability. Scores between 10 and 12 obtained after appropriate conservative treatment indicate that the individual needs surgical treatment (M. Lequesne; M. G. Lequesne, 1997; M. G. Lequesne, Mery, Samson, & Gerard, 1987). There is no Turkish version of the questionnaire. Activities Scale Knee Questionnaire (ADLS): It evaluates functional limitations and symptoms in daily living activities resulting from knee pathologies or disorders (Irrgang, Snyder-Mackler, Wainner, Fu, & Harner, 1998). ADLS includes 17 items that question certain symptoms (7 items) and functional limitations (10 items) such as pain, crepitus, stiffness, swelling, instability / shift, rotation and weakness (Rogers & Irrgang, 2003). The scores obtained from all items are summed and divided by 80 and then multiplied by 100, resulting in a scoring system scored between 0 and 100 (Irrgang et al., 1998). OA, treated in the United States, has been developed in patients with various knee pathologies such as meniscal injuries, ligamentous injuries, and patellofemoral pain syndrome (Rogers & Irrgang, 2003). The Turkish version of the questionnaire was conducted by Evcik et al. (Evcik, Ay, Ege, Turel, & Kavuncu, 2009). Intermittent and Constant Osteoarthritis Pain Questionnaire (ICOAP): ICOAP is a multi-faceted assessment tool designed specifically for OA and provides a comprehensive assessment of people's pain experience in the knee or hip joint. It includes pain severity, frequency, type according to the activity, sleep and quality of life and the effects of independence and quality of life subscores (G. A. Hawker et al., 2008). Each score is scored between 0 and 4, with a total score of 0 (no pain) and 100 (the most severe pain) (G. A. Hawker et al., 2008). The Turkish version of the questionnaire was conducted by Erel et al. on patients diagnosed with knee OA (Erel, Şimşek, & Özkan, 2015). Hospital for Special Surgery (HSS): The HSS knee scoring system is a questionnaire developed for the evaluation of some knee pathologies, especially TKA patients. This assessment tool, which includes both Fatih ÖZDEN, Özgür Nadiye KARAMAN • 121 objective and subjective assessment, is scored with a total of 100 points. It includes pain, functionality, range of motion, strength, deformity and instability subscores. The score of 85 and above is rated as excellent, scores between 70 and 84 are rated as good, scores between 60 and 69 are rated as moderate and scores below 60 are considered as poor. The Turkish validity and reliability study of HSS was conducted by Narin et al. (Narin, Unver, Bakirhan, Bozan, & Karatosun, 2014). Patient-Specific Functional Scale (PSFS): PSFS is a questionnaire to record patients' perceptions of their own disability. In this questionnaire, patients describe three main complaints (eg, difficulty in performing some activities) and were asked to transfer the degree of difficulty to an 11-point numerical scale or visual analog scale (Stratford, Gill, Westaway, & Binkley, 1995). This method was followed to record small differences between the skills of patients in performing these activities (Beurskens et al., 1999). These three main complaints should be as specific to the patient as possible and should be related to their knee OA status. The validity and reliability study of this questionnaire was conducted in patients diagnosed with knee OA and who were scheduled for TKA (Berghmans, Lenssen, van Rhijn, & de Bie, 2015). There is no Turkish version of the questionnaire. Knee Society Clinical Rating System: The Knee Society Clinical Rating System has provided a more comprehensive approach to grading the knee joint in general. This system is divided into two subgroups which include the clinical knee score including the grading of the knee joint and the general functional status of the patient such as walking and climbing stairs and a maximum total score of 100 can be obtained (Insall, Dorr, Scott, & Scott, 1989). In the clinical knee score section, pain (50 points), stability (25 points), and range of motion (25 points) are evaluated. In functional status score, walking distance (50 points), stair climbing and stroke (50 points) are evaluated (Martimbianco et al., 2012). The Turkish version of this questionnaire has not been studied. The New Knee Society Scoring System (The New KSS): Some uncertainties and deficiencies in the Knee Society’s old scoring system have led to difficulties with the use of the questionnaire (Ghanem et al., 2010). The Knee Society Clinical Rating System does not include parameters that evaluate radiographic findings (Ewald, 1989). This scoring system, which evaluates the results of total or partial knee arthroplasty, has 122 • Health Sciences Research Papers been shown to be the most popular assessment tool worldwide, but it is also stated that there are some problems regarding the validity and reliability of the original score. In addition, the uncertainties and deficiencies in the Knee Society Clinical Rating System, especially those that are operated by TKA. Different expectations, satisfactions and functional status features that are not compatible with the current patient profile (Noble et al., 2012; Scuderi et al., 2012). The New KSS is a scale developed to evaluate TKA patients in the pre-op and post-op period. It consists of four sub-sections. Objective knee score (7 items, 100 points), Satisfaction score (5 items, 40 points) expectation score (3 items; 15 points), functional activity score (19 items; 110 points) (Noble et al., 2012; Scuderi et al., 2012). The Turkish version of the study was conducted by Özden et al. (Özden, Tuğay, Tuğay, & Kılınç, 2019).

Conclusion In this review, we reviewed the most commonly used questionnaires for osteoarthritis and arthroplasty of the knee. In addition to objective evaluations with PROMs, we can see that subjective evaluations can be done especially regarding patient expectations and satisfaction. We believe that these questionnaires, where they can express their perceptions and expressions about the clinical situation more clearly, can contribute to the monitoring of treatment by adding a different dimension to the objective evaluations of the clinicians. Cultural adaptation and validation is important in the use of such surveys. We think that some of the questionnaires with and without Turkish version can be used more frequently in the clinic. As a result, we hope that this review will contribute greatly to health professionals in ensuring the accuracy of selection and use of PROMs.

Fatih ÖZDEN, Özgür Nadiye KARAMAN • 123

REFERENCES Bellamy, N. (1995). Outcome measurement in osteoarthritis clinical trials. The Journal of rheumatology. Supplement, 43, 49-51. Bellamy, N., Buchanan, W. W., Goldsmith, C. H., Campbell, J., & Stitt, L. W. (1988). Validation study of WOMAC: a health status instrument for measuring clinically important patient relevant outcomes to antirheumatic drug therapy in patients with osteoarthritis of the hip or knee. The Journal of rheumatology, 15(12), 1833-1840. Berghmans, D. D. P., Lenssen, A. F., van Rhijn, L. W., & de Bie, R. A. (2015). The patient-specific functional scale: its reliability and responsiveness in patients undergoing a Total knee arthroplasty. Journal of Orthopaedic & Sports Physical Therapy, 45(7), 550-556. Beswick, A. D., Wylde, V., Gooberman-Hill, R., Blom, A., & Dieppe, P. (2012). What proportion of patients report long-term pain after total hip or knee replacement for osteoarthritis? A systematic review of prospective studies in unselected patients. BMJ open, 2(1), e000435. Beurskens, A. J., Henrica, C., Kökeb, A. J., Lindeman, E., van der Heijden, G. J., Regtope, W., & Knipschild, P. G. (1999). A patient-specific approach for measuring functional status in low back pain. Journal of manipulative and physiological therapeutics, 22(3), 144-148. Bijlsma, J. W. J., Berenbaum, F., & Lafeber, F. P. J. G. (2011). Osteoarthritis: an update with relevance for clinical practice. The Lancet, 377(9783), 2115- 2126. Brandt, K. D., Dieppe, P., & Radin, E. L. (2008). Etiopathogenesis of osteoarthritis. Rheumatic Disease Clinics of North America, 34(3), 531- 559. Dawson, J., Fitzpatrick, R., Murray, D., & Carr, A. (1998). Questionnaire on the perceptions of patients about total knee replacement. The Journal of bone and joint surgery. British volume, 80(1), 63-69. Englund, M., Roos, E. M., Roos, H. P., & Lohmander, L. S. (2001). Patient‐ relevant outcomes fourteen years after meniscectomy: influence of type of meniscal tear and size of resection. Rheumatology, 40(6), 631-639. Erel, S., Şimşek, İ. E., & Özkan, H. (2015). Analysis of the reliability and validity of the Turkish version of the intermittent and constant osteoarthritis pain questionnaire. Acta Orthop Traumatol Turc, 49(5), 508-512. Evcik, D., Ay, S., Ege, A., Turel, A., & Kavuncu, V. (2009). Adaptation and validation of Turkish version of the knee outcome survey-activities for 124 • Health Sciences Research Papers

daily living scale. Clinical Orthopaedics and Related Research®, 467(8), 2077-2082. Ewald, F. C. (1989). The Knee Society total knee arthroplasty roentgenographic evaluation and scoring system. Clinical orthopaedics and related research(248), 9-12. Felson, D. T., & Neogi, T. (2004). Osteoarthritis: is it a disease of cartilage or of bone? Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, 50(2), 341-344. Garratt, A. M., Brealey, S., & Gillespie, W. J. (2004). Patient-assessed health instruments for the knee: a structured review. Rheumatology, 43(11), 1414- 1423. Ghanem, E., Pawasarat, I., Lindsay, A., May, L., Azzam, K., Joshi, A., & Parvizi, J. (2010). Limitations of the Knee Society Score in evaluating outcomes following revision total knee arthroplasty. JBJS, 92(14), 2445-2451. Gonçalves, R. S., Tomás, A. M., & Martins, D. I. (2012). Cross-cultural adaptation and validation of the Portuguese version of the Oxford Knee Score (OKS). The Knee, 19(4), 344-347. Hamamoto, Y., Ito, H., Furu, M., Ishikawa, M., Azukizawa, M., Kuriyama, S & Matsuda, S. (2015). Cross-cultural adaptation and validation of the Japanese version of the new Knee Society Scoring System for osteoarthritic knee with total knee arthroplasty. Journal of Orthopaedic Science, 20(5), 849-853. Hawker, G. A., Davis, A. M., French, M. R., Cibere, J., Jordan, J. M., March, L & Dieppe, P. (2008). Development and preliminary psychometric testing of a new OA pain measure–an OARSI/OMERACT initiative. Osteoarthritis and Cartilage, 16(4), 409-414. Hawker, G. A., Mian, S., Kendzerska, T., & French, M. (2011). Measures of adult pain: Visual analog scale for pain (vas pain), numeric rating scale for pain (nrs pain), mcgill pain questionnaire (mpq), short‐form mcgill pain questionnaire (sf‐mpq), chronic pain grade scale (cpgs), short form‐36 bodily pain scale (sf‐36 bps), and measure of intermittent and constant osteoarthritis pain (icoap). Arthritis care & research, 63(S11), S240-S252. Hirschmann, M. T., Testa, E., Amsler, F., & Friederich, N. F. (2013). The unhappy total knee arthroplasty (TKA) patient: higher WOMAC and lower KSS in depressed patients prior and after TKA. Knee Surgery, Sports Traumatology, Arthroscopy, 21(10), 2405-2411. Insall, J. N., Dorr, L. D., Scott, R. D., & Scott, W. N. (1989). Rationale of the Knee Society clinical rating system. Clin Orthop relat res, 248(248), 13-14. Fatih ÖZDEN, Özgür Nadiye KARAMAN • 125

Irrgang, J. J., Snyder-Mackler, L., Wainner, R. S., Fu, F. H., & Harner, C. D. (1998). Development of a patient-reported measure of function of the knee. JBJS, 80(8), 1132-1145. Katz, J. N., Brophy, R. H., Chaisson, C. E., De Chaves, L., Cole, B. J., Dahm, D. L, & Jones, M. H. (2013). Surgery versus physical therapy for a meniscal tear and osteoarthritis. New England Journal of Medicine, 368(18), 1675- 1684. Kennedy, D. M., Stratford, P. W., Wessel, J., Gollish, J. D., & Penney, D. (2005). Assessing stability and change of four performance measures: a longitudinal study evaluating outcome following total hip and knee arthroplasty. BMC musculoskeletal disorders, 6(1), 3. Khurana, D., Sankhala, S. S., Malik, M., Shekhawat, V., & Rathore, D. S. (2015). Comparative study of high tibial osteotomy using dynamic axial fixator and locked low-profile plate in medial osteoarthritis of knee. European Journal of Orthopaedic Surgery & Traumatology, 25(4), 763-773. Lequesne, M. (1991). Indices of severity and disease activity for osteoarthritis. Lequesne, M. G. (1997). The algofunctional indices for hip and knee osteoarthritis. The Journal of rheumatology, 24(4), 779-781. Mancuso, C. A., Sculco, T. P., Wickiewicz, T. L., Jones, E. C., Robbins, L., Warren, R. F., & Williams-Russo, P. (2001). Patients’ expectations of knee surgery. JBJS, 83(7), 1005-1012. Martel-Pelletier, J., & Pelletier, J.-P. (2010). Is osteoarthritis a disease involving only cartilage or other articular tissues. Eklem Hastalik Cerrahisi, 21(1), 2- 14. Martimbianco, A. L. C., Calabrese, F. R., Iha, L. A. N., Petrilli, M., Lira Neto, O., & Carneiro Filho, M. (2012). Reliability of the" American Knee Society Score"(AKSS). Acta ortopedica brasileira, 20(1), 34-38. Michael, J. W. P., Schlüter-Brust, K. U., & Eysel, P. (2010). The epidemiology, etiology, diagnosis, and treatment of osteoarthritis of the knee. Deutsches Arzteblatt International, 107(9), 152. Milne, A. D., Evans, N. A., & Stanish, W. D. (2000). Nonoperative management of knee osteoarthritis: Diagnosis, behavior modification, and pharmacologic options. WOMEN’S HEALTH in primary care, 3(12), 841- 846. Mota, R. E. M., Tarricone, R., Ciani, O., Bridges, J. F. P., & Drummond, M. (2012). Determinants of demand for total hip and knee arthroplasty: a systematic literature review. BMC health services research, 12(1), 225. 126 • Health Sciences Research Papers

Murray, D. W., Fitzpatrick, R., Rogers, K., Pandit, H., Beard, D. J., Carr, A. J., & Dawson, J. (2007). The use of the Oxford hip and knee scores. The Journal of bone and joint surgery. British volume, 89(8), 1010-1014. Naal, F. D., Impellizzeri, F. M., Sieverding, M., Loibl, M., Von Knoch, F., Mannion, A. F., . . . Munzinger, U. (2009). The 12-item Oxford Knee Score: cross-cultural adaptation into German and assessment of its psychometric properties in patients with osteoarthritis of the knee. Osteoarthritis and Cartilage, 17(1), 49-52. Narin, S., Unver, B., Bakirhan, S., Bozan, O., & Karatosun, V. (2014). Cross- cultural adaptation, reliability and validity of the Turkish version of the Hospital for Special Surgery (HSS) Knee Score. Acta Orthop Traumatol Turc, 48(3), 241-248. Noble, P. C., Scuderi, G. R., Brekke, A. C., Sikorskii, A., Benjamin, J. B., Lonner, J. H., & Bourne, R. B. (2012). Development of a new Knee Society scoring system. Clinical Orthopaedics and Related Research®, 470(1), 20-32. Oliveria, S. A., Felson, D. T., Reed, J. I., Cirillo, P. A., & Walker, A. M. (1995). Incidence of symptomatic hand, hip, and knee osteoarthritis among patients in a health maintenance organization. Arthritis & Rheumatism: Official Journal of the American College of Rheumatology, 38(8), 1134-1141. Ornetti, P., Parratte, S., Gossec, L., Tavernier, C., Argenson, J. N., Roos, E. M., . . . Maillefert, J. F. (2008). Cross-cultural adaptation and validation of the French version of the Knee injury and Osteoarthritis Outcome Score (KOOS) in knee osteoarthritis patients. Osteoarthritis and Cartilage, 16(4), 423-428. Özden, F., Tuğay, N., Tuğay, B. U., & Kılınç, C. Y. (2019). Psychometrical properties of the Turkish translation of the new knee society scoring system. Acta orthopaedica et traumatologica turcica, 2019;53: 184-188. Paker, N., Buğdaycı, D., Sabırlı, F., Özel, S., & Ersoy, S. (2007). Diz incinme ve osteoartrit sonuç skoru: Türkçe sürümünün güvenilirlik ve geçerlilik çalışması. Turkiye Klinikleri J Med Sci, 27(3), 350-356. Papakostidou, I., Dailiana, Z. H., Papapolychroniou, T., Liaropoulos, L., Zintzaras, E., Karachalios, T. S., & Malizos, K. N. (2012). Factors affecting the quality of life after total knee arthroplasties: a prospective study. BMC musculoskeletal disorders, 13(1), 116. Ranawat, C. S., Ranawat, A. S., & Mehta, A. (2003). Total knee arthroplasty rehabilitation protocol: Whatmakes the difference? The Journal of arthroplasty, 18(3), 27-30. Fatih ÖZDEN, Özgür Nadiye KARAMAN • 127

Rogers, J. C., & Irrgang, J. J. (2003). Measures of adult lower extremity function: The American Academy of Orthopedic Surgeons Lower Limb Questionnaire, The Activities of Daily Living Scale of the Knee Outcome Survey (ADLS), Foot Function Index (FFI), Functional Assessment System (FAS), Harris Hip Score (HHS), Index of Severity for Hip Osteoarthritis (ISH), Index of Severity for Knee Osteoarthritis (ISK), Knee Injury and Osteoarthritis Outcome Score (KOOS), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC™). Arthritis Care & Research: Official Journal of the American College of Rheumatology, 49(S5), S67-S84. Roos, E. M., Roos, H. P., Lohmander, L. S., Ekdahl, C., & Beynnon, B. D. (1998). Knee Injury and Osteoarthritis Outcome Score (KOOS)—development of a self-administered outcome measure. Journal of Orthopaedic & Sports Physical Therapy, 28(2), 88-96. Roos, E. M., & Toksvig-Larsen, S. (2003). Knee injury and Osteoarthritis Outcome Score (KOOS)–validation and comparison to the WOMAC in total knee replacement. Health and quality of life outcomes, 1(1), 17. Scuderi, G. R., Bourne, R. B., Noble, P. C., Benjamin, J. B., Lonner, J. H., & Scott, W. N. (2012). The new knee society knee scoring system. Clinical Orthopaedics and Related Research®, 470(1), 3-19. Stratford, P., Gill, C., Westaway, M., & Binkley, J. (1995). Assessing disability and change on individual patients: a report of a patient specific measure. Physiotherapy canada, 47(4), 258-263. Theodoulou, A., Bramwell, D. C., Spiteri, A. C., Kim, S. W., & Krishnan, J. (2016). The use of scoring systems in knee arthroplasty: a systematic review of the literature. The Journal of arthroplasty, 31(10), 2364-2370. Tugay, B. U., Tugay, N., Guney, H., Kinikli, G. I., Yuksel, I., & Atilla, B. (2016). Oxford Knee Score: cross-cultural adaptation and validation of the Turkish version in patients with osteoarthritis of the knee. Acta Orthop Traumatol Turc, 50(2), 198-206. Tüzün, E. H., Eker, L., Aytar, A., Daşkapan, A., & Bayramoğlu, M. (2005). Acceptability, reliability, validity and responsiveness of the Turkish version of WOMAC osteoarthritis index. Osteoarthritis and Cartilage, 13(1), 28-33. Uysal, F. G., & Basaran, S. (2009). Knee osteoarthritis/diz osteoartriti. Turkish Journal of Physical Medicine and Rehabilitation, 1-8. Van Der Straeten, C., Witvrouw, E., Willems, T., Bellemans, J., & Victor, J. (2013). Translation and validation of the Dutch new knee society scoring system©. Clinical Orthopaedics and Related Research®, 471(11), 3565-3571.

VIOLENCE PHENOMENON IN EARLY MARRIAGES

CHAPTER 9

Funda ÖZPULAT1

1 Asst. Prof., Selcuk University, Aksehir Kadir Yallagoz School of Health, [email protected]

Funda ÖZPULAT • 131

INTRODUCTION In most areas of the world, one of the fundamental human rights violations women and female children encounter is early and forced marriages. Early marriage, or child marriage, in other words, means two people's, at least one of the involved parties being underage, union with an official, religious, or traditional marriage contract. Since most of these marriages happen without the informed consent of the child, they are also defined as "early and forced marriages" (Aktepe & Atay, 2017; Doğan Köroğlu, 2014). According to the latest report of the United Nations Population Fund (UNFPA), in South Asia, 46% of women in the 20-24 age range had either been married or had lived together with someone before they turned 18. The ratio of the marriage of female children is significantly higher than males. Still, in South Asia, 30% of female children in the 15-19 age range are either married or live together with someone, compared to only 5% of male children in the same age range. While South Asia constitutes the highest percentage of early marriages, this practice is prevalent in most of the other countries that appear in this report. For example, 18% of women in the 20-24 age range in East Asia and Pacific, and 11% in Eastern Europe and Central Asia had either been married or had lived together with someone before they turned 18 (UNFPA, 2012). According to the 2010 data of WHO, globally, 36.4 million women in the 20-24 age range had had their first live birth experience before they turned 18. 5.6 million of these women had given birth before they were 15 years of age. These numbers show that every year, 7.3 million female children under the age of 18 give birth. In other words, 20 thousand births per day (World Health Organization, 2006). According to one research conducted in India, mortality risk among babies within their first year on earth is 50% higher for those born to underage mothers compared to those born to mothers older than 19 years of age (Raj et al, 2010). The main reason early marriages pose a problem in a lot of ways can be considered a result of this period coinciding with the adolescence period. The adolescence period is a stressful and psychologically challenging period in which rapid physical, psychological, and cognitive changes and experiences are gained. In a person's life, this period is considered as the second growth and evolvement period in which the individual can face difficulties. At times, the adolescent cannot adjust to the many rapid 132 • Health Sciences Research Papers changes. On the one hand, while the adolescent grapples with physical changes, growth and evolvement troubles, and with sexual identity development in this period, on the other hand, they face problems related to psycho-social development. In order to get through this period healthily, there are several developmental tasks an adolescent has to fulfill. Developing a distinctive identity leads these tasks. Developing an identity is not an easy task for the disoriented adolescent. Marriages formed in this period can cause the adolescent to lose confidence in herself and can make developing a distinctive identity harder (Aktepe & Atay, 2017; Roman & Frantz, 2013; İçağasıoğlu Çoban, 2009). This situation aggravates undertaking the roles and responsibilities of an early marriage too (Le Strat, Dubertret & Le Foll, 2011). Though it poses a problem for both sexes, female children, in particular, are affected by these types of marriages to a greater extent (Ozpulat, 2016). Early marriages cause the formation of young and unhealthy mothers by affecting the physical and psychological developments of female children, in particular, and can culminate with them encountering the harsh facts of life unprepared. As a result of adolescent children's, who are at disadvantaged positions both materially and spiritually, marriage, their education is interrupted, their development is affected negatively, and their opportunities in social and professional areas. Children are separated from their families and friends, they are exposed to domestic violence and oppression. In addition to these, the "child mothers" face severe dangers such as risks in childbirth or permanent diseases, besides depression and suicide, psychological problems are also encountered (Orçan & Kar, 2008; Aktepe, Atay, 2017). Early marriage, or child bride, phenomenon, for many years, has been drawing attention as one of the major social problems in Turkey. Frequently encountered cases, particularly in closed societies, continue their existence, even now, as a result of the ongoing practice of traditions such as; custom, bride price, bride exchange, and being betrothed in the cradle (Yağbasan & Tekdemir, 2017). Usually, women in patriarchal societies are not able to have a say in a matter concerning them, and they are forced to accept the marriage arrangement that is decided on their behalf. Occasionally, they involuntarily help the continuation of patriarchal tradition (Abukan & Yıldırım, 2017). Women, who were exposed to patriarchal authority by their fathers before marriage, may submit to the powers of their spouses after marriage (Belhorma, 2016). Funda ÖZPULAT • 133

Prevention of early marriages is the first step on the prevention of adolescent pregnancies. For women, who were married at an early age, utilizing health services is essential in the way of actively performing family planning counseling and services for the prevention of adolescent pregnancies, and in preventing several life-threatening complications for the mother and the baby. Furthermore, conducting studies that aim to raise awareness and mindfulness about the negative consequences of early marriages, and conducting grassroots studies by ensuring the active participation of education officers, religious officials, other related institutions and organizations, and health care officials in every rank can be efficient in reducing early marriages and its possible negative consequences (Ozpulat, 2016).

VIOLENCE PHENOMENON In its most simplistic form, the violence phenomenon is described as "forcing someone to do something by applying power or pressure" (Harcar et al., 2008). World Health Organization, on the other hand, defines violence as “the intentional use of physical force or power, threatened or actual, against oneself, another person, or against a group or community, that either results in or has a high likelihood of resulting in injury, death, psychological harm, maldevelopment or deprivation” (WHO, 2002). Violence phenomenon, which is observed in various magnitudes on individual and societal levels throughout human history, is, unfortunately, encountered as an inseparable part of life. This phenomenon, which sometimes is demonstrated physically and at other times psychologically, 134 • Health Sciences Research Papers substantially harms people's quality of life. Women, unfortunately, are the main objects of violence, which is tackled in almost every society as the leading social problem (Harcar et al., 2008). The main reason for violence against women is the patriarchal social system, and the subsidiary role assigned to women by the gender roles that define the rules of this system. Men's superior position in the patriarchal social system, which demonstrates women as the dependent side, in an attempt to control women, and ensure the order and the discipline of the system, is the root cause of men's use of violence (Aşkın & Aşkın, 2018). The following cases could be included among the reasons for violence against women: i. Gender discrimination, ii. Wrong beliefs of the society, iii. Lack of love and inability to express love, iv. Miscommunication, v. Early marriages, vi. Cultural differences, vii. Absence of legally sufficient infrastructure and punishments against the violent perpetrator viii. Deviation from values, ix. Low self-respect, x. Lack of education and the individual having adverse character traits (Tatlılıoğlu & Küçükköse, 2015). Funda ÖZPULAT • 135

Violence against women, regardless of their development level, is a crucial social problem in almost every country around the world. Although this violence is encountered in different forms in different countries, generally, it can be categorized as physical, psychologic, sexual, and economic violence (Ediz & Altan, 2017). Physical Violence: The first one of the violence types, physical violence, is the most encountered one by women and contains acts that cause bodily harm to women, and is directed at women's bodies. This type of violence brings psychological violence along (Ediz & Altan, 2017). Physical violence, although being the most encountered type, is a continually hidden phenomenon. It also causes women, in an attempt to not disrupt the order at home, to avoid being abandoned by their husbands, to avoid feeling shame or disturbing the peace, or because of women's fear, to stay silent (Yanık et al., 2014). Physical domestic violence usually demonstrates itself in forms of being shoved, being abused, being beaten (kicks, punches, slaps, etc.), or being physically harmed by sharp objects (Baydur & Ertem, 2006). Because physical violence is the most visible form of violence against women, there is a frequently encountered misconception about violence, which makes it seem like an act that harms women just physically. However, violence against women harms and hurts women in sexual, psychologic, or economic ways too (Sargın, 2010). Besides, there is a higher possibility of child brides, as a result of the violence they had encountered, justifying their husbands' actions as rightful (ICRW, 2007). 136 • Health Sciences Research Papers

Psychological violence: Psychological violence is consistent exploitation of an individual's feelings in an attempt to apply pressure, or being used as a means of suppression, punishment, or intimidation. Every type of pressure applied to an individual, such as, consistently putting psychological pressure on them, emotionally suppressing and humiliating, employing punishments towards them or isolating them from the society as a means of punishment is known as psychological violence (Tatlılıoğlu & Küçükköse, 2015). Physical violence leaving more permanent and visible marks on a woman's body, and despite the negative effect of psychological violence on women being as intense as physical violence, places physical violence at the forefront in society compared to psychological violence (Ediz & Altan, 2017). While the early marriage of women is discussed in socio-cultural and economic contexts, the psychological violence aspect of it must be handled separately. For psychological violence is a part of every other aspect of gender-based societal violence. In other words, psychological violence may occur as either the result of other violence types or the cause of them (Abukan & Yıldırım, 2017). Serious expectations, obligations, and problems brought by being married early (forming a family at an early age, undertaking responsibilities such as home and childcare) can cause these women to experience mental disorders and attempt suicide in their advanced ages (Soylu & Ayaz, 2013). Economic Violence: Owned physical resources or money being used as a means of threat or punishment on women is known as economic violence. Female children, who are kept on the background in the socialization and socializing process, taught to settle for what they have, deprived of economic freedom, are not let into business life, mostly by being left behind in the educational process. Women, who are deprived of education or lack education, are also exploited inside their homes, they are made to work home-based and can not experience economic freedom (Gökkaya, 2011). When used as a means of pressure and control over women, this violence type establishes the ground for other types of violence to be implemented (Ediz & Altan, 2017). Sexual Violence: Sexual violence is described as sexuality being used as a threat, punishment, and a means of control towards women. Sexual violence arises as women being forced to have sexual relations in unwanted, risky, and shameful ways. Being forced to have sexual relations with others, being forced to birth children without women's consent or Funda ÖZPULAT • 137 obstruction of their right to birth, forcing them to marry someone they do not desire to, early marriages, obstruction of their right to birth control, forcing them to have abortions, exhibiting sexually explicit behavior via telephones, letters, or social media, sexual harassment and rape are frequent examples of this violence type (Tatlılıoğlu & Küçükköse, 2015). Female children being killed before or after their birth, them being married off at a young age, female circumcision, early pregnancies, obstruction of the use of family planning services, committing crimes in the name of honor against women are also evaluated under the title of sexual violence by the United Nations (KSGM, 2003). The age range of women exposed to sexual violence is vaster than the age range of women exposed to other violence types (Ediz & Altan, 2017). When the situation in the European Union (EU) countries is analyzed, it can be seen that violence against women is a widespread problem. The results of a research conducted in person by the EU Agency for Fundamental Rights (FRA), in 28 EU countries with 42 thousand women reveal this. Research results show: - One in three women being exposed to physical or sexual violence since the age of fifteen and this ratio stands at 8% in the last twelve months, - Women are commonly abused yet only a low percentage of these abuses are ever recorded, only 14% of domestic violence cases and 13% of other violence cases being reported, - Women, who had ended abusive relationships, being still at risk, - Two out of every five women, around 43%, being exposed to psychological violence from either their current or former husbands or partners, - 18% of women being victims of stalking since the age of 15, - Approximately 12% of women have suffered the sexual abuse of an adult before the age of 15, - 42% of women, who were exposed to the violence of their husbands or partners, being exposed to violence even when they were pregnant (FRA, 2014). 138 • Health Sciences Research Papers

According to research conducted by the International Center for Research on Women (ICRW) in 2005, female children who marry at an early age are exposed to twice as much of physical violence and three times as much of sexual violence compared to women in other age groups. Due to being exposed to physical violence, the possibility of married female children accepting their husbands' positions as rightful is higher. According to the national results of violence against women research conducted in Turkey in 2014; when violence against women by their husbands or partners is evaluated based on age it can be seen that 25% of women in the age range of 15-24 being exposed to physical violence, 10% of them being exposed sexual violence, 28% of them being exposed to both physical and sexual violence, 38% of them being exposed to emotional violence and abuse, and 29% of them being exposed to economic violence and abuse (Republic of Turkey, Family and Social Services Provincial Directorate, 2014). According to another research conducted in Turkey, it was determined that 14,6% of female children, who were married off at an early age, were exposed to physical violence and abuse, while 27,1% of them were exposed to emotional violence and abuse by their husbands (Soylu & Ayaz, 2013). According to a different research conducted in Turkey, cases involving early marriages were significantly more exposed to physical and sexual violence compared to the control group (Güneş et al., 2016). According to research conducted in Pakistan, a significant raise in being exposed to severe physical and emotional violence in early marriages was discovered, compared to adult marriages (Nasrullah et al., 2014). According to another research conducted in Bangladesh, despite, the lack of a meaningful association between sexual violence and adolescent marriages, a significant connection between physical violence and early marriage connection had been discovered (Rahman et al, 2014). Battered woman, in addition to several other problems, also has to cope with health problems. Violence-related primary health problems affecting women are as follows:

✓ Depending on the severity of the applied violence, lethal results could transpire. The definition of death by physical violence in the legal system is a homicide.

✓ Violence could result in women committing suicide due to depression. Funda ÖZPULAT • 139

✓ As a result of violence, the loss of a limb and continued disability or injury situations could occur.

✓ Constant stress, generated by psychological violence, causes intense pressure and depression. This situation brings along a wide range of illnesses, from chronic pain to hypertension.

✓ Sexual violence negatively affects reproductive health. It causes unwanted pregnancies.

✓ Pregnancy terminations in unwanted and unfavorable conditions and unhealthy miscarriages could occur.

✓ Sexually transmitted diseases could occur.

✓ Complications in pregnancies and preterm delivery could occur.

✓ Several gynecologic diseases could occur in women exposed to violence.

✓ Developmental disorders could occur in the fetus, and the baby could be born with low birth weight.

✓ Psychologic problems such as psychological breakdown, depression, anxiety disorder, and fear could occur.

✓ Sexual behavior problems could transpire.

✓ The individual could lose self-respect.

✓ A feeling of insecurity could be developed.

✓ A resentment of life could emerge.

✓ A dependency on medication and alcohol usage can emerge.

✓ Isolation from the social environment could aggravate the victim's health. It could lead to negative behavior, such as the use of harmful substances.

✓ Constant weight gain or excessive weight loss could occur.

✓ In addition to eating disorders, irregular sleep can also be listed among the harmful factors for health. 140 • Health Sciences Research Papers

✓ All these could trigger situations as heart attack or stroke (Çetin et al., 2011; Güler et al., 2005). When the reality of a child being married in early marriages considered, it is better to remember the child, who is exposed to violence, faces the risk of death or injury. In addition to this, there is no possibility of the child not being affected by violence in regards to her psychological development, all acts of violence leave psychological marks on the child's psyche in varying degrees. Even years later, the effects of violence on children could disrupt the child's quality of life. These effects include the following:

✓ Physical health problems, such as alterations in brain development, physical injuries, bruises, and fractures.

✓ Problems related to learning and prevention of development of intelligence.

✓ Severe mental disorders, such as post-traumatic stress disorder, depression, anxiety disorders, etc.

✓ Difficulty in socializing.

✓ Being more inclined to the exhibition of risky behavior, such as alcohol consumption, drug usage, or sexual activity at a very young age.

✓ Problems and deviations in personality development.

✓ Mental health problems including the need to kill could occur (UNICEF, 2013). The majority of female children married off at a young age are forced to live together with their husband's family. Specifically, in almost all marriages, in which the age gap is smaller between the marrying parties, this situation occurs. While the traditional values could affect this, there are other factors involved related to being a child or being financially dependent too. In these types of situations, the domestic violence that child brides are exposed is not limited to the person they were married off to, and they may be subjected to the assault of the other members of the family too. This situation, at times, can cause severe trauma to child brides that can affect them for life (Anık & Barlin, 2017). Funda ÖZPULAT • 141

Battered women think domestic conflict and violence could not be shared with everybody and that these type of experiences could be shared with only trustworthy family members that can hide this information. Additional support systems available to women include friends, neighbors, police officials, women's shelters, and health care professionals. Being exposed to repeated abuse, not believing that they can get support, thinking about their children, not breaking-up of the family, to avoid being a talking point to others, not knowing what to do, accepting violence as the norm or as a frivolous offense, emotional attachment to their partners, fear of revenge, not having financial independence, a lack of support from their family and friends, the fear of being separated from their children, believing that their partners would eventually change, not having a place to return to and the prejudice against divorced women play a crucial role among the reasons why women do not share their experiences with violence (Başkale & Sözer, 2015).

CONCLUSION In addition to being a human rights violation, early marriages are also a sign of violation of children's rights. Additionally, early marriages and related violence phenomena are encountered as an epidemic in several developing countries, including Turkey. Legal regulations are certainly essential, however, reducing the problem just to the inadequacy of official regulations, or looking for this problem's solution in the dissuasiveness of official regulations alone, is an insufficient attempt at abolishing this problem. The fight against a problem, which includes multiplicity concerning its causes and consequences, is also required to include multiplicity. Therefore, one of the main actions, which needs to be taken to resolve this problem, is to activate different units related to this topic and ensure coordination between these units. It is extremely important to ensure the coordination between these units, specifically in education, health, and security departments and units in the Ministry of Family, Labor, and Social Services. Still, on this topic, being in productive cooperation with local government units, and additionally, coordinating with non-governmental organizations (NGOs) that work on this topic are rather important (Anık & Barlin, 2017). 142 • Health Sciences Research Papers

Additionally, projects and health education programs, which are actively attended by the universities and health care officials in every rank, specifically primary healthcare professionals, can be efficient in reducing the number of early marriages and violence associated with these types of marriages.

Funda ÖZPULAT • 143

REFERENCES 1. Abukan B, Yıldırım F. Empowerıng Women to Prevent Early Marrıages as A Form of Gender-Based Vıolence. The Journal of International Social Research, 2017; 10(53): 425-432. 2. Aktepe E, Atay İ.M. Child Marriages and Psychosocial Outcomes. Current Approaches in Psychiatry, 2017; 9(4): 410-420. 3. Anık M, Barlin R. The Problem of Child Brides in Turkey: Balıkesir Example. Journal of Human and Social Sciences Research, 2017; 6(3): 1827-1841. 4. Aşkın E.Ö, Aşkın U. Relatıonshıp Between Poverty and Domestıc Vıolence Agaınst Women: A Research on Domestıc Vıolent Women. Cappadocıa Academıc Revıew, 2018; 1 (2); 16-37. 5. Başkale H, Sözer A. Violence Perception and Victimization Reasons of Women Exposed to Spouse/Partner Violence. TAF Prev Med Bull, 2015; 14(6): 468-474. 6. Baydur E, Ertem B. Legal Aspects of Domestic Violence Against Women. An Examination in the Scope of the Criminal Code, Civil Code and Law on the Protection of the Family. Journal of TBB, 2006; 65: 89- 118. 7. Belhorma S . Two Months of Marriage Were Sufficient to Turn My Life Upside Down: Early Marriage as a Form of Gender-Based Violence, Gender & Development, 2016; 24(2), 219-230. 8. Council of Europe. Number of cases of violence against women increasing, warns PACE, 2012. http://www. assembly.coe.int/nw/xml/News/News- View-EN. asp?newsid=4222&lang=2&cat=17 (Accesed Date: 15.09.2019). 9. Çetin M, Bilgiç Selman Ş, Umur Y.F. Marriage and Health. T. C. Ministry of Family and Social Policies General Directorate of Family and Community Services. Nakış Offset. Ankara, 2011. 10. Doğan Koroğlu A. Combating Early Marriages in Least Developed and Developing Countries and Turkey Applications. T.C. Ministry of Family and Social Policies Directorate General on the Status of Women. Ankara, 2014. 11. Ediz A, Altan Ş. A Field Study on Violence Against Women in Turkey. Journal of TBB, 2017; (Special Issue): 397-410. 12. European Union Agency for Fundamental Rights (FRA). Violence Against Women: An EU-Wide Survey, 2014. 144 • Health Sciences Research Papers

http://fra.europa.eu/sites/default/files/fra-2014-vaw-survey-main- results-apr14_en.pdf (Accesed Date: 08.09.2019). 13. Gökkaya, V.B. Economic Violence Against Woman in Turkey. Cumhuriyet University Journal of Economics and Administrative Sciences, 2011; 12(2):103. 14. Güler N, Tel H, Özkan Tuncay F. The View of Womans’ to The Violence Experienced Within the Family. Cumhuriyet University Journal of Medical Faculty, 2005; 27 (2): 51- 56. 15. Güneş M, Selçuk H, Demir S, İbiloğlu A.O, Bulut M, Kaya M.C, Yılmaz A, Atli A, Sır A. Marital Harmony and Childhood Psychological Trauma in Child Marriage. Journal of Mood Disorders, 2016; 6:63-70. 16. Harcar T, Cakır O, Surgevil O, Budak G. Violence against women and violence against women in Turkey Status. Society and Democracy, 2008; 2(4): 51-70. 17. ICRW. Development Initiative on Supporting Health Adolescents (DISHA) Project. Analysis of quantitative baseline survey. Washington, D.C.: ICRW, 2005. 18. ICRW (2007). New Insights on Preventing Child Marriage: A Global Analysis of Factors and Programs. http://www.icrw.org/files/publications/New- Insights-on-Preventing- Child-Marriage.pdf (Accesed Date: 08.09.2019). 19. İçağasıoğlu Çoban A. Adolescent Marriages. Family and Society, 2009; 11(4):37-50. 20. KSGM. Pekin+5 Political Declaration Result Document and Platform for Action. 2nd Edition, Cem Offset. Ankara, 2003. 21. Le Strat Y, Dubertret C, Le Foll B. Child Marriage in the United States and Its Association with Mental Health in Women. Pediatrics, 2011; 128(3): 524- 530. 22. Nasrullah M, Zakar R, Zakar MZ. Child Marriage and its Associations with Controlling Behaviors and Spousal Violence Against Adolescent and Young Women in Pakistan. Journal of Adolescent Health 2014; 55(6):804-909. 23. Orçan M, Kar M. The Early Marriage and the Perception of Risk in Turkey: The Case of Bismil. Family and Society, 2008; 14(14): 97-112. 24. Ozpulat F. Neglected Aspect Of Socıety: Child Marriages And Their Reflection On Women's Health. Online Turkish Journal of Health Sciences, 2016; 2(1): 11-22. Funda ÖZPULAT • 145

25. Rahman M, Hoque MA, Mostofa MG, Makinoda S . Association between Adolescent Marriage and Intimate Partner Violence: A Study of Young Adult Women in Bangladesh. Asia Pac J Public Health, 2014; 26 (2): 160- 168. , 26. Raj A, Saggurti N, Winter M, Labonte A, Decker M. R, Balaiah D, Silverman J.G. The Effect of Maternal Child Marriage on Morbidity and Mortality of Children Under 5 in India: Cross Sectional Study of a Nationally Representative Sample, BMJ, 2010; 340:b4258. 27. Roman N.V, Frantz J.M. The Prevalence of İntimate Partner Violence in the Family: A Systematic Review of the İmplications for Adolescents in Africa. Fam Pract, 2013; 30(3): 256–265. 28. Sargın A. Combating Violence Against Women and Support Mechanisms for Violence for Public Institutions and Women's NGOs. Strengthening Women and Women NGOs Project, 2010-2012. 29. Soylu N, Ayaz M. Sociodemographic Characteristics and Psychiatric Evaluation of Girls Who Were Married At Younger Age and Referred for Criminal Evaluation. Anatolian Journal of Psychiatry, 2013; 14: 136- 144. 30. Tatlılıoğlu K, Küçükköse İ. (2015). THE Vıolence Agaınst the Women ın Turkey: The Reasons, Protectıons, Preventıon and Interventıon Servıces. Dicle University Journal of Institute of Social Sciences, 2015; 7.13, 194-209. 31. Republic of Turkey, Family and Social Services Provincial Directorate. Research on Domestic Violence against Women in Turkey. Hacettepe University, 2014. 32. UNICEF. Guidelines for Monitoring Violence Against Children. March, 2013. 33. UNFPA. Marrying Too Young: End Child Marriage, New York, UNFPA, 2012. Available at http://www.unfpa.org/sites/ default/files/pub-pdf/MarryingTooYoung.pdf (Accesed Date: 08.09.2019). 34. World Health Organization. Guidelines on Preventing Early Pregnancy and Poor Reproductive Outcomes Among Adolescents in Developing Countries, Geneva, World Health Organization, 2011. http://www.who.int/immunization/hpv/target/preventing_early_pregnan cy_and_poor_reproductive_ outcomes_who_2006.pdf (Accesed Date: 12.09.2019). 146 • Health Sciences Research Papers

35. WHO, World Report On Violence And Health: In Krug EG, Dahlberg LL, Mercy JA, Zwi AB, Lozano R (editors). Geneva: World Health Organization, 2002, 3-22. 36. Yağbasan M, Tekdemir N. Trauma of Child Brides from a Sociological Perspective (A Field Study in the Context of Mother-Child Relationship and Communication in Eastern and Southeastern Anatolia). The Journal of Social Science, 2017; 4(16): 18-32. Yanık A, Hanbaba Z, Soygür S, Ayaltı B, Doğan M. Evaluation of Vıolent Behavıor Agaıst Women: Evıdence from Turkey. Electronic Journal of Vocational Colleges, 2014; 104- 111.

THE DUTIES AND RESPONSIBILITIES OF THE NURSE IN SEXUAL ABUSE CASES TOWARDS CHILDREN

CHAPTER 10

Funda ÖZPULAT1

1 Asst. Prof., Selcuk University, Aksehir Kadir Yallagoz School of Health, [email protected]

Funda ÖZPULAT • 149

INTRODUCTION Child abuse is a crucial problem that affects the child's physical, psychological, and emotional health negatively, and that is not associated with the child's rights and humanity (Geçkil, 2017). In addition to having rights such as; living, developing and growing up healthy, having educational opportunities, being happy and at ease, and not being exposed to abuse and neglect while exercising these rights is the natural right of children (Gökler, 2009). However, every year, millions of children in the world are exposed to physical, sexual, or emotional violence, or they witness these types of situations (WHO, 2006). The 1999 definition of child abuse, by World Health Organization, established its extent and child abuse was defined as "all forms of physical and/or emotional ill-treatment, sexual abuse, neglect or negligent treatment or commercial or other exploitation, resulting in actual or potential harm to the child's health, survival, development or dignity in the context of a relationship of responsibility, trust and power" (Runyan et al., 2002). Child abuse, implemented by individuals, could be examined in the following four groups: Physical abuse Sexual abuse Emotional abuse Neglect (Runyan et al., 2002). Even though child abuse is said to be as old as the history of humanity, the conversation around the notion of abuse and conducting systematic studies concerning that started only in the last century (Polat, 2007). The society's values, beliefs, societal norms they had adopted, society's knowledge about child development and family relationships determine which behaviors towards a child are perceived as child abuse and neglect. As a result, it seems unfeasible to universally determine the definition of child abuse and neglect (Gökler, 2006). One of the sources of many problems individuals experience in childhood or adulthood, such as loneliness, mental health deterioration, stress, and depressive symptoms, is neglect and abuse experienced in childhood (Pekdoğan & Bozgün, 2018). In the adolescence period of children who were abused and neglected, physical grievances such as increased gastrointestinal complaints and persistent headaches may occur 150 • Health Sciences Research Papers

(Spertus et al., 2003). The long-term effects of experiencing child abuse and sexual trauma include cognitive, emotional disorders, continuous dependence on drugs and alcohol, eating disorders, suicide attempts, major depression, anxiety disorders and other mental illnesses, risky sexual intercourses, tendency to commit crimes in childhood and adolescence (Alpaslan, 2014; Jacobi et al., 2010). Abused children can be encountered as adults, who are prone to violence while experiencing self-esteem and communication problems, exhibiting behaviors that are not accepted by the society, inclined to commit crimes, addicts, inclined to commit suicide and self-harm. The child that was raised in an abusive environment can adopt a vicious circle, which can accept the treatment they had received as standard over time (Altıparmak et al., 2013).

SEXUAL ABUSE AND ITS EFFECTS ON CHILDREN Sexual abuse in children was first described by Ruth and Henry Kempe (1978) as "children and adolescents, who are dependent and developmentally immature, not being able to consciously give consent, not being able to fully comprehend, or being a party to sexual activities that contradict social taboos related to family roles" (Kempe, 1984). Sexual abuse is the hardest one to detect among the types of child abuse. Sexual abuse, which remains hidden and is not ascertained easily, is an important phenomenon, especially in terms of its short and long-term effects. The most important reason why sexual abuse remains hidden is that it is usually committed by members of the family or by individuals close to the family (Polat, 2007; Topbaş, 2004). Sexual abuse is encountered as a result of the interaction of multiple factors in different areas. Risk factors, which are causing sexual abuse, can be examined at personal, relational, environmental, and societal levels (Aydemir & Yurtkulu, 2012). The younger the children are, the higher their possibility of being abused and neglected, economic, environmental, and socio-cultural risk factors such as poverty, low education levels, unemployment, violence, and substance abuse can lead to child abuse and neglect (Kazez, 2010; Ducharme, Atkinson & Poulton, 2001). In addition, mental and physical disabilities of children and adolescents, and psychiatric disorders such as schizophrenia, bipolar disorder, impulse control disorder, attention deficit hyperactivity disorder (ADHD) are among factors that increase the risk of sexual abuse (Zoroğlu, 2001). The patriarchal family structure in Turkey, having many children and continue Funda ÖZPULAT • 151 living in one-roomed houses, low level of education of parents, acceptance of all types of violence against children and women, gender inequality in the society, early marriages of female children can also be counted among the situations that increase child abuse (Tirali, Oğuz & Soydan, 2014). Additionally, the increasing number of conflict environments that have invaded the world in recent years can also be seen as one of the most crucial factors causing child abuse (Hartley, Mullings & Marquart, 2013). Sexual abuse could occur in different forms: a. Non-contact Sexual Abuse: Sexually explicit speech, exhibitionism, and voyeurism b. Sexual Fondling: Abuser could touch the victim, for sexual gratification, or force the victim to touch themselves. c. Oral - Genital Sex d. Intercrural sex (Indecent Assault): A type of abuse with friction, without penetration. e. Sexual Penetration (Rape): It could be any form of genital sex, anal sex, oral sex, penetration with objects, and penetration with fingers. f. Sexual Exploitation: It involves child pornography and child prostitution (Polat, 2001). It is harder to define sexual abuse compared to physical and emotional abuse. Because, in societies that are strictly adhered to their traditions and customs, the expression of sexual abuse may deepen the problems. Notably, while women should be receiving more support in these issues than men, the opposite can occur, and they can be excluded (Pekdoğan & Bozgün, 2018). Sexual abuse towards children can be committed by parents, the child's older friends, siblings, step-parents, neighbors, teachers, other familiar individuals, or any unfamiliar strangers. Despite the consensus, sexual abuse is reported to have been committed by an adult member of the family, or an individual known by the child rather than a stranger outside the family (Aydemir & Yurtkulu, 2012). Abuse is an acute and chronic trauma that weakens the child's emotional and sexual development, interpersonal relationships, and self-esteem (Nurcombe, 2000). Clinical features of sexual abuse and its effects on 152 • Health Sciences Research Papers children change according to the child's relationship with the abuser, the type and duration of the abuse, the use of violence, the existence of physical harm, the age and the developmental period of the child, the child's psychological characteristics, and psychological development prior to the trauma. The family's reaction to the incident also has an influential role (Alpaslan, 2014). When the children experience abuse:  They may think the world as a dangerous and unsecured place.  Feelings of helplessness and defenselessness can develop.  They may perceive threats and challenges more prominent than they are in actuality.  They may feel worthless and experience apathy and hyperalertness, which are symptoms of depression and post- traumatic stress disorder (Dinleyici & Şahin Dağlı,2016). The experience of sexual abuse can result in psychiatric disorders, including anxiety, depression, substance abuse, suicidal behavior, personality disorders, and post-traumatic stress disorder (Alpaslan, 2014). Night terrors and other sleeping disorders such as difficulties in falling asleep and maintaining the state of sleep, introversion, fear, depression, being overly aggressive for no reason, being disobedient, acting out manifested by temper tantrums, refusing to go home or attempting to escape from home are among the behavioral symptoms of sexual abuse (Lowenthal, 2001). Attention deficit, hyperactivity disorder, secondary enuresis, and encopresis are more commonly seen in victims of sexual abuse (Taner & Gökler, 2004). Sexual abuse also affects children's ability to establish relationships with society and maintain social relationships in a negative way. It is observed that these children may avoid establishing relationships at times, while at other times, they may form an excessive number of controlling relationships with high expectations in need of extreme intimacy. Both types of these relationships are far from functional and often result in the loneliness of the individual (Tackett, 2002). In addition, they can experience the fear of reliving and repeating the sexual trauma they had experienced, depression, anger, hostility, sexual identity disorders, fear, and sexual dysfunctions, as well as possibly developing behaviors in opposite direction, such as, being sexually active, frequently masturbating, exhibiting sexual behaviors that are not suitable for their age, Funda ÖZPULAT • 153 playing sexual games, exhibiting seductive behaviors towards adults (Ovayolu, Uçan & Serindağ, 2007; Topbas, 2004). There is a consensus on the prevention of child abuse and neglect being a complex and multifaceted issue. Also, the cyclical nature of violence and the possible reality of current victims being the potential abusers of the future are accepted by everyone. This situation makes it necessary to prioritize the prevention of child abuse and neglect (Theodore, 2002).

ASPECTS TO PAY ATTENTION TO IN SEXUAL ABUSE PHENOMENONS Sexual abuse of the children is a remarkably thorough and complicated issue that has physical, mental, social, cultural, moral, and legal dimensions. The early diagnosis of abuse reduces the children's burden that they have to bear, stops the children's exposure to abuse, enables the support systems to get involved quickly, prevents their mental health from being gravely affected, and helps to maintain their functionality as healthy individuals in the long term (Dağlı & İnanıcı, 2010). The story is very significant in abuse phenomenons. Usually, the abuse diagnosis can be made with the story alone. A good story contains clues. Children over the age of three can provide credible stories (Selby, 2007). The other important point is the thorough and systematic physical examination of the entire body. In this examination, inspection should be done first, then the palpation, if there is an injured area, it should be examined last (Ayvaz & Aksoy, 2004). Also, all of the evidence must be gathered within the 72 hours after the attack, and the biologic evidence must be examined within 120 hours (Murphy et al., 2010). In over 90% of the cases that are reported within 24 hours, forensic evidence can be found, the evidence, in particular, can be gathered from undergarments. While successful results cannot be gathered from sperm and pre-seminal fluid analyses done after 24 hours, the undergarments must be withheld for analyzation (Christian et al., 2000). In physical examinations of abused female children, it is necessary to determine the sexual development stage they are in. It is crucial for these pieces of information to be determined and recorded because there can be discrepancies in the child's sexual development stage between the time the abuse takes place and the court date. In the gynecological examination, the child's general physical condition should be recorded first. The condition of the patient's clothes 154 • Health Sciences Research Papers

(blood stains, etc.), general state, emotional state, and hygiene status should be recorded in detail (Sakelliadis, Spiliopoulou & Papadodima, 2009). Wounds in the genital area, bleeding, irritation, and ulcers can be counted among the signs and symptoms of sexual abuse. The presence of blood in undergarments, torn and dirty clothes, the children feeling discomfort while urinating, swelling in the genital area, nonspecific vaginitis, sexually transmitted diseases, oozing from the tip of the penis, difficulty in walking and sitting down, bad odour in the genital area, recurrent infections, presence of sperm in the genital area or on the clothes, pregnancy at a young age can be signs of sexual abuse (Hockenberry & Wilson, 2009). Sexual abuse usually does not leave traces on male children, or despite being easily spotted, the importance of those traces is not apparent. There can be abrasions, bite marks, ecchymoses, and edema in various parts of the penile penis. Genital injuries in male children may occur as a result of non-sexual punishment rather than sexual abuse. In female children, they are, hymenal laceration or scarring, thinning co- existing with the loss of hymenal tissue, and lacerations and scarring extending from the back of the anal edge to the perianal surface in anus mucosa. Pregnancy, sexually transmitted diseases, or positive forensic pieces of evidence should bring abuse into the question (Polat, 2004). Additionally, the child's excessive interest or excessive indifference to issues related to sexuality, exhibiting sexual behavior, frequently requesting to kiss, attempting to touch their genitals, rubbing, the effort to show their genitals to other individuals, stating their bodies are defiled, the presence of features that might suggest that they have been exposed to sexual abuse in their drawings or games, wetting themselves or their beds in potty-trained children, exhibiting infantile behaviors are signs that indicate the children are sexually abused (Ulukol, 2013).

Funda ÖZPULAT • 155

Table 1. Signs of Sexual Abuse in Children and Adolescents

AGES 6 – 11 AGES 12 - 17 Exhibiting openly sexual Sexually exploitative behavior behaviors with their peers. with little children. Verbally expressing their sexual Overly sexual behavior or experiences. complete sexual abstinence. Being excessively into their The attempt to get rid of the Female genitalia. feeling of guilt, shame, and Children Sexual relations with adults. denigration. Distrust and fear of the Running away from their homes. individuals around them. Knowledge of sexual behavior unsuitable for their age. Eating disorders. Sleep-related problems (night terrors). Exhibiting openly sexual behaviors with their peers. Sexually exploitative behavior Distrust and fear of the with little children, and having an Male individuals around them. aggressive stand against them. Children Aggressive behavior. Regressive behavior. Losing interest in previous fields Risky sexual behavior. of interest. Regressive behavior. The attempt to get rid of the Sleep-related problems (night feeling of guilt, shame, and terrors). denigration. Source: UNICEF, 2019.

While approaching the abuse phenomenon, it is necessary to evaluate both the child's situation and the state of parents or the individual that committed the abuse. The abuse tendency could, usually, be related to being young parents, either one of the parents' having prior exposure to abuse as a child, alcohol or substance addiction, financial problems related to external factors, death of one of the partners, mental or physical illness in family history, having a large number of children, or a broken family (Hancı, 2002). Children often do not express the sexual abuse they experience to anyone. The reasons for this are, being afraid of the abuser's threat of harm against themselves, or their family, experiencing a feeling of shame and guilt, fearing their family's reaction and people not believing them (Caringfork, 2009). When the sexual abuse phenomenon is encountered, the children's safety should be ensured immediately, their state of health should be evaluated, examination 156 • Health Sciences Research Papers findings that could be related to the abuse should be recorded, and the abuse situation should be reported to the respective judicial authorities. The treatment process of sexual abuse cases, after the first and immediate evaluation, is done in this way, varies depending on the period of the abuse, whether the abuser is a member of the family, the age of the children, and the state of the symptoms (Vatansever, 2004). The information that will be gathered from the child, who is alleged to have been sexually abused, after the application is of utmost importance. The interview should be free from prejudice and should not be leading the victim. While insufficient information could obstruct legal adjudication, in other words, preventing the child's protection, leading of the victim during the interview will result in the harming of an innocent person. (Runyan et al., 2002). In case of suspicion of child abuse and neglect, the notification done by the health care professional is the first step towards the rescue of the abused child. It should be remembered that if the individuals, who witness child abuse, do their part, they can prevent the children from being exposed to repeated abuse (Yalçın et al., 2014).

Funda ÖZPULAT • 157

THE DUTIES AND RESPONSIBILITIES OF THE NURSE IN SEXUAL ABUSE CASES Child abuse and neglect are significant public health concerns with medical, legal, and social aspects that cause severe injuries and deaths. To tackle the issue of child abuse at the international, national, regional, and local levels, different sectors need to intervene at different stages. Health care, social services, and law sectors are at the forefront of these sectors. (Beyaztaş, Çelik & Bütün, 2011; Mian & Boothby, 2016). Social service specialists, doctors, nurses, teachers, child development specialists, in the teams, in these sectors, have crucial roles (Tıraşçı & Gören, 2007). Every individual that comes into contact with children in their professional lives should know to have a suspicion of child abuse and what to do when they observe that possibility (Geçkil, 2017). However, the slow behaviors of teachers at schools, medical or forensic science personnel, and social service specialists, in abuse cases causes a delay in reporting of these cases. Additionally, responsible individuals, involved in the matter, ignoring certain aspects of abuse can cause many cases to go unnoticed (Polat, 2000). Also, cases are not being reported as a result of several reasons, such as the employees not being familiar with the facts, hesitating to report due to not knowing the legislation, avoiding to attend to the court, and child protection being a critical decision (Geçkil, 2017). Preventive and therapeutical healthcare services have an essential part in the prevention of child abuse and the protection of children from abuse. In this context, along with other experts, nurses have vital duties (Arıkan, Yaman & Çelebioğlu, 2000). Nurses are often the first to come into contact with the abused children, and they have legal responsibilities to report on their suspicions about child abuse or neglect (Piltz & Wachtel, 2009). Nurses are an essential member of the team due to being the person to reach out to in emergencies at all times. Nurses are the ones, most of the times, who come into contact with the individuals, who have been exposed to abuse and neglect, the materials that will be considered as evidence, record these materials in a way they may be formed as evidence to the court, and that can and will immediately contact the patient's family (Çolak, 2003). Nurses find the opportunity to observe the children and their families for a more extended period both during child health follow-ups and during the diagnosis, treatment, and rehabilitation of the children in the hospital environment. Thus, nurses are in a key position, specifically in 158 • Health Sciences Research Papers cases of abuse that have not been discovered yet, to obtain essential pieces of evidence. However, nurses working in pediatric and emergency services should be able to approach the pediatric patients in the right way, and spot the abuse and neglect cases that are mandatory to be reported, body image differences, shyness, or deformities apparent on the body, and should know the necessary procedure to follow, their awareness of physical symptoms of child abuse may remain inadequate (Fraser et al., 2010; Zoroğlu et al., 2001; Gölge, Hamzaoğlu & Türk, 2012). Controlling and recording the physical and emotional responses of the children, immediate recognization, initiating co-operation with other members of the team, identification of psychosocial factors, and increasing self-regard are among nursing initiatives that can be performed. Providing an appropriate environment for the safety of children, supporting them to talk and to think, and therapeutic plays can be counted among other nursing initiatives (Koyun, Taşkın & Terzioğlu, 2011). However, many nurses have limited information about child abuse and neglect, and need to receive an education on mandatory reporting and child protection services (Piltz & Wachtel, 2009).

PREVENTİNG CHİLD ABUSE AND NEGLECT STRATEGY APPROACH Strengthen economic Strengthening household financial security supports to families Family-friendly work policies Change social norms to Public engagement and education campaigns support parents and Legislative approaches to reduce corporal positive parenting punishment Provide quality care Preschool enrichment with family engagement and education early in Improved quality of child care through licensing life and accreditation Enhance parenting Early childhood home visitation skills to promote Parenting skill and family relationship approaches healthy child development Intervene to lessen Enhanced primary care harms and prevent Behavioral parent training programs future risk Treatment to lessen harms of abuse and neglect exposure Treatment to prevent problem behavior and later involvement in violence Source: Fortson et al., 2016. Funda ÖZPULAT • 159

Nurses working in institutions providing preventive healthcare services, pediatric nurses, forensic nurses must play an active role in early diagnosis of abuse and neglect, treatment of it, assessment of risk groups, tackling the risk factors, educating the family and the society about this issue, and providing a safe environment for the children to receive healthcare services (Çatık & Çam, 2006; Selph, Bougatsos & Blazina Nelson, 2013). Education and counseling are a component of preventive healthcare services, and regular home visits are effective in reducing the abuse and neglect cases (Block & Palusci, 2006).

CONCLUSION AND SUGGESTIONS The prevention of abuse is more economical than its treatment. These types of societal problems have a high cost to society, and they are preventable problems. Early intervention prevents children from running away from home, having problems at school, delinquency, having alcohol and drug addiction, and being harmed by grave emotional and developmental difficulties that can lead to prostitution, and cutting back on financial expenses. Since the presence of the history of child abuse in the family is often linked with the abuse of the mother, looking for domestic abuse is also very important. Specifically, primary care nurses being attentive and sensitive can prevent abuse cases that may develop. If screening techniques can be used to eliminate the threat and intervene in the first few years of a children's life, severe trauma can be prevented. Education and screening programs that will be conducted on a regional level with the cooperation of universities, primary healthcare personnel with nurses at the forefront, and local government units can be influential in raising the level of public consciousness. The inclusion of nurses, who are working in critical units such as emergency services and secondary healthcare services, in training programs where they can acquire the efficiency in detecting the abuse, can be crucial in the early discovery of abuse cases and prevention of their repetition. Additionally, nurses can professionally contribute to the development of a national action plan for the prevention of child abuse and the implementation and evaluation of this action plan.

REFERENCES 1. Alpaslan A.H. Childhood Sexual Abuse. Kocatepe Medical Journal, 2014;15(2):194-201. 2. Altıparmak S, Yıldırım G, Yardımcı F, Ergin D. Child Abuse and Neglect Based on The İnformation Obtained from Mothers, and The Factors Affecting Child Abuse. Anatolian Journal of Psychiatry, 2013; 14: 354- 361. 3. Arıkan D, Yaman S, Çelebioğlu S. Knowledge Of Nurses on Child Neglectand Abuse. Journal of Atatürk University School of Nursing, 2000; 3(2): 29- 35. 4. Aydemir İ, Yurtkulu F. Fıgtıng Sexual Abuse Agaınıst Chıldren: Chıld Fallow Up Center. Ankara Journal of Health Sciences, 2012 1(2):151-165. 5. Ayvaz M, Aksoy M.C. Child Abuse and Neglect. Hacettepe Medical Journal, 2004; 35: 27-33. 6. Beyaztaş F.Y, Çelik M, Bütün C. The İmportance of İmaging Techniques in Child Abuse. Forensic Medicine Bulletin, 2011; 16(1): 25-31. 7. Block R.W, Palusci V.J. Child Abuse Pediatrics: A New Pediatric Subspecialty. J Pediatr, 2006; 148: 711-712. 8. Caringfork D.S. Child Sexual Abuse Fact Sheet April 2009. https://www.nctsn.org/sites/default/files/resources/fact- sheet/caring_for_kids_what_parents_need_know_about_sexual_abuse.p df. (15. 11.2019) 9. Christian C.W, Lavelle J.M, De Jong A.R, Loisell J, Brenner L, Joffe M. Forensic Evidence Findings in Prepubertal Victims of Sexual Assault. Pediatrics, 2000; 106:100-104. 10. Çatık A.E, Çam O. Determınıng Nurses’ and Mıdewıves’ Level of Knowledge on Symptoms and Rısk of Chıld Abuse And Neglect. Journal of Ege University School of Nursing, 2006; 22 (2): 103-119. 11. Çolak B. İnanıcı M.A. Yaycı N. Child Sexual Abuse and Forensic Nursing. Nursing Forum, 2003; 6: 7-13. 12. Dağlı T, İnanıcı M.A. Hospital-Based Child Protection Centers Manual for Universities, 2010. https://cokmed.org/_ekurs/uploads/4_universiteler_ckm.pdf. (11.10.2019) 13. Dinleyici M, Şahin Dağlı F. Emotıonal Abuse, Neglect And The Role of Pedıatrıcıan. Osmangazi Journal of Medicine, 2016; 38(2), 18-27. Funda ÖZPULAT • 161

14. Ducharme J.M, Atkinson L, Poulton L. Errorless Compliance Training with Physically Abusive Mothers: A Single-Case Approach. Child Abuse Negl, 2001; 25: 855- 68. 15. Fortson B.L, Klevens J, Merrick M.T, Gilbert L.K, Alexander S.P. Preventing Child Abuse and Neglect: A Technical Package for Policy, Norm, and Programmatic Activities. Atlanta, GA: National Center for Injury Prevention and Control, Centers for Disease Control and Prevention, 2016. 16. Fraser J.A, Mathews B, Walsh K, Chen L, Dunne M. Factors İnfluencing Child Abuse and Neglect Recognition and Reporting by Nurses: A multivariate Analysis. International Journal of Nursing Studies, 2010; 47(2): 146-53. 17. Geçkil E. Physıcal Abuse ın Chıldren and Nursıng Interventıons. Gümüşhane University Journal Of Health Sciences, 2017; 6(1): 129 – 139. 18. Gökler R. Peer Bullying in Schools. International Journal of Human Sciences (JHS), 2009; 6(2): 511-537. 19. Gökler R.A General Overview The Abuse and Negligence of Children In Education. Turkey Social Studies Journal, 2006; 10 (3): 47-76. 20. Gölge Z.B, Hamzaoğlu N, Türk B. Assesment of Medical Staff Awareness about Child Abuse and Neglect. J For Med, 2012; 26(2): 86-96. 21. Hancı İ.H. Forensic Medicine and Ali Sciences. Seçkin Publishing. 1st Edition. Ankara, 2002; 263- 284. 22. Hartley D.J, Mullings J.L, Marquart J.W. Factors İmpacting Prosecution of Child Sexual Abuse, Physical Abuse, and Neglect Cases Processed Through A Children’s Advocacy Center. Journal of Child & Adolescent Trauma, 2013; 6: 260–273. 23. Hockenberry M, Wilson D. Wong’s Essentilals of Pediatric Nursing. English: 8th ed. Mosby Elseviver, 2009. 24. Jacobi G, Dettmeyer R, Banaschak S, Brosig B, Herrmann B. Child Abuse and Neglect: Diagnosis and Management. Dtsch Arztebl Int, 2010; 107: 231- 40. 25. Kazez A. Trauma in Children. Turk Arch Ped, 2010; 45 Suppl: 12-16. 26. Kempe, R.S, Kempe C.H. The Common Secret: Sexual Abuse of Children and Adolescents. 1984: WH Freeman New York. 27. Koyun A. Taşkın L. Terzioglu F. Women Health and Psychological Functioning in Different Periods of Life: Evaluation of Nursing Approach Psikiyatride - Current Approaches in Psychiatry, 2011; 3: 72. 162 • Health Sciences Research Papers

28. Lowenthal, B. (2001). Abuse and Neglect: The Educator’s Guide to the Identification and Prevention of Child Maltreatment. Baltimore, Maryland: Paul H. Brookes Publishing Co, Inc. 29. Mian M, Boothby M.R. Cross-Sectoral Approach to Child Abuse Prevention. Dağlı T, ed. 30. Centers (ÇOKMED); 2016; p. 38-51. 31. Murphy S.B, Potter S.J, Stapleton J.G, Wiesen-Martin D, Pierce-Weeks J. Findings from Sexual Assault Nurse Examiners (SANE): A Case Study of New Hampshire’s Pediatric SANE Database. Journal of Forensic Nursing 2010; 6: 163-169. 32. Nurcombe B. Child Sexual Abuse I: Psychopathology. Aust N Z J Psychiatry, 2000; 34(1): 85-91. 33. Ovayolu N, Uçan O, Serindag S. Sexual Abuse in Children and Its Effects. Fırat Health Services Journal, 2007; (2): 4: 13-22. 34. Pekdoğan S, Bozgün K. Investıgatıon of Teacher's Vıews About Chıld Neglect and Abuse. Manas Journal of Social Studies, 2018; 7(2): 433- 443. 35. Piltz A, Wachtel T. Barriers that İnhibit Nurses Reporting Suspected Cases of Child Abuse and Neglect. Australıan Journal of Advanced Nursıng, 2009; 26(3): 93-100. 36. Polat O. Child Abuse in All Dimensions. 1. Printing. Ankara: Seçkin Publishing, 2007: 25-58. 37. Polat O. Clinical Forensic Medicine. Seçkin Publishing. 1st Edition. Ankara, 2004; 85-131. 38. Polat O. Sexual abuse. Child and violence. İstanbul: Der Publications, 2001; 207-314. 39. Polat O. Relationship Between Abuser and Victim in Child Abuse. Children's Forum Journal,2000; 3(4): 1- 17. 40. Runyan D, Corrine W, Ikeda R, Hassan F. Child Abuse And Neglect By Parents And Other Caregivers. In: World Report On Violence And Health. Krug EG, Dahlberg L.L, Mercy J.A. (eds) World Health Organization, Geneva, 2002; 57-86. 41. Sakelliadis E, Spiliopoulou C, Papadodima S. Forensic Investigation of Child Victim with Sexual Abuse. Indian Pediatrics, 2009; 46:144-151. 42. Selby J.B. Child Abuse. Textbook of Family Practice. Ed. Rakel RE. 7. Printing. Philadelphia, WB Saunders, 2007; 55-57 Funda ÖZPULAT • 163

43. Selph S, Bougatsos C, Blazina Nelson H. Behavioral Interventions and Counseling to Prevent Child Abuse and Neglect Systematic Review to Update the U.S. Preventive Services Task Force Recommendation Evidence Syntheses, 2013; (3):179-183. 44. Spertus I.L, Yehuda R, Wong C.M, Halligan S, Seremetis S.V. Childhood Emotional Abuse and Neglect as Predictors of psychological and Physical Symptoms in Women Presenting to A Primary Care Practice. Child Abuse Negl, 2003; 27: 1247-1258. 45. Tackett K.K. The Health Effects of Child Abuse: Four Pathways by Which Buse Can İnfluence Health. Child Abuse Negl, 2002; 26: 715-729. 46. Taner Y, Gökler B. Child Abuse and Neglect: Psychiatric Aspects. Hacettepe Medical Journal, 2004; 35: 82-86. 47. Theodore A.D. Child Abuse. Child Development. The Macmillan Psychology Reference Series. (Ed: Neil J. Salkind). New York: Gale Group, 2002. 48. Tırasçı Y, Gören S. Child Abuse and Neglect. Dicle Medical Journal, 2007; 34(1): 70-74. 49. Tıralı E, Oğuz Y, Soydan S.S. Oral Symptoms of Chıld Abuse And Neglect. J Dent Fac Atatürk Uni, 2014; 154-157. 50. Topbaş M. A Great Shame of Humanity: Child Abuse. TAF Preventive Medicine Bulletin, 2004: 3: 7-80. 51. Ulukol B. Child Abuse (Abuse and Neglect). Ankara University: Faculty of Medicine, Department of Social Pediatrics, Ankara Child Protection Unit. İzmir, 2013. 52. UNİCEF. Signs of Sexual Abuse in Children and Adolescents. https://www.unicef.org/teachers/protection/prevention.htm. (12.10.2019). 53. WHO. Preventing Child Maltreatment: A Guide to Taking Action and Generating Evidence. WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva , 2006, Switzerland. 54. Vatansever Ü. Pediatric Emergency Medicine Book.Pediatric Emergency Medicine and Intensive Care Society publication, 2004. 55. Yalçın N, İnce D, Acar Y, Kandırıcı A, Atılgan H. Management of Child Abuse as Related to Three Cases. Turkish Journal of Pediatric Disease, 2014; 2: 86-89. 56. Zoroğlu S.S, Tüzün Ü, Şar V, Öztürk M, Eröcal M.K, Alyanak B. Probable Results of Childhood Abuse and Neglect. Anatolian Journal of Psychiatry, 2001; 2(2): 69-78.

EVALUATION OF INFERTILITY ETIOLOGY IN WOMEN AND MEN AND TREATMENT OPTIONS

CHAPTER 11

Gizem KÖPRÜLÜLÜ1

1 T.C. İstanbul Şişli Meslek Yüksekokulu, gizem.köprülülü@sisli.edu.tr

Gizem KÖPRÜLÜLÜ • 167

INTRODUCTION The question of how gender is determined in the embryonic period has been an enigma since ancient times. Many theories about this issue have been produced. For example, Aristotle claims that environmental factors are influence on gender stereotyping. He belived that if a baby develops in the hot uterus, it will be a boy and if this baby develops in moist and cold uterus, it will be a girl ( Troedsson HT. and Madill S., 2004). But at the beginning of the 20th century, It was understood that Chromosomes have a key role on sex determination. Thus, the thesis in which the environmental conditions of gender are determined has been refuted.

Formation of Sexual Cell in Human The first occurrence of sexual reproduction is the formation of sex cells. Sexual cells are sperm and eggs. These are generally called as gametes which are prıduced in gonads. Gamet formation process is gametogenesis and which has four important stages: 1. Embriyonic roots of sex cells and migration of these cells to gonads 2. Increase the number of sex cells by mitosis division 3. Reduce by half the number of sex cells by meiosis 4. Maturation of sex cells Earliest forms of gametes are primordial germ cells (PGC). They form outside the Gonads and then migrate to the gonads to form the sex cells. PGCs, which reach the gonads, increase their numbers by mitosis. Active germ cells that pass mitosis, are called spermatogonia in men. Mitosis in male sex cells beging earlier than females and male sex cells divide life-long. Spermatogonia undergo periodic mitotic division during puberty and new cells enter the meiosis division stage and this continue throughout life. The production process of the egg cell begins with the differentiation of primordial germ cells in the female embryo and transformation of oogony. Oogoniums (oogonia) are stem cells which will form eggs and then they differentiate to give primary oocytes. Oogoniums reproduce in developing female embryos and enter the meiosis division process. However, this process pauses in the prophase I phase of the meiosis 168 • Health Sciences Research Papers division I. Primary oocytes wait in this phase until they are reactivated during adolescence an this process is called .

Genetic Control of Sex Determination Human genotype has X and Y chromosomes. Y chromosome is dominate factor to generate tha male sex and which has long and short arms that demonstrate respectively as Yq and Yp. Yq has AZF (Azoospermic Factor) gene on the distal end which manages the spermatogenesis process and it relates to male infertility. In the third quarter of the 20th century, ıt is finded that Y chromosome has a role on determination of male sex.

Figure 1: Y chromosome and gene regions (Kido T., & Lau YFC. (2015). Roles of the Y chromosome genes in human cancers. Asian Journal of Andrology, 17(3), 373–80.)

Female and Male Sex Formation

SRY (Sex Determination region on Y gene) The end of last century SRY gene (Sex Determination region on Y gene) isolation was occured on Y chromosome. SRY gene is responsible for the development of testes which is located on short arm of Y chromosome and in a position very close to the region called pseodoautosomal region 1 (PAR1). PAR1 is the region where the crossing over event frequently occurs during the prophase I of the meiosis division and sometimes the SRY also participates in this gene exchange. For this reason the embryo will develop as a female because it has lost the SRY gene even though it has XY chromosome and an embryo, which has XX chromosome, will develop as a male to gains SRY gene (Seda O and et. al., 2006). SRY is in the nuclei of support cells of undifferentiated gonads. First six week period in human is undifferentiated stage in which biopotential Gizem KÖPRÜLÜLÜ • 169 organ structures are shaped. In this process germ cells have not differentiated (Hassa O. and Aştı RN., 2003). SRY will start differentiation. After sexual act in humans, SRY becomes active in 6th week. When this chromosome part loosenes, the relevant gene part is stimulated for transcription and sport cells in undifferentiated gonads in XY living transform Sertoli cells. Sertoli cells stimulates the conversion of germ cells in gonad draft to spermatogoniums and product the MIF (Mullerian Inhibiting Factor) which causes atrophy of Müllerian channel and provides the conversion of the mesenchymal cells to the Leydig cells which actively secrete testosterone. Thus testes are formed to biopotential gonads under the control of SRY. SRY gene synthesizes TDF (Testis- determining factor) protein. TDF protein provides testis formation. After 7th week, with the TDF effect, gonads begin to differentiate in the direction of the testes. In the absence of SRY, gonads will automatically turn into ovariums. If TDF is not coded, ovary will occur. In human, development of sexual cells directly associated with genetic control mechanism and hormonal control mechanisms.

SOX9 (SRY Box 9)

SOX9 is a transcription factor which plays a critical role fort both sex and skeletal development. SRY gene of transient expression initiates a cascade of gene interactions orchestrated by SOX9, leading to the formation of testes from bipotential gonads (Cox et al., 2011). XX humans have an extra copy of SOX 9 develop as males although they have no SRY gene. Sox 9 protein binds ti promoter site on the Amh gene and this linkage is critical to development of male phenotype.

170 • Health Sciences Research Papers

Figure 2: Interaction of SOX 9 and Sf1 to activate the expression of the Amh gene. (A) The binding of Sox9 to the Amh promoter initiates transcription of the Amhgene in the Sertoli cells. (B) After Sox9 binds, the expression of AMH is upregulated by the binding of SF1 and Wt-1. AMH is believed to position SF1 on its DNA-binding site, and Wt-1 is joined to the Sf1 protein. (C) Gata (a transcription factor common to many cell types) upregulates Amh expression further. Neither Sf1 nor Gata can function if Sox9 is absent. (Arango N A, Lovell- Badge R, Behringer R E. Targeted mutagenesis of the endogenous mouse Mis gene promoter: In vivo definition of genetic pathways of vertebrate sexual development. Cell. 1999;99.409–419.)

Sf1 (Steroidogenic Factor 1) Sf1 plays an important role to maket he biopotential gonad. Sf1 works with Sox9 in Sertoli cells and which is needed to elevate the levels of Amh transcription ( see Figure 2; Arango et al. 1999). In Leydig cells, Sf1 activates the gene which encode enzymes that make testosterone. SRY directly or indirectly activates the Sf1 gene and Sf1 protein then stimulates and activates Sertoli-Amh and Leydig- testosterone which are both components of the male sexual differentiation pathway.

Dax 1 ( X'e bağlı antitestis geni) It is encoded by the NR0B1 gene in humans that is a nuclear receptor protein. The role of Dax 1 in sex determination is enigmatic. It plays a role to formation of adrenal gland. It is thought that DAX1 is a gene that is involved in ovary determination. Gizem KÖPRÜLÜLÜ • 171

If there is overexpression of Dax1 it can inhibit the normal functioning of Sf1 in testes development and thereby causes the sex-reversal phenotype.

Figure 3: Phenotypic sex reversal in humans having two copies of the DAX1 locus. (The Genetics Review Group. (1995). Sex Determination: One for a boy, two for a girl? Current Biology, 5(1), 37–39)

Wnt4 (Wingless-type MMTV integration site family member 4)

Wnt4 gene is critical in ovary determination. Sry respress Wnt4 expression in the genital ridge to forms testes and also promotes the Sf1 gene.

Figure 4: Mechanism of primary sex determination in mammals (Gilbert, S. F. (2000). Chromosomal Sex Determination in Mammals. Developmental Biology, 2, 1–12) 172 • Health Sciences Research Papers

FGF-9 (Fibroblast growth factor 9) FGF-9, which is expressed in embryos and fetuses, plays an important role for male sex determination, testicular cord formation and Sertoli cell differentiation.

WT1 (Wilms’ Tumor 1) WT1 is a transcription factor which regulates SRY expression during the initial stages of sex determination in humans. WT1 activates gene cascade that leads to testis differentiation. Mutations in WT1 are responsible for urogenital defects in men. DMRT-1 (Doublesex and mab-3 related transcription factor 1) It is an autosomal gene which is essential for development of the testis. DMRT-1 plays a role for differentiation of male germ cells and maintaining of male sexual cell fates of somatic gonad cells.

FOXL-2 (Forkhead Box L2) FOXL2 is a transcription factor. It is active in multiple tissues, such as eyelids, the ovaries and pituitary. FOXL2 proteib regulates the growth and division of hormone producing ovarian cells which is called granulosa cells.FOXL2 protein also plays imprortant roles to brakedown of fats, steroid hormones and harmful molecules in the ovaries and also has a role in apoptosis in the ovaries.

RSPO1 (R-spondins 1) RSPO 1 is a small family of growth factors. RSPO1 is required for Wnt4 expression in XX gonads and regulates β-catenin activation in females. Absence of RSPO1or deletion RSPO1 precludes the upregulation of Wnt4 which is necessary for ovarian development and results in the development of a male-typical coelomic vessel and migration of steroidogenic cells into the gonad. RSPO1, like Wnt4, represses male sex development with activation of β-catenin signaling pathwayn necessary for female development. Parma P. and colleagues demonstrated that first time degredation of a single gene causes to complete female-to-male sex reversal in the absence of the testis-determining gene, SRY (Parma P. Et al., 2006).

Gizem KÖPRÜLÜLÜ • 173

Figure 5: In the embryonic testis (top), SRY promotes SOX9 which induces Sertoli cell differentiation. In fetal ovaries (bottom), RSPO1, which is a direct WNT4/β-catenin pathway activator, regulates granulosa cell differentiation. Male genes are upregulated to trigger the development of testes but female genes are repressed. In contrast, when ovary develops, male genes are respressed while female genes are upregulated (Jameson et al. 2012).

INFERTILITY Infertility is defined as a condition in which a couple in the age of reproduction does not have any gestational age despite regular sexual intercourse for at least one year without any contraception. Factors that can cause infertility in female and male:  Genetic Factors  Enviromental Factors  Hypothalamic-pituitary factors  General Factors

Chromosome disorders

Translocations Translocation is a chromosome anomaly thas is defined as adherence of a missing or broken part of a chromosome to another chromosme. Reciprocal translocations can lead to reduced fertility spontaneous abortions or birth defects. Translocation lead to the production of 174 • Health Sciences Research Papers genetically unbalanced gametes and produce errors in meiosis and cell death. Males with reciprocal X-autosome translocations can exist severe spermatogenic arrest that results in azoospermia (Madan, 1983). At the reciprocal Y-autosome translocations, abnormal sex chromosome pairs and may result in abnormal spermatogenesis. Because of the X- inactivation elements have been removed from control of the translocated segment, in females, the translocated X segment is assumed to remain transcriptionally active. Robertsonian translocation observed in infertile males which is the most common translocation. Acrocentric chromosomes carry the nucleolar organizer regions (NOR) on the heterochromatic short arms. In the Robertsonian translocations that have lost their NOR, the likelihood of cell disruption and germ-cell death can increase, thus decreasing fertility. (Robez, 1986).

Klinefelter syndrome (47,XXY) Klinefelter syndrome is associated with 47,XXY that is seen on in 1000 males. It can be in all cells (full-blown trisomy) or in ‘mosaic’ form. Unless males are mosaic form, they are sterile (Rives et al., 2000). It is a syndrome caused by the fact that X is not separated from sex chromosomes during cell division. It occurs when an egg cell carrying two X chromosomes is fertilized with a normal sperm. This people,which are seen male, have long arms and legs, the feminine hip protrusions, are the first things that make a difference. His testicles are small, feminine chest (gynecomastia) and muscle development. The beard and mustache developments are minimal, and the body hair is a feminine appearance. Spermatogenesis is absent and infertile individuals.

47 XYY Male It is seen at 1/1000 frequency in male births. 47 XYY appears in paternal meiotic II separation of Y chromosome.

Turner Syndrome Turner syndrome; the absence of one of the sex chromosomes in a female. The phenotype of Turner syndromes is female; sexual organs and sex cells do not develop. They are infertile individuals. Turner syndrome occurs an egg cell that does not carry an X chromosome is fertilized with a sperm carrying an X chromosome Gizem KÖPRÜLÜLÜ • 175 or a sperm that does not carry a sex chromosome is fertilized with a normal egg cell. One of the sex chromosomes is absent in female or is in disorder. It is seen in 1/2000 live-born female babies. Karyotype is shown as 45, X or 45, XO or has different genetic variations.

47 XXX It is occured one in 1000 females. %95 of X chromosome is of maternal origin (Hassold et al., 1996). Most of 47 XXX females have normal weight, height, mental function and pre-pubertal development and have an early onset of menopause at about 30 years of age.

What is the infertility? Infertility is defined as a condition in which a couple in the age of reproduction is not using any contraceptive method and does not have gestation despite regular sexual intercourse for at least one year (Rowe and Griffin, 2002). Infertility is effected 13-18% of couple in a year. only 20% of the infertile couples, are responsible for the male factor while 30% to 40% are associated with the female factor. When today's conditions are evaluated, infertility has become a common health problem. Success in infertility treatment depends on accurate diagnosis, detailed examination and correct intervention.

Table 1: Factors affecting on fertility (WHC, 2009). Male Infertility There has been considerable progress in the treatment of male infertility with development of the medical genetics. Infertile males are assessed by physical examination in the light of laboratory tests and the etiology and causes of male infertility are defined and appropriate treatment method is determined (Pazarba, 2011). Routine testing is not recommended for all infertil males because it is waste of time and economic resources (Akdere and Burgazlı, 2013). 176 • Health Sciences Research Papers

One of the most important factors related to male infertility is such as sperm motility, the quality and quantity of sperm function and sperm production (Güney et al., 2012). Azoospermic patients with no semen in semen fluid are divided into two groups as obstructive and nonobstructive. The sperm cells produced in the testis are expelled from the penis via the carrier channels. The continuity of these channels.

Diagnostict Test For Male Infertility Semen Analysis: It must be performed according to World Health Organization (WHO) procedure. Assesment of a couple, semen analysis is mandatory (WHO,1992). After semen analysis, azoospermia and types of oligozoospermia are determined and then assisted reproductive techniques (ART) are selected for therapy. FSH,LH and testosterone measurement: Serum FSH is the most important measurement for diagnostic and prognostic. Low levels of FSH, LH, and testosterone suggest prolactinoma, hypogonadotropic hypogonadism may cause deterioration due to various causes. Operations, testis defects, sexually transmitted diseases, infections such as tuberculosis, and some genetic diseases are the most important causes of obstructive azoospermia (Obstructive Azoospermia). Nonobstructive Azoospermia, which is a azoospermia type, is not dependent obstruction. If there is no sperm in the ejaculate and this is caused by a problem of testicle, it is defined as Nonobstructive Azoospermia (NOA). Oligozoospermic patients; The number of sperm in the ejaculate is less than 5 million/ml, which is considered as a severe oligozoospermia. Genetic testing is appropriate because your pregnancy will not be normal in this group of patients and will require assisted reproductive techniques (Akdere and Burgazlı, 2013). Genetic assessment: Y chromosome defects cause infertility such as microdeletions on AZF gene. Chromosome abnormalities for example sex chromosome aneuploidies such as the 47,XXY and the 47, XYY karyotype, Robertsonian translocations and other type of defects. Gizem KÖPRÜLÜLÜ • 177

Spermatogenesis is a process for sperm production. Genes which are associated with spermatogenesis located on Y chromosome. SRY gene is tasked with differentiation of gonads and located on short arm of Y chromosome. This gene is important for testicular formation but it is not enough for testicular development. AZF gene has also a role in spermatogenesis process. When a defect occurs on AZF gene regions which is AZFa, AZFb and AZFc, this results in sperm production problems. For this reasons genetic assesment is important to detect male infertility. By molecular analysis and methods, gene defects are detected.

TERMINOLOGY NUMBER OF SPERM (million/ml) Azoospermia 0 Severe Oligozoospermia < 1 million Moderate Oligozoospermia 1-5 million Slight Oligozoospermia 5-20 million Normal >20 million

Table 2: Classification of oligozoospermia (Poongothai et. al., 2009) The main pathologies leading to male infertility are: 1- Microdeletions of Y chromosome 2- Chromosomal anomalies 3- Genetic mutations 4. Changes in gene expression profile

Assisted Reproductive Techniques in Male Infertility Treatment Microinjection (ICSI) is the direct introduction of spermatozoa into an oocyte to achieve fertilization and pregnancy when there are few or no spermatozoa in the ejaculate. In the latter case, ICSI can be administered using testicular spermatozoa extracted directly from the tissue or obtained from the epididymis. Furthermore, spermatide injection technique can be applied in oocytes and first-term pregnancies can be obtained in humans. Although ICSI has spread worldwide in recent years, there are potential risks arising from random use of the ICSI only in recent periods. These concerns have recently emerged as a result of recent advances in genetically determined male infertility. ICSI is more fearful in transmitting genetic anomalies for emerging offspring than other forms of supported 178 • Health Sciences Research Papers reproductive techniques. Because an active spermatozoa which is needed to start all of mechanism for oocyte penetration an normal capacitation and to be exposed to the acrosome reaction, skip all physiological steps associated with fertilization. With the skipping of these steps, the ICSI permits egg fertilization of a modified spermatozoon. This will increase the risk of genetic damage. Mulhall et al reported for the first time that they obtained fertilization and pregnancy using ICSI with testicular spermatozoa from azoospermic patients with deletions in the DAZ region. This suggests that the spermatozoa carrying the deleted Y chromosome is completely effective on fertilization. Subsequently, the same group reported boys birth through an ICSI from a male with AZFc deletion, and they obtained similar findings as a result of their work in other investigators. Following Mendel's probabilities, all of the boys inherit the Y chromosome which has deletion coming from their father. In vitro fertilization (IVF) is a technique based on synthetic fertilization of an egg by sperm in vitro conditions. IVF is an important method of treatment when other assisted reproductive techniques in infertility treatment are unsuccessful. Summarize IVF operation; to follow a woman's ovulation process, to obtain eggs from ovaries and to fertilize them with sperm in in vitro conditions in the laboratory, to transfer the fertilized egg, called zygote, to a successful pregnancy in the uterus. Spermatozoa carrying a deleterious Y chromosome also transmits deletions to male children through in vitro fertilization (IVF). In fact, this indicates that the spermatozoan obtained from an oligozoospermic test subject which has deletion in the long arm of the Y chromosome can fertilize the oocyte under in vitro conditions. The acrosome reaction can be performed when sperm which carries a deletion that affects all of characteristic enter the oocyte for fertilization. Very rarely, there are findings to support that in spontaneously developing pregnancies Y chromosome deletions transfer from the father to the son.

Female Infertility Ovulation disorders, uterine abnormalities, tubal obstruction and peritoneal factors can cause female infertility. Cervical factors are also play a minor role for female infertility. Gizem KÖPRÜLÜLÜ • 179

Previous use of contraception, intake alcohol, tobaco use, medication use, previous surgery on reproductive organs, previous pregnancies and pelvic infections can cause female infertiliy. According to the Center of Disease Control (CDC, 2013) there are three categories for causes of female infertility. These categories are defective ovulation, transport and implantation. Female

1. Defective Ovulation Endocrine disorders: In case of dysfunction of hypotalamus and pituitary gland, prolactin can be secreted in excess amount and this may prohibit ovulation. Adrenal glands and thyroid glands may also decelerate ovulation. Physical disorders: Obesity, anorexia, nervosa and excessive exercise may cause overweight and malnurition and menstrual cycle. Ovarian disorders: Polycystic ovarian disease (PCO) causes female infertility Because of an increased amount of LH and decrease intake glucose. Low FSH levels damage the production of eggs from ovarian follicles. 2. Defective Transport Ovum: Occurrence of Pelvic Inflammatory Disease (PID), previous tubal surgery, peritonitis can cause tubal obstruction Scar tissue after abdominal surgery: In this case can cause altering the movement of ovaries, fallopian tubes and uterus, resulting infertility. Sperm: Vaginismus, dyspareunia may damage fertilization Cervix: Surgery, infections, trauma in cervical mucus may delay pregnancy. 3. Defective Implantation Congenital anomaly and fibroids: Bicornuate uterus and uterine fibroids may alter implantation of the zygote and cause infertility

180 • Health Sciences Research Papers

Table 3: Etiology and Evaluation of Infertility

Assisted Reproductive Techniques (ART) in Female Infertility Treatment Women with endometriosis are usually teated with ovarian suppression either with GnRH agonists or with danazole, medroxyprogesterone acetate, oral contraceptives, and gestrinone (Crosignani and Rubin, 1996). Tubal obsturction, ovarian endometriomas, adhesions are associated with severe endometriosis and surgical treatment has an important role for therapy. Infection or inflammation of vagina or cervix are treated with medical therapy. Electroresection or laser surgery is recommended in this case. In case of negative (abnormal) Post-coital test (PCT) intrauterine insemination (IUI) is recommended to patients. Uterus abnormalities such as intrauterine septa or submucosal fibroids should be treated by hysteroscopic electroresection. Myomas must be done by macrosurgery. Gizem KÖPRÜLÜLÜ • 181

Premature Overian Failure (POF) is a table with caracterizing impairment of over function before age 40 years, seconder amenorrhea and elevated gonadotropin levels and consequent decline in blood estrogen levels. POF; in young women aged 40 years are occurred with 4 months process of amenorrhea, by detecting follicle-stimulating hormone (FSH) serum levels of 40 IU / L and decreased in sex steroids. Adoption or ovum donation could be an option for these couples ( Forti and Krausz, 1998). Women with normogonadotropic anovulation have normal FSH Level but LH level may be increased. In obese patient, treatment is an antiestrogen such as clomiphene citrate or tamoxifen combined with diet. If women is not response the clomiphene, laparoscopic ovarian electrocautery can be proposed (Dunaif et al., 1996). Most commonly used thearpy is application of pulsatile GnRH or human purified recombşnant FSH and following hCG or an ART. Hypogonadotropic anovulation should be recommended for underweight patients (BMI< 19). In case of primary pituitary failure is peresent, ovulation may be induced with gonadotropins.

Figure 6: Algorithm for infertility evaluation (Tammy et al., 2015).

182 • Health Sciences Research Papers

After couples are evaluated, treatment models are explained. This models are select according to couples problems. This treatment modalities are: Intra-uterine Insemination (IUI): This model generally could be used for female infertility, unexplained infertility and mild male factor infertility problems. Normal (healthy) sperms are collected and placed directly in the uterus on the time of ovulation. In vitro Fertilization (IVF): Mature eggs are collected from woman and fertilized with healthy sperm outside the womb and inside a laboratory. After fertilization, fertilized embryos are implanted in the uterus. Zygote Intra-fallopian Transfer (ZIFT): Fertilized egg is transfered into fallopian tube. Gamete Intra-fallopian Transfer (GIFT): Sperms and eggs are mixtured and mix are placed into the fallopian tube and fertilization occurs in fallopian tube. Donor Eggs and Sperms: This model uses for married couple’s who have problems on their egg and sperms. Donor sperms or embryo are taken for enhancing fertility. Gestational Carrier: This is called as surrogate pregnancy. If woman have not uterus or uterus are not functional and her pregnancy can endanger health, in this situation couple can decide to have gestational carrier. Adoption: This is an option for infertile couples. If couples have multiple unexplained IVF failure cycles, they can prefer this option.

Gizem KÖPRÜLÜLÜ • 183

REFRENCES 1. Crosignani PG, Rubin B. (1996). The ESHRE Capri Workshop. Guidelines to the prevalence, diagnosis, treatment and management of infertility.Hum Reprod. 11:1775–1807 2. Hanley NA., Ball SG., Clement-Jones M., Hagan DM., Strachan T., Lindsay S.,Wilson, DI. (1999). Expression of steroidogenic factor 1 and Wilms’ tumour 1 during early human gonadal development and sex determination. Mechanisms of Development, 87(1–2), 175–180. https://doi.org/10.1016/S0925-4773(99)00123-9 3. Hassa O., Aştı RN. (2003). Embriyoloji.133–134. Yorum Basın Yayın Sanayi Ltd Şti, Ankara 4. Jameson SA., Lin YT. & Capel B. (2012). Testis development requires the repression of Wnt4 by Fgf signaling. Developmental Biology 370 24–32. doi:10.1016/j.ydbio.2012.06.009 5. Jost A. (1972). A new look at the mechanisms controlling sexual differentiation in mammals. Johns Hopkins Med. J., 130: 38-53. 6. Kobayashi A., Behringer RR. (2003). Developmental genetics of the female reproductive tract in mammals, Nature Reviews Genetics, 4: 969–980 7. Madan K. (1983). Balanced structural changes involving the human X: effect on sexual phenotype Human Genetics 63 216–221s 8. Matson C. K., Murphy, M. W., Sarver A. L., Griswold M. D., Bardwell, V. J., & Zarkower, D. (2011). DMRT1 prevents female reprogramming in the postnatal mammalian testis. Nature, 476(7358), 101–105. https://doi.org/10.1038/nature10239 9. Pannetier M., Chassot AA., Chaboissier MC., & Pailhoux E. (2016). Involvement of FOXL2 and RSPO1 in Ovarian Determination, Development, and Maintenance in Mammals. Sexual Development. S. Karger AG. https://doi.org/10.1159/000448667 10. Parma P., Radi O., Vidal V., Chaboissier MC., Dellambra E., Valentini S., Camerino G. (2006). R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nature Genetics, 38(11), 1304–1309. https://doi.org/10.1038/ng1907 11. Rives N, Joly G, Machy A, Sim´eon N, Leclerc P and Mac´e B (2000). Assessment of sex chromosome aneuploidy in sperm nuclei from 47,XXY and 46,XY/47,XXY males: comparison with fertile and infertile males with normal karyotype Molecular Human Reproduction 6 107– 112 184 • Health Sciences Research Papers

12. Robez Z. (1986). Meiotic association between the XY chromsomes and the autosomal quadrivalent of a reciprocal translocation in 2 infertile men, 46XY t(19:22) and 46XY t(17:21) Cytogenetics and Cellular Genetics 43 154. 13. Schambony A., Kunz M., & Gradl D. (2004). Cross-regulation of Wnt signaling and cell adhesion. Differentiation. Blackwell Publishing Ltd. https://doi.org/10.1111/j.1432-0436.2004.07207002.x 14. Seda O., Liska F., Sedova L. (2006). Sex Determination. In: Multimedia E- textbook of Medical Biology, Genetics and Genomics. Czech Republic, Prague. 15. Spiller C., Koopman P., & Bowles J. (2017). Sex Determination in the Mammalian Germline. Annual Review of Genetics, (August), 265–285. https://doi.org/10.1146/annurev-genet-120215-035449 16. Tagliarini E. B., Assumpção J. G., Scolfaro M. R., de Mello M. P., Maciel- Guerra A. T., Guerra Júnior G., & Hackel C. (2005). Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis. Brazilian Journal of Medical and Biological Research, 38(1), 17–25. https://doi.org/10.1590/S0100- 879X2005000100004 17. Troedsson H. T., Madill S. (2004). Pathophsiology of the Reproductive System. 214–220. In: R. H. Dunlop, C. H. Malbert (Ed), Veterinary Pathophysiology. 18. World Health Organization. (1992). WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 3rd ed. Cambridge, UK: Cambridge University Press; 44–45.

INVESTIGATION OF THE PERCEPTION / THE ATTITUDE LEVELS OF DIFFERENT GENERATION FREE / INDEPENDENT PHARMACISTS FOR PHARMACEUTICAL CARE: ADIYAMAN CASE CHAPTER 12

Gulsen KIRPIK1, Erkan YILMAZ2, Sezen DEMIR3

1 Dr., Adiyaman University, Pharmacy Faculty, Pharmacy Management, [email protected] 2 Dr., Adiyaman University, Pharmacy Faculty, [email protected] 3 Adiyaman University, Pharmacy Faculty Senior Student, [email protected]

Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 187

INTRODUCTION Pharmaceutical care is a newly introduced concept in pharmacy and it is a patient-oriented pharmacy service based on providing information and consultancy services by the pharmacist for the patient to make the best use of the drugs, monitoring the drug treatment and taking care of the patient's health primarily by the pharmacist. Therefore, there are not many studies on this subject. Although the term pharmaceutical care has been defined long ago; The term has been misinterpreted by most pharmacists. In fact, Hutchinson and Schumock (1994) stated that if each pharmacist defines pharmaceutical care for their short-term or political purpose, pharmaceutical care will fail. The misinterpretation and interpretation of the term may also vary by different generations of pharmacists. It is common for different generations of pharmacists with different characteristics, attitudes and beliefs to be different from each other in terms of birth years, and it is expected that the level of pharmaceutical care perception that is the subject of the study will differ from different generations. In this context, the aim of this study is to determine the positive or negative attitudes of different generations of independent pharmacists in the province of Adiyaman towards pharmaceutical care. Although there are a limited number of studies on pharmaceutical care in the literature, no other study comparing the attitudes of pharmaceutical pharmacists to different generations has been found. Therefore, this study is important in terms of revealing the positive or negative direction of perceptions / attitudes of independent pharmacists towards pharmaceutical care and whether the perceptual / attitudes differ significantly in terms of both generational differences and socio-demographic variables such as gender and seniority level. In the light of the findings to be obtained, suggestions for independent pharmacists and other interests will be developed, and the fact that it is a study to guide other interested researchers and contributing to the knowledge in the literature is among the secondary objectives of the study.

1. Pharmaceutical Care Pharmaceutical care has become an important topic in the world and in our country, especially in recent years. Article 4 (g) of the Guide to Good Pharmacy Practices, prepared in accordance with Article 50 of the Regulation on Pharmacists and Pharmacies; It is defined as a patient- 188 • Health Sciences Research Papers oriented pharmacy service based on providing information and consultancy services by the pharmacist in order to make the best use of the drugs, monitoring the drug treatment and taking the health care of the pharmacist first. The concept of pharmaceutical care accepted and applied in many countries is a new concept in terms of pharmacy in our country. The quality of pharmacy services continues to change in Turkey as well as in the whole world, and the scope of pharmaceutical care and clinical pharmacy services in the pharmaceutical services offered in pharmacies in Turkey is also to include developing intensively. However, there is no routine pharmaceutical care programs registered for the relatively new concept for Turkey. In a study conducted in Turkey in terms of good pharmacy practice; It was reported that the pharmacy practices in the free pharmacies in the region where the research was conducted were insufficient (Ertuna & Arun, 2019: 21). Probably, the earliest published use of the term drug care was made by Brodie (1973 and 1980) in the context of considerations about drug use control and drug-related services. It is a widely used term in the pharmacy profession, and is a term that has been widely written and discussed about, especially after the article published by Hepler and Strain in 1990 (URL-1; 19.06.2019). Pharmaceutical care is defined as the provision of specific (specific to patient / individual) drug treatment by the pharmacist in order to achieve precise results that improve the quality of life of a patient by Hepler and Strand (1990: 533), Franklin and Van Mill (2005), and the American Society of Health Pharmacists-ASHP (URL-1; 19.06.2019: 331). Hepler and Strand stated that it is not enough for pharmacies to distribute the right drug or to provide complex pharmaceutical services, or to develop new technical applications. It was also emphasized that pharmacists should adopt patient- centered pharmaceutical care as their working philosophy. Directing the focus of the study from products and biological systems to the best drug treatment and patient safety will increase the pharmacist's responsibility and will require philosophical, organizational and functional changes. In 1998, the International Pharmaceutical Federation (FIP) defined pharmaceutical care (PCP) as the responsibility of providing pharmacotherapy to achieve conclusive results that maintain or improve the patient's quality of life. It is a collaborative process that identifies, prevents or solves medical product and health problems. It is a continuous quality improvement process for the use of medicinal products. The Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 189 definition of pharmaceutical care by FIP is a slightly modified version of the most commonly used definition of pharmaceutical care in Hepler and Strand (1990) (Suleiman & Onaneye, 2011: 92). According to Hepler and Strand (1990: 533), pharmacists and institutions should begin to direct their energy to greater social benefit rather than their internal affairs. According to the FDA (Food and Drug Administration) report, in 1987 there were 12,000 deaths and 15,000 hospitalized patients due to some drug reactions (ADRs). Drug-related morbidity and mortality can often be prevented, and pharmaceutical services can reduce the number of ADRs, length of hospital stay, and maintenance costs. Therefore, there is a need to establish new working standards, to establish a cooperative relationship with other healthcare professionals and to determine strategies for pharmaceutical care. The re- professionalisation of the pharmacy will only be completed when all pharmacists accept the social duties of the individual patient to ensure safe and effective drug treatment. Clark et al. (2007) reported that clinical compliance of pharmaceutical pharmacists increased patient compliance and reduced prescription errors. Pharmaceutical care was examined in three main aspects, respectively:  First, the patient's pharmaceutical care needs should be identified.  Following this, a patient-specific pharmaceutical care plan should be prepared taking into account the patient's knowledge, general health belief and motivation.  Finally, the targeted results in the care plan should be evaluated. By the other; As best known and recommended by Hepler and Strand (1990), the pharmaceutical care program consists of the following steps:  Establishing the pharmacist-patient relationship,  Patient data collection, analysis and interpretation,  Finding drug-related problems,  Determination of therapy results and targets with the patient,  Identifying possible pharmacotherapy alternatives and selecting the best plan, 190 • Health Sciences Research Papers

 Preparation of a monitoring plan,  Implementation of individual pharmacotherapy and monitoring the plan,  Following. Although the program have been shown above and a pharmaceutically acceptable in general, Turnaci et al (2009), as indicated by the pharmaceutical care is a relatively new concept in Turkey; yet, there are no registered routine pharmaceutical care programs. However, some of the studies in the literature on pharmaceutical care are briefly summarized below. In a study conducted by Cordina et al. (1999) in Malta, it was stated that 72% of independent pharmacists were eager for pharmaceutical care. In addition, the factors that prevent pharmaceutical care are listed in order of importance, such as reimbursement conditions, lack of time, the need for qualified support personnel, and inadequate communication between physicians and pharmacists. In a study by Uema et al. (2008) on pharmacists working in different work environments in Argentina, the most important obstacle to pharmaceutical care was seen as lack of time. In addition, the lack of training in pharmaceutical care, the lack of patient communication skills and the lack of sufficient space in the pharmacy for pharmaceutical care were among other barriers. Suleiman and Onaneye (2011) surveyed 120 hospital and pharmacy pharmacists to evaluate the attitudes, perceptions, knowledge and practices of pharmacists in Ogun state. 92.2% of respondents were aware of the concept of pharmaceutical care, but only 22.9% identified it correctly. In this study, it was determined that pharmacists had good attitude towards pharmaceutical care but did not know how to apply pharmaceutical care. Sancar et al (2013) study carried out by the perspectives are evaluated regarding the application of pharmaceutical care pharmacists in Turkey. In this context, the questionnaire was administered to 385 pharmacists who participated in the Clinical Pharmacy and Pharmaceutical Care Continuing Education Program which organized by the Academy of Turkish Pharmacists Association. It was found that 97.3% of pharmacists wanted to provide pharmaceutical care services and 83.5% thought that this service was a duty of pharmacist. The major barriers to pharmaceutical care services were the lack of technical knowledge of how to provide Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 191 pharmaceutical care, lack of knowledge of medicine and disease, lack of communication with clinicians, and government workload. In the studies reviewed above, it was found that the difficulties encountered in providing pharmaceutical care were the difficulties encountered in accessing medical history, clinical and laboratory data of patients, as well as lack of materials, equipment, pharmacotherapy / therapeutic information and motivation (Farris, Fernandez-Llimos and Benrimoj, 2005; Ngorsuraches and Li, 2006; Uema et al., 2008). Kiran and Taskiran (2015: 164) stated that the reflections of the understanding of the importance of clinical pharmacy in the world are gradually being felt in our country. According to the findings of the study conducted between the first year students of Ege University Faculty of Pharmacy, one third of the students considered free pharmacy as a field of study after graduation, and about one fifth thought of clinical pharmacy, instead of intense orientation to the field of free pharmacy in the previous years; the interest of clinical pharmacy, industrial pharmacy, academic started to shift to areas such as pharmacy. In addition, in another study conducted by Kiran (2015: 222), it was stated that 275 vocational training programs were organized by Turkish Pharmacists Association Pharmacy Academy between 2009-2013; the highest level of education was provided in 2010 (25.5%). Among the education subjects, pharmaceutical care and disease information subjects were predominantly (56.7%). On the other hand, with the support of the Ministry of Health (URL-3, 2019), training programs that increase pharmacists' knowledge of pharmaceutical care and clinical pharmacy and define and realize the role of pharmacists in the treatment of different diseases are continuing. Ertuna and Arun (2019: 26) stated that free pharmacies can communicate with patients frequently and easily. In addition, due to the immediate access to prescription information and the immediate access to the times when medicines need to be reprinted, they have a unique place in the healthcare system because they are places where reminders can be made and where patients can receive support, such as training, adherence to treatment, without appointment. However, in pharmacies in Turkey, many of which are associated with chronic illness and medicine as presented in informal format, the quality of pharmaceutical care services, the content of pharmaceutical 192 • Health Sciences Research Papers care services and of how the provision of pharmaceutical care services, it is stated that a very limited number of studies found. It is observed that the studies conducted in the literature are generally directed to the level of knowledge about pharmaceutical care, awareness, positive / negative point of view and the reasons that prevent pharmaceutical care. In general, pharmacists are familiar with the definition but do not fully understand the concept of pharmaceutical care, but are eager to provide pharmaceutical care. However, there is no study examining the level of pharmacists' perception of pharmaceutical care in terms of generational or socio-demographic variables such as gender and seniority. Therefore, since this study is the first study in this field, it is thought that it will guide both the accumulation of knowledge in the literature and other researchers.

2. Generation Concept and Characteristics of Different Generations On the official website of the Turkish Language Association; The group of individuals that make up the age groups of approximately twenty-five to thirty years is defined as group of individuals, belly, abdomen, generation (URL-3, 2019). When the concept of generation is taken together with other definitions, the generation can be defined as the average time interval between the births of parents and their children, and people with similar behaviors that have been affected by similar economic and social movements (Goksel & Gunes, 2017: 809). In the study conducted by Gurbuz (2015: 40), it is stated that the perception and belief that the attitudes and values of the generations are different are kept on the agenda especially by a certain group of researchers, educators, management consultants and gurus. In addition, according to this belief, it is stated that the basic values of each generation in working life, their attitudes towards work and organization differ. It was stated by Aksu (2015) that experiences in business life shaped the professional life and that gender, education, socio-economic level and world view affect the opinion and communication style. However, it was emphasized that one of the important factors in shaping perceptions and behaviors was when we were born and raised. This reveals the importance of “generation” differences. Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 193

A particularly important difference for the generations is that it involves very striking events in which one generation lives but another generation either does not live or lives outside critical years of socialization. As each generation matures with such events, the characteristics that distinguish it from the predecessors and the latters develop (Aksu, 2015: 63). In this study, 6 generations, which were formed by Andrea, Gabriella and Times (2016), are considered in the classification of the generations in chronological terms. These are the Traditionalist Generation (1925-1945), Baby Burst Generation (1946-1960), Generation X (1961-1980), Generation Y (1981-1995), Generation Z (1996-2010) and Alpha Generation (2010 +). Information about the characteristics of some of these generations in today's business world and important developments in their lives are given below. Table 1. Characteristic features of generations in the world literature and important developments in their lifes

The Birth Years of Traditionalist Baby Burst Generation Generation X (1965- Generation Y (1981- Generations Generation (1946-1964) 1980) 2000) (1925-1945) Important Car Television Personal Computer Tablet / Smartphone Productions Communication Written Letter Phone E-mail / Message Social Media Ways General Practical Optimist Skeptical Hopeful / Optimistic Appearance Professional Ethics Dedicated to Work Ambitious Balanced Pretentious Affected Leader Hierarchy Consensus Competence / Skill Success Style Attitude in Self-forgetful Satisfactory Reluctant to be loyal Low Loyalty Relationships “A good job Awards in the satisfaction is “Money, title, “Freedom is the “Do your job with Workplace provided by job appreciation” highest reward.” purpose.” security.” Perspectives on “I learned the “If you train too much, “The more they learn, “Continuous learning is Education in the hard way, and you they'il leave the the longer they stay.” a way of life.” Workplace can do it.” office.” 1967: Che Guevara's 1989: The Fall of the 1950: The emergence murder Berlin Wall 1929: Economic of television 1968: Assassination of 1981: MTV Depression 1955: Civil Rights Martin Luther King 1984: AIDS 1930: Cinema Movement 1968: Sorbonne 1986: Challenger Important Events in 1940: World War 1957: Launch of the University Rebellion Disaster the World II Soviet Union's Sputnik 1969: Landing on the 1995: Oklahoma City 1945: The Korean satellite into space First Moon attack War 1960: The Vietnam 1969: Woodstock 1990s: Clinton-Lewinsky 1930-1960: War 1970: Women's scandal Labour Unions 1962: The Cuban freedom movement 1999: Columbine High Missile Crisis 1973: Watergate and School massacre 1963: The the energy crisis 1980s-90s: The assassination of John 1976: Tandyand Apple emergence of the Internet F. Kennedy Personal Computers and ESPN Source: Kelgokmen & Yalcin, (2017). 194 • Health Sciences Research Papers

Since the events seen in the above tables will affect the same generation members who were born and raised at the same time, the same generation members will have similar characteristics. Aksu (2015) stated that these experiences shared with peers during a certain period - historical, cultural and social - have an impact on perspective and perception. Especially social events and important social and political events affect the characteristics of generations. However, since it is not clear which important events affect more people or societies, there are differences in the date ranges especially in the generation classifications. Therefore, generations are not strictly divided, but in general the characteristics of the generations are sufficient for all age groups (Andrea et al., 2016: 92). The aim of this study is to determine whether positive or negative perception levels of pharmacists of different generations working as free / independent pharmacists differ according to generation differences. In addition, it will be examined whether the perception levels of pharmaceutical care differ in terms of socio-demographic variables such as gender and seniority of independent pharmacists.

3. Research In this part of the study, the aim of the research, hypotheses, population and sample, analysis method, reliability of pharmaceutical care scale are given.

3.1. Purpose and Limits of Research The aim of this study was to determine the positive or negative perception levels of different generations of free / indeendent pharmacists in Adiyaman. In addition, the second objective of the study is to examine whether the perception levels of pharmaceutical care differ in terms of other socio-demographic variables that independent pharmacists have. On the other hand, according to the results of this study, comparisons will be made with the results of other similar studies and evaluations will be made and other researchers will be guided. This study only deals with a small sample of independent pharmacists operating in Adiyaman province. The findings may not be generalized for independent pharmacists in other regions and provinces where socio- demographic, technological, cultural and economic characteristics differ. Because the variables that affect the pharmacist's perception of pharmaceutical care may vary between regions. Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 195

In addition, there is a limitation of the study whether the free pharmacists participating in the study understand the questionnaire questions correctly and in the same direction. However, assuming that the participants understand the questions correctly and in the same direction, it is assumed that the answers given reflect the real situation.

3.2. Research Hypotheses The hypotheses developed depending on the purpose of the research are given below.

1. H0: The level of perception of pharmaceutical care does not differ significantly in terms of generational status of free pharmacists.

2. H1: The level of perception of pharmaceutical care differ significantly in terms of generational status of free pharmacists.

3. H0: The level of perception of pharmaceutical care does not differ significantly in terms of gender status of independent pharmacists.

4. H1: The level of perception of pharmaceutical care differ significantly in terms of gender status of independent pharmacists.

5. H0: The level of perception of pharmaceutical care does not differ significantly in terms of seniority level of independent pharmacists.

6. H1: The level of perception of pharmaceutical care differ significantly in terms of seniority level of independent pharmacists.

3.3. Population and Sample The population of the study is the free pharmacists in Adiyaman. There are 164 free pharmacists in Adiyaman (http://www.adiyamaneo.org.tr/Eczaneler.aspx, 25.10.2019). The sample was consisted of 80 volunteer free pharmacists selected by simple random method. 48% of the population was reached. In terms of the adequacy of the sample, the information given by Yazicioglu and Erdogan (2004) and the time and cost constraints were taken into consideration and the sample was found to be sufficient. 196 • Health Sciences Research Papers

3.4. Data Collection and Analysis Method The research was carried out on the individuals who made free pharmacy in Adiyaman province and districts. Face-to-face survey method was used to determine the positive or negative differences in the attitude / perception of pharmaceutical care in terms of generational status of the free pharmacists participating in the study. First of all, a pilot application was made for the questionnaires prepared. As a result of the pilot application, the reliability level of the pharmaceutical care attitude / perception scale was analyzed and it was decided to remove some questions from the scale by factor analysis. At the beginning of the study, reliability and factor analysis were performed on the scale which consisted of 14 questions. After the factor analysis, it was decided to exclude the 4 questions from the scale which had a low variance rate (<0.50). The study continued with 10 questions. The questionnaire used in the study consisted of questions about demographic characteristics (3 questions) of the free pharmacist such as sex, birth years interval and seniority level, and 10-item pharmaceutical care attitude / perception scale variables. Sancar et al. (2013) and Suleiman & Onaneye (2011) and the opinions of academicians were used in the establishment of the scale of pharmaceutical care perception. Likert-style scales were used to measure the perception of pharmaceutical care. In this context, it was prepared with a 5-point Likert-type scale ranging from 1 “Strongly Disagree” to 5 “Strongly Agree”. The questionnaire was applied between March-April 2019. SPSS 22.0 package program was used to analyze the data set and the research hypotheses were analyzed.

3.5. Reliability of the Scale The Cronbach's Alpha coefficient of the Pharmaceutical Care Attitude / Perception Scale used in this study was calculated and its reliability value was reached. Table 2. Reliability Analysis Application Test Type Result Pharmaceutical Care Attitude Scale Cronbach’s Alpha (10) 0,899

As can be seen in Table 2, the Cronbach's Alpha value of the Pharmaceutical Care Perception Scale consisting of 10 variables was found Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 197 to be 0.899. According to the information provided by Kalayci (2016), it can be said that the scale is highly reliable.

3.6. Research Findings In this part of the study, descriptive statistical findings related to birth intervals, gender and seniority level and pharmaceutical care perception levels showing the generational status of the free pharmacists participated in the study are included.

3.6.1. Descriptive Findings The distribution of socio-demographic characteristics such as birth intervals, gender and seniority level of the free pharmacists participating in the study are shown in the table below. Table 3. Descriptive Findings

Socio-Demographic Characteristics n % Female 31 38,8 Gender Male 49 61,2 Total 80 100,0 Baby Burst Generation (1946-1960) 7 8,8 Generation Status (Birth Generation X (1961-1980) 24 30,0 Intervals) Generation Y (1981-1995) 49 61,2 Total 80 100,0 0-5 years 14 17,5 6 years -10 years 13 16,2 Seniority Level 11 years -15 years 27 33,8 16 years and over 26 32,5 Total 80 100,0

It was seen that 61.2% were male and 38.8% were female pharmacists, and 61.2% were from the Y generation, 30% were from the X generation and 8.8% were from the Baby Burst generation related with free / independent pharmacists participated in the study. In addition, it has been seen that 33.8% of them had seniority between 11-15 years, and 32.5% of them had 16 years and over, and 17.5% of them had 0-5 years, and 16.2% of them had seniority level between 6-10 years. When the statistical results of the descriptive variables were evaluated, it was observed that the majority of the free pharmacists participating in this study had male (61.2%), Generation Y (61.2%) and seniority level over 10 years (66.3%).

198 • Health Sciences Research Papers

3.6.2. Findings Regarding Pharmaceutical Care Perception Levels of Free Pharmacists Participating in the Research In this part of the study, the findings obtained for defining the level of pharmaceutical care perception of the independent pharmacists participating in the research are given. The intervals in Table 4 were used for the scoring of the answers given. The intervals were assumed to be equal, and the score range for the arithmetic means was 0.80. The evaluation range of the arithmetic means of the answers given to the questions in the scale of pharmaceutical care perception is given in the table below. Table 4. Value Ranges of Perceptual Means Interval Option 1,00-1,80 Totally Negative 1,81-2,60 Rarely Negative 2,61-3,40 Neutral (Unstable) 3,41-4,20 Mostly Positive 4,21-5,00 Always Positive The ranges of values given in Table 4 were used to define the average perceptions of the pharmacists involved in the study. Accordingly, both the average and perceptual average of each variable of the pharmaceutical care scale of the participating pharmacists are defined in Table 5 below. Table 5. Identification of Mean Values of Pharmaceutical Care Perceptions of Participating Free Pharmacists

Std. Definition of Pharmaceutical Care Attitude / Perception Scale N Mean Deviation Mean Perception Pharmacists in Adiyaman provide pharmaceutical 80 2,69 ,86557 Neutral (Unstable) care. The main responsibility of pharmacists is to apply 80 3,31 1,10915 Neutral (Unstable) pharmaceutical care. The application of pharmaceutical care is 80 4,00 ,99365 Mostly Positive important. I offer pharmaceutical care to my patients. 80 3,53 ,96710 Mostly Positive Providing pharmaceutical care is professionally 80 4,04 ,93381 Mostly Positive beneficial. Pharmaceutical care reduces the cost of 80 3,54 1,37743 Mostly Positive medication. Pharmaceutical care benefits pharmacists. 80 4,14 ,95126 Mostly Positive Pharmaceutical care improves patient health. 80 4,25 1,01258 Mostly Positive The pharmaceutical care app benefits your 80 4,20 1,01133 Mostly Positive professional pharmacy career. Providing pharmaceutical care is worth the 3,90 ,97565 80 Mostly Positive additional workload in the sector. General Attitude Towards Pharmaceutical Care 80 3,76 ,74402 Mostly Positive Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 199

Considering the above table, when the means of all variables were examined, it was seen that free pharmacists always had a very high positive attitude (4.25> 4.20) in terms of improving the health of the patient. Furthermore, in terms of the importance of pharmaceutical care, the delivery of pharmaceutical care, the reduction of drug costs, and other benefits (> 3.40), it was observed that free pharmacists were often in a positive attitude. However, the independent pharmacists who participated in the study showed a neutral (unstable) attitude regarding the variables that pharmacists in Adiyaman performed pharmaceutical care (2.69 <3.40) and that pharmacists were primarily responsible for applying pharmaceutical care (3.31 <3.40). However, as a result of the analyzes, it was determined that the free pharmacists in Adiyaman had a generally high level of positive attitude towards pharmaceutical care (3.76> 3.40).

3.6.3. Testing Hypotheses Kolmogrov-Smirnov Test was applied to the data in order to decide which parametric or non-parametric tests to be applied to test the research hypotheses. As a result of the normality test, it was found that the data did not show a normal distribution (p≤0.01). Therefore, Mann-Whitney U and Kruskal Wallis H tests, which are non-parametric tests, were used to test the research hypotheses. Testing the research hypotheses is shown below respectively.

3.6.3.1. Testing Mean Rank Value Attitudes / Perception Differences for Pharmaceutical Care in terms of Generation Status of Independent Pharmacists Kruskal Wallis H test was used to determine whether there is a significant difference between the levels of perceived pharmaceutical care in terms of generation status of the free pharmacists participating in the study. Table 6. Analysis of Pharmaceutical Care Attitude / Perception Differences According to Generation Status of Free Pharmacists by Kruskal Wallis H Test

Generation Mean Chi- Asymp. N df General Average Status Rank Square Sig. Pharmaceutical Baby Burst 7 13,71 10,842 2 ,004* Care Attitude / Generation Perception Generation X 24 40,04 Generation Y 49 44,55 200 • Health Sciences Research Papers

As a result of Kruskal-Wallis Test analysis of the attitude towards pharmaceutical care for different generation of pharmacists, it was determined that there was a very high level of difference between the attitudes in 99% confidence interval (p = 0.004 <0.01). According to this, the free pharmacists with the highest positive attitude towards pharmaceutical care in Adiyaman were the free pharmacists from Generation Y, which is the youngest generation as of today; the lowest positive attitudes were the free pharmacists from the Baby Burst Generation. Free pharmacists from Generation X following the Baby Boom Generation had a moderate positive attitude. Therefore, it can be said that there are significant differences between the levels of attitudes towards pharmaceutical care of different generation pharmacists in

Adiyaman. In this context, the first hypothesis of the study, “1.H1: The level of perception of pharmaceutical care differ significantly in terms of generational status of free pharmacists”, was accepted. The 1.H0 hypothesis was rejected.

3.6.3.2. Testing the Differences in Attitude / Perception of Pharmaceutical Care in Terms of Gender Status of Independent Pharmacists The Mann-Whitney U test was used to determine whether there is a significant difference between the levels of perceived pharmaceutical care of the free pharmacists participating in the study in terms of gender status. Table 7. Analysis of Pharmaceutical Care Attitude / Perception Differences in Terms of Gender Status of Free Pharmacists by Mann-Whitney U Test

Mean Mann- Asymp. Sig. General Average Gender N z Pharmaceutical Rank Whitney U (2-tailed) Care Attitude / Female 31 39,55 730,000 -,292 ,770 Perception Male 49 41,10

The differences between the level of attitudes towards pharmaceutical care in terms of gender status of the free pharmacists participated in the study were analyzed by Mann-Whitney U test and no significant differences were found in the 95% confidence interval (p = 0.770> 0.05). Accordingly, it can be said that there is no difference between the attitudes of female pharmacists or male pharmacists towards pharmaceutical care.

In this context, the second hypothesis of the study, “2.H1: The level of Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 201 perception of pharmaceutical care differ significantly in terms of gender status of independent pharmacists”, was rejected; The 2.H0 hypothesis was accepted.

3.6.3.3. Test of Differences in Attitude / Perception of Pharmaceutical Care in Terms of Seniority of Independent Pharmacists Kruskal Wallis H test was used to determine whether there is a significant difference between the levels of perceived pharmaceutical care in terms of seniority of the free pharmacists participating in the study. Table 8. Analysis of Attitudes / Perceptions Differences in Pharmaceutical Care in Terms of Seniority of Free Pharmacists by Kruskal Wallis H Test

Mean Chi- Asymp. Seniority N df General Average Rank Square Sig. Pharmaceutical 0-5 years 14 52,96 10,802 3 ,013* Care Attitude / 6 years -10 years 13 50,65 Perception 11 years -15 years 27 37,80 16 years and over 26 31,52

The differences between the level of seniority of the pharmacists participating in the study in terms of seniority levels towards pharmaceutical care were analyzed by Kruskal-Wallis Test and a significant difference was found in 95% confidence interval (p = 0.013 <0.05). Accordingly, the highest positive attitude level was observed in free / independent pharmacists with seniority of 0-5 years, and the lowest attitude level value was observed in free / independent pharmacists with seniority of 16 years and more. Therefore, it can be said that as the seniority level increases, the level of positive perception towards pharmaceutical care decreases. In this context, the third hypothesis of the study, 3.H1: The level of perception of pharmaceutical care differ significantly in terms of seniority level of independent pharmacists, was accepted; The 3.H0 hypothesis was rejected.

Conclusions and Recommendations Today, it should be remembered that the pharmacist is the main factor in the access of patients to the drug in the health system, regardless of the generation. Within the scope of the social responsibilities fulfilled by the pharmacist, it is necessary to improve patient health, to increase the quality of service and to make the service rendered visible with the contribution of 202 • Health Sciences Research Papers effective use of public resources. In order to achieve these results, Pharmaceutical Care is one of the most important stepping stones. Continuity of this type of studies is also important for better monitoring of pharmacists, which is one of the most important pillars of pharmaceutical care. This study was conducted to determine the positive or negative attitudes of the different generation of pharmacists in the province of Adiyaman towards pharmaceutical care. In addition, it was examined whether the attitudes of the independent pharmacists differed in terms of socio- demographic characteristics such as gender and seniority level. Considering the findings of the study, when the means of all variables were examined, it was seen that free pharmacists always had a very high positive attitude (4.25> 4.20) in terms of improving the health of the patient. Furthermore, in terms of the importance of pharmaceutical care, the provision of pharmaceutical care, the reduction of drug costs, and similar benefits (> 3.41), it was observed that free pharmacists were often in a positive attitude. However, the independent pharmacists who participated in the study showed an unstable attitude regarding the variables that pharmacists in Adiyaman performed pharmaceutical care (2.69 <3.41) and that pharmacists were responsible for applying pharmaceutical care (3.31 <3.41). As a result of the analysis of the attitudes towards pharmaceutical care for different generations of independent pharmacists, it was determined that there was a very high significant difference between the attitudes in the 99% confidence interval (p = 0.004 <0.01). According to this, the free pharmacists with the highest positive attitude towards pharmaceutical care in Adiyaman are the free pharmacists from Generation Y, which is the youngest generation as of today; the lowest positive attitudes were the free pharmacists from the Baby Burst Generation. Free pharmacists from Generation X following the Baby Burst Generation had a moderate positive attitude. Therefore, it can be said that there are significant differences between the levels of attitudes towards pharmaceutical care of different generation pharmacists in Adiyaman. On the other hand, there was no difference between the attitudes of pharmacists towards pharmaceutical care in terms of gender variable (p = 0.770> 0.05). Accordingly, the attitudes of female pharmacists or male pharmacists towards pharmaceutical care are similar, and the difference between attitudes is not statistically significant. Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 203

A significant difference was found between the attitudes / perceptions of pharmaceutical pharmacists in terms of seniority of the independent pharmacists participating in the study (p = 0.013 <0.05). Accordingly, the highest positive attitude level was observed in free / independent pharmacists with seniority of 0-5 years, and the lowest attitude level value was observed in pharmacists with seniority of 16 years and more. Therefore, it can be said that as the seniority level increases, the level of positive perception towards pharmaceutical care decreases. Generation Y is a generation with high self-confidence, open to learning, change and innovation, and a high level of technological competence (Aksu, 2015; Yüksekbilgili, 2015; Goksel & Gunes, 2017). The characteristics of the Y generation support the fact that, as can be seen in these study findings, it has a higher level of positive perception in terms of pharmaceutical care compared to the previous generations. In issues related to the Pharmaceutical Care, Generation Y pharmacist has an important feature that distinguishes itself from other generations ago. This important feature is that were beginned Clinical Pharmacy Education in 1991 in Turkey. Today, Clinical Pharmacy and Pharmaceutical Care Courses continue to be offered in Faculties of Pharmacy (URL-4, 2019; URL-5, 2019; URL-6, 2019). This generation has grown up to be more conscious and knowledgeable in this regard. Nevertheless, it is noteworthy that the highest positive attitude level for pharmacists with seniority of 0-5 years and the lowest level of attitude for pharmacists with seniority of 16 years and more were determined. The importance of 0-5 years is remarkable even if it is evaluated in the same generation up to 16 years. Therefore, it can be assumed that the information acquired after a while is gradually lost. Once again, one can infer how important post-graduate education is. The aim of the study was achieved by testing the hypotheses mentioned in the study. However, although the findings obtained from this study are statistically valid, this study is limited to the free / independent pharmacists in Adiyaman province. On the other hand, although the studies in the literature have stated that despite the use of the term pharmaceutical care in pharmacy for many years, most pharmacists still have insufficient knowledge about the meaning of this term, it is assumed that the pharmacists participating in this study are familiar with the concept of pharmaceutical care and correctly understand and answer the questionnaire questions. 204 • Health Sciences Research Papers

Recommendations for this study are listed below:  Increasing patient-oriented training programs supported by the Ministry of Health and conducted by the Turkish Pharmacists' Association, and supporting postgraduate training programs provided by higher education institutions can improve both the knowledge of pharmacists and the quality of pharmaceutical care practices.  Other studies can be conducted for provinces that have easy access to pharmaceutical maintenance trainings and provinces that cannot access training. In this way, comparative research results will guide the planning and programming of future pharmaceutical care trainings.  When the parameters used in this study are taken into consideration for the dissemination of such training programs, it can be considered that gender will not have any effect, but generation and seniority cases may affect the results of such programs and trainings. The mentioned parameters can be evaluated regionally before starting the mentioned training programs and can be used as a guide to prevent the operation, continuity, obstacles and job losses of the program. For example, in a region, when it is desired to advance in the field of pharmaceutical care with a group of pharmacists who are elderly in generation and who have worked in seniority for many years; more efforts should be made to raise awareness and raise awareness before starting such intensive training in terms of time, money and labor. Only in this way will the success of such investments be guaranteed.  In this study, the level of attitude / perception of pharmaceutical care of free / independent pharmacists in Adiyaman province were examined in terms of differences of generational, gender and seniority. However, socio-economic structure, regional differences, income level of pharmacists, patient profile are other parameters that should be examined. Therefore, it would be beneficial to include both other provinces and other parameters in future studies. Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 205

 As stated by Kiran (2019: 229), the education of large masses can be realized independently from time and place by distance education method. Therefore, as suggested by Kiran (2019), TEB Academy should agree with universities experienced in this field. In the virtual classrooms where pharmacists can participate instantaneously through both TV and web-based applications, they should plan training on pharmaceutical care by using interactive methods and deliver this training to all pharmacists regardless of location. The video recordings of these courses can be made accessible to colleagues with their passwords so that they can be watched again at any time. Thus, trainings should be made accessible to all pharmacists regardless of location and time.  On the other hand, it is questionable whether the education system, which is independent of space and time, is suitable for all pharmacists. It can be thought that distance education will not be a problem for generation Y, which is related to social media and whose lifestyle is continuous learning. In addition, it is possible to determine whether such training is suitable for the generations before generation Y and the success of the program can be followed by various analyzes.  As stated by Hepler and Strand (1990: 533), free / independent pharmacists should begin to direct their energy to greater social benefit rather than their internal affairs.

206 • Health Sciences Research Papers

RESOURCES Aksu, B. (2015). Importance of generational differences in business life, Pharmacy Management, Editors: Ozcelikay, G., Sencan, N., Ankara: Academician Medicine Bookstore. Andrea, B., Gabriella, H. C., ve Tímea, J. (2016). “Y and Z Generations at Workplaces”, Journal of Competitiveness, 8(3), 90-106. Clark, P. M., Karagoz, T., Apikoglu-Rabus, S. and Izzettin, F. V. (2007). “Effect of pharmacist-led patient education on adherence to tuberculosis treatment”, American Journal of Health-System Pharmacy, 64, 497-505. Cordina, M., McElnay, J. C. and Hughes, C. M. (1999). “The importance that community pharmacists in Malta place on the introduction of pharmaceutical care, Pharm World Sci, 21, 69-73. Ertuna, E. and Arun, M. Z. (2019). “In free pharmacies in Turkey presented related to antithrombotic therapy, evaluation of pharmaceutical care services”, DEU Journal of Medical Faculty, 33(1), 19-32. Farris, K. B., Fernandez-Llimos, F. and Benrimoj, S. I. (2005). “Pharmaceutical care in community pharmacies: Practice and research from around the World”, Ann Pharmacother, 39, 1539-1541. Franklin, B. D., Van Mil J. W. (2005). “Defining clinical pharmacy and pharmaceutical care”, Pharm World Sci, 27 (3), 137. Goksel, A. ve Gunes, G. (2017). “Differentiation Between Generations: Analysis of Generations X and Y in the Context of Organizational Silence Behavior”, Gazi University Journal of Faculty of Economics and Administrative Sciences, 19/3, 807-828. Gurbuz, S. (2015). “Generation Differences: Myth or Truth?”, Job and Human Journal, Volume-Number 2 (1), 39-57. Hepler, C. D., Strand, L. M. 1990). “Opportunities and responsibilities in pharmaceutical care”, American Journal of Hospital Pharmacy, Volume 47, Issue 3, Pages 533–543. Hutchinson, R.A. and Schumock, G. T. (1994). “Need to develop a legal and ethical base for pharmaceutical care”, Ann Pharmaco, 28, 954-956. Kalayci, S. (2016). SPSS Applied Multivariate Statistical Techniques, (7th Edition), Ankara: Asil Publication Distribution Company. Kiran, B. (2015). “Turkish Pharmacists Association Pharmacy Academy Analysis from Professional Development Perspective”, Marmara Pharm J, 19: 222- 231. Gulsen KIRPIK, Erkan YILMAZ, Sezen DEMIR • 207

Kiran, B. and Taskiran, E. G., (2015). “Factors Affecting the Vocational Choice of First Year Students of Ege University Pharmacy Faculty”, Marmara Pharmaceutical Journal, 19, 159-167. Kelgokmen I., D. and Yalcin, B. (2017). “Y Generation's Differing Business Values and Leadership Perceptions”, Journal of Yasar University, 12(46), 136-160. Ngorsuraches, S. and Li, S. C. (2006). “Thai pharmacists' understanding, attitudes, and perceived barriers related to providing pharmaceutical care”, American Journal of Health-System Pharmacy, 63, 2144-2150. Sancar, M., Okuyan, B., Apikoglu-Rabus, S. and Izzettin, F. V. (2013). “Opinion and Knowledge Towards Pharmaceutical Care of The Pharmacists Participated in Clinical Pharmacy and Pharmaceutical Care Continuing Education Program”, Turk J Pharm Sci, 10 (2), 245-254. Suleiman, I. A. and Onaneye, O. (2011). “Pharmaceutical Care Implementation: A Survey of Attitude, Perception and Practice of Pharmacists in Ogun State, South-Western Nigeria”, International Journal of Health Research, 4(2): 91-97. Turnacilar, M., Sancar, M., Apikoglu-Rabus, S., Hursitoglu, M. and Izzettin, F. V. (2009). “Improvement of diabetes indices of care by a short pharmaceutical care program, Pharmacy World & Science, 31, 689-695. Uema, S. A., Vega, E.M., Armando P.D. and Fontana D. (2008). “Barriers to pharmaceutical care in Argentina”, Pharm World Sci, 30, 211-215. Yazicioglu, Y. and Erdogan, S. (2004). “SPSS applied scientific research methods”, Detay Publishing, Ankara. Yuksekbilgili, Z. ve Akduman, G. (2015). “Workaholicism according to generations”, Adiyaman Univercity Journal of Institute of Social Sciences, 8(19), 415-440. USED INTERNET RESOURCES URL-1,https://www.ashp.org/-/media/assets/policy- guidelines/docs/statements/pharmaceutical-care.ashx Date of access: 19.06.2019 URL-2,https://www.teb.org.tr/news/8049/Rehber-Eczanem-Program%C4%B1- itle-Eczac%C4%B1-Eczac%C4%B1ya-G%C3%BC%C3%A7- Kat%C4%B1yor/ Date of access: 22.06.2019 URL-3, http://sozluk.gov.tr/ Date of access: 20.06.2019 URL-4, http://www.pharmacy.ankara.edu.tr/ders-bilgileri/, Date of access: 02.07.2019 208 • Health Sciences Research Papers

URL-5,http://eczacilik.istanbul.edu.tr/tr/content/egitim/lisans- (turkce~2Fdisiplin), Erişim Tarihi: 02.07.2019 URL-6,https://eczacilik.ege.edu.tr/tr-3068/farmakoloji_anabilim_dali.html Erişim Tarihi: 02.07.2019

TOOTH EXTRACTION REASONS AND TECHNIQUES IN PEDIATRIC PATIENTS

CHAPTER 13

Muammer Çağrı BURDURLU1 Volkan Çağrı DAĞAŞAN2

1 Yeditepe University Faculty of Dentistry Department of Oral and Maxillofacial Surgery - Asistant Professor 2 Yeditepe University Faculty of Dentistry Department of Oral and Maxillofacial Surgery - Asistant Professor

Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 211

Medical Evaluation Medical anamnesis should be carefully taken and it should be evaluated whether there is a contraindication for tooth extraction or local anesthesia application. In the presence of systemic diseases, consultation should be written to patient’s physician if necessary. Allergic situations are among the most important issues to be considered in pediatric patients. Particular attention should be paid to the reactions that may develop during the first local anesthesia experience (Wilson, 2013).

Dental Evaluation Following medical history, dental anamnesis should be taken. Dental evaluation consists of clinical and radiological examination. Clinical examination consists of intraoral and extraoral evaluations. The number, position and arrangement of the teeth should be examined according to the age range. Presence of swelling, asymmetry, lesion, color, shape and consistency should be evaluated separately for hard and soft tissues seperately. Mouth opening and jaw movements should also be evaluated. The number and location of permanent and primary teeth, follicle structures, presence of pathology, growth and development patterns should be evaluated with radiological examination. If necessary, advanced imaging techniques such as computed tomography can be used (Wilson,2013).

Cooperation The successful completion of the treatment process including; examination, anesthesia and tooth extraction is through a well established communication. The child's social, emotional and psychological condition should be carefully evaluated. Sound, odor and equipment that can create stress in the child should be removed from the environment (AAPD,2015).

Growth and Development Traumatic interventions may lead to asymmetry or defects by disrupting the ongoing growth and development pattern, resulting in loss of function and aesthetics. Erupting permanent teeth, adjacent teeth and follicles should be preserved during extractions (AAPD,2015).

212 • Health Sciences Research Papers

Differences of Primary Teeth From Permanent Teeth Physiology: Resorption in primary teeth is physiological where as in permanent teeth it is pathological. Supporting Tissue: Alveolar bone is more flexible; however, as the age progresses, the flexibility of the alveolar bone decreases. Size: Both crown and root volume are smaller than the permanent teeth. Shape: The crowns have a more domed anatomy. Furcations of the molars are located more cervically. All of these differences have resulted in a number of different approaches for the extraction of primary teeth:  Due to the cervical position of furcation, the forceps should be positioned more cervically.  Due to the wide angle of the molars' roots, more expansion is required in the extraction.  The mouth and handle of the forceps are smaller. In accordance with the domed structure of the molars,mouths of the forceps are more curved.  Roots may be thinner due to physiological root resorption. Therefore, there is a high probability of root fracture during extraction.  Care must be taken against residual root fragments (Costa, 2019).

Principles of Extraction  Evaluation of the developmental stage and position of the erupting permanent teeth and avoidment of aggressive curettage to prevent damage to permanent tooth follicle.  Evaluation of possible pathologies.  Considering the number, shape and angle of the roots.  Avoiding soft tissue traumas such as tongue, gingiva, cheek, lips and the floor of the mouth.  Avoiding hard tissue traumas such as developing permanent teeth, adjacent teeth, and alveolar bone. Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 213

 Alveolar compression is not recommended after extraction of primary teeth, to eliminate the risk of contamination and possible damage to the underlying permanent tooth follicle (Kaban, 2004).

Dental Extraction Tools

Elevators Tooth and root elevation, seperation of the interradicular septum are the main functionf of the elevators. There are 3 types of elevators according to their designs and functions: 1. Bein Elevator: It is the most commonly used elevator and first instrument used in tooth extraction (Fig. 1). It is used for seperation of the gingiva and movement of the tooth. Bein elevator is vertically effective.

Figure 1: Bein elevator

2. Apexo Elevator: Used to remove the upper jaw roots (Fig 2). Sagittally effective. 214 • Health Sciences Research Papers

Figure 2: Apexo elevators

3. Cryer Elevator: It is used to break the septum in the lower jaw and remove the roots (Fig 3). Movement starts in the transverse direction and continues sagittally.

Figure 3: Cryer elavators

Forceps They are the instruments where the tooth is held for luxation and rotation after elevation movement. Consists of 3 parts; mouth part (holds the teeth), joint part (the middle part which is movable) and handle part (the back part which is rough to prevent slipping). Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 215

Movements Used in Tooth Extraction Elevation is the starting movement. It is used to separate the circular fibers in the neck of the tooth, seperate the periodontal fibers and mobilize the tooth. Single rooted or conical rooted teeth are extracted with Rotation. The lower premolars and upper anterior teeth, which do not have a curved root structure, are held by the neck and extracted with this movement. Luxation is the movement of the tooth in the vestibulolingual or vestibulopalatinal direction. Multi-rooted teeth or teeth with curved root structure can be extracted with this movement. In the lower anterior region, the upper premolars, the lower and the upper molars are extracted with this movement. Traction is the movement of pulling out the luxated tooth

Stages of Tooth Extraction 1. Separation of the soft tissue attachment from the tooth. 2. Elevation of tooth with dental elevators. 3. Adapting the forceps to the tooth. 4. Luxate tooth with forceps. 5. Separation of the tooth from the socket (Kaban, 2004).

Extraction Techniques Extraction techniques, patient and physician positions and hand tools can be classified according to the location of the teeth: 1. Maxillary Teeth: a. Maxillary anterior b. Maxillary premolar c. Maxillary molar 2. Mandibular Teeth: a. Mandibular anterior b. Mandibular premolar c. Mandibular molar

4. Maxillary Teeth a. Maxillary anterior: Includes central incisor, lateral incisor and canine extraction techniques. Root anatomy, which is suitable for rotational movements, makes them easier to extract than other 216 • Health Sciences Research Papers

teeth. They usually have single conical root. The patient's head is at or above the physician's elbow. The seat is inclined 15-30 degrees to the ground. Patient's head turned towards the dentist. When the patient opens his mouth, the occlusal plane of the upper jaw is positioned at an angle of 60 degrees with the ground plane. Right-handed dentists are located on the right front of the patient (7 O’clock), and left-handed dentists are located on the left front of the patient (5 O’clock). Unused hand supports alveolar socket. Elevation-luxation-rotation-traction movements are performed respectively. Extraction starts with a bein elevator. The tooth is elevated by entering the gum pocket at an angle of about 45 degrees from the neck to the long axis. With the maxillary primary anterior incisor forceps, the socket is expanded and the periodontal ligaments are teared. Movement startes by luxation by first applying force in the vestibular direction. Due to the cortical structure of the palatal bone, no force is given in that direction. After the flexion of the socket by luxation, rotation is performed and then the tooth is removed from the socket by traction movement. b. Maxillary Premolar: Technically they are similar to the extraction of the maxillary anterior teeth. The positions of the patient and the dentist are the same as the extraction of the maxillary anterior teeth. Root number and shape may vary.They can have two or three roots.Especially in the first premolars, the prevalence of double roots is quite high. In double-rooted premolars, rotation is not performed. Extraction performed with luxation in the vestibular direction. c. Maxillary Molar: Technically they are similar to the extraction of the maxillary premolars. Attention should be given to the righ-left seperation of the forcepses. While the convex side of the forceps handle is directed towards the dentist, the notched part of the mouth grasps the vestibule of the tooth and the flat part grasps the palatinal region (AAPD, 2015). Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 217

Figure 4: Maxillary molar forceps

Figure 5: Maxillary root forceps

2. Mandibular Teeth a. Mandibular Anterior: Technically they are similar to the extraction of the maxillary anterior teeth. When the patient opens 218 • Health Sciences Research Papers

his mouth, the occlusal plane of the lower jaw is positioned parallel to the ground plane. Dentist next to the patient (7-8 O’clock). The seat is inclined 60-75 degrees to the ground. The patient's head is at or below the physician's elbow. Patient’s head looks forward. The dentist stands in front of the patient. The dentist can pass to the back of the patient for canine extraction. Right-handed dentists are located on the right back of the patient (12-3 O’clock), and left- handed dentists are located on the left back of the patient (9-12 O’clock). Following the elevation, the mandibular primary incisor forceps is used. Force is applied in vestibule direction to perform luxation. b. Mandibular Premolar: The mandibular premolar forceps is used. The patient position is the same as the extraction of the mandibular anterior teeth. Right-handed dentists are located on the right back of the patient (9-12 O’clock), and left-handed dentists are located on the left back of the patient (12-3 O’clock). c. Mandibular Molar: Technically they are similar to the extraction of the mandibular premolars. The mandibular molar forceps is used. The patient position is the same as the extraction of the mandibular anterior teeth (AAPD, 2015).

Figure 6: Mandibular molar forceps

Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 219

Indications For Tooth Extraction 1. Deep caries which can not be restorated: Primary and secondary caries plus all sequelae including periapical abcess and failed pulpotomy 2. Orthodontic indications: Totth removed to prevent or correct malocclusion 3. Trauma: Broken teeth that cannot be restored. 4. Periodontal disease. 5. Primary tooth that blocks permanent tooth eruption. 6. Teeth without root canal treatment option. 7. Supernumerary and malpositioned teeth. 8. Loss: Tooth extracted because of mobility time for exfoliation 9. Pathological lesions. 10. Teeth in the fracture line that may cause infection or prevent the reduction and stabilization of fracture parts. 11. Teeth that may be a source of infection before radiotherapy or chemotherapy. 12. Esthetic reasons. 13. Economical problems. 14. General medical resons: Prophylactic extraction (Alsheneifi, 2001). Contraindications For Tooth Extraction 1. Acute infection. 2. Teeth adjacent to tumoral pathologies. 3. Teeth on the radiotherapy field. 4. Presence of wound healing disorder. 5. Presence of malignant diseases and chemotherapy or radiotherapy receiving patient should not be exposed to extraction without consultation. 6. Uncontrolled metabolic diseases (diabetes, hyperthyroidism, uremia developing kidney diseases etc.). 7. Previous heart disease or operation in the last 6 months. 8. In the presence of life-threatening health problems. 220 • Health Sciences Research Papers

Complications of Tooth Extraction Complications during the extraction 1. Damaging of the neighboring tooth. 2. Wrong tooth extraction. 3. Tooth fracture. 4. Fracture of the jaws 5. Fracture of the alveolar bone 6. Fracture of the tuber maxilla 7. Gingival or mucosal lacerations. 8. Burns or abrasions of the gingiva or mucosa. 9. Tooth or foreign body migration to upper respiratory tract 10. Damage to nerves 11. Tooth / root migration to fascial spaces 12. Subcutaneous emphysema 13. Maxillary sinus perforation 14. Tooth/root leakage to maxillary sinus 15. Trauma 16. Dislocation Complications After Tooth Extraction 1. Ecchymosis and hematoma 2. Pain, loss of sensation 3. Septic periostitis 4. Alveolar osteitis 5. Bleeding (primary, secondary) 6. Infection 7. Swelling 8. Trismus Muammer Çağrı BURDURLU, Volkan Çağrı DAĞAŞAN • 221

Post-operative Care Following Dental Extraction Post-operative care recommendations should also be shared with the child's family and made sure that they are understood. It is generally not recommended to eat before numbness has passed. Considering that soft tissues such as tongue and lips are also numb, it is possible for the child to bite soft tissues and cause serious wounds while chewing. Mouth rinsing should be avoided. Suction and spitting should be avoided. Some pain, swelling and bleeding should be informed that it is normal. It should be advised not to consume very hot foods. Warm and soft nutrition should be recommended on the first day, granular foods such as snacks should be avoided. The wound should not be disturbed with any object. Oral hygiene should be provided. In situations which family is not sure of what to do, denitst should be consulted (AAPD, 2015).

222 • Health Sciences Research Papers

REFERENCES Alsheneifi T, Hughes CV. Reasons for dental extractions in children (2001). American Academy of Pediatric Dentistry. 2000; 23: 2: 109-112. American Academy of Pediatric Dentistry. Management Considerations for Pediatric Oral Surgery and Oral Pathology (2015). Reference Manual. Pediatr Dent; 40: 618-619. Costa VPP, de Querioz IQD, Lia EN. Primary and permanent dentitions; Charecteristics and differences (2019). Pediatric Restorative Dentistry. Cham, Springer. Kaban L, Troulis M. Behavior Management and conscious sedation of pediatric patients in the oral surgery Office (2004). In: Pediatric Oral and Maxillofacial Surgery. Philedelphia, Saunders: 75-85. Wilson S. Montgomery RD. Local anesthesia and oral surgery in children (2013). Pediatr Dent: Infancy through Adolescence. 5th ed. St. Louis, Elsevier Saunders: 398-410.

CONSCIOUSNESS LEVEL ANALYSIS OF HEALTH PERSONNEL TOWARDS INTERNATIONAL PATIENT SAFETY GOALS: A Case Study Of Private Hospital In Turkey1

CHAPTER 14

Nuray GİRGİNER2,

Mine İSKENDEROĞLU3

1 This study derived from master thesis titled “ uluslararası Hasta Güvenliği Hedeflerine yönelik Sigma Seviyesi Analizi ve Personel Bilinç Düzeyleri Analizi: Özel Hastane Örneği “ completed by Mine İskenderoğlu under the supervision of Nuray Girginer on 22-05-2019 in Eskişehir Osmangazi University Institute of Social Sciences. 2 Prof. Dr., Eskişehir Osmangazi University – Business Department, [email protected] 3 Expert Quality, City Hospital in Eskişehir, [email protected]

Nuray GİRGİNER , Mine İSKENDEROĞLU • 225

1. INTRODUCTION The basic principle of health services is patient safety. Because the patient is the most basic element of the patient care process, it requires responsibility awareness, up-to-date information and attention to be valid throughout the process. Various problems occurring in the processes and systems in patient-oriented treatment practices can harm the patient. Patient safety is a complex system that includes performance improvement, environmental safety and risk management as well as service improvements, infection control, safe use of medicines, equipment safety, safe clinical practices and safe care environment. Some institutions that are aware of the importance of patient safety have made patient safety a standard. One of them is Joint Commission International (JCI). JCI has set 6 International Patient Safety objectives in terms of standards. These goals are; accurate identification of the patient, increasing effective communication, improving the safety of high-risk drugs, providing the right side-procedure-patient surgery, reducing health- related infections, and reducing the risk of patients falling from injury. (Joint Commission International , 5th Ed. 2014). Each of the International Patient Safety Goals (IPSG) is important for patients. An error in any of these targets may cause unwanted results. Therefore for each goal, process definitions should be made and standardization should be ensured. To adopt improvement and safety culture in patient safety primarily based on the adoption by the employees of the organization into a corporate culture and the implementation of system improvements with a systematic and planned approach. The greatest responsibility for not making any mistakes in the Institution towards the International Patient Safety Goals belongs to the healthcare workers. If health professionals know the procedures related to each target and act in accordance with the procedures, errors that may occur in the process can be prevented. The higher the awareness and awareness of the employees in the institution, the more they will affect the groups they work with and the more they will contribute to the formation and spread of the patient safety culture. Therefore, it is important to determine the level of awareness of employees in a health institution in these goals and processes. When the literature on patient safety studies reviewed; these studies dealing with patient safety as a cultural change, basics of patient safety, 226 • Health Sciences Research Papers patient safety from the perspective of health professionals, reasons for the occurrence of errors that violate patient safety, providing patient safety on the basis of departments of the hospital (intensive care, clinics, operating room, etc.), patient safety costs of inferiority, etc. studies (Astı ve Acaroglu (2000); Aslan ve Ünal (2005); Özata vd., (2008); Intepeler ve Dursun (2012); Candaş ve Gürsoy (2015); Korkmazer vd. (2016); Yurttas vd., (2016); Biskin ve Cebeci (2017); Barıs ve İntepeler (2017); Karayurt vd. (2017); İskenderoğlu vd. (2018); Menken (2018); Cook vd. (2000); Gaba (2000); Vincent ve Coulter (2002); Leape vd. (2002); Burke (2003); Rogers vd. (2004); Davis vd. (2007); Hellings vd. (2007); Stone vd. (2007); Khuri vd. (2007); Longtin vd. (2010); Vries vd. (2010); Marx (2019)). Due to literature review, few studies aimed at determining the level of cognition and awareness of health workers in relation to IPSG are mostly focused on the awareness of these goals. This deficiency in the literature constituted the idea stage of the planning of this study. The aim of this study is to determine the level of adoption of the International Patient Safety Goals by the employees of an institution that adopts patient safety as a culture, routinely provides training to patients on patient safety and tries to produce solutions by focusing on the system in case of any error. The findings of the study are expected that health institutions focused on increasing quality by reaching IPSG will be guiding in terms of policies and trainings for their employees. It is consider that a health care institution can only provide quality service together with employees who are focused on quality and on the safety of their clients.

2. METHODOLOGY This study was conducted in a private hospital accredited by JCI in order to determine the personnel awareness levels for the International Patient Safety Objectives. The hospital is an institution with a capacity of 103 beds and 400 employees. At the same time, the importance of lean applications in this hospital has been the reason of choice for this study. A survey was conducted with health workers to determine their consciousness of IPSG. In this study, a full census was targeted and 127 of the 150 health personnel working in the institution were administered a questionnaire prepared by us. The first part of the two-part questionnaire includes questions such as gender, occupational group and working time to determine the demographic characteristics of the employees. In the second part of the Nuray GİRGİNER , Mine İSKENDEROĞLU • 227 questionnaire, there are 12 questions to determine the consciousness levels of health personnel regarding IPSG. Questionnaires were prepared in accordance with the parameters related to IPSG and taking into consideration the opinions and suggestions of the experts in the institution. The distribution of the questions in the second part according to the objectives and their parameters is as follows: for parameters for identification goal 1th, 2th and 3th questions, for parameters for effective communication 4th and 5th questions: for parameters for safety of high- risk drugs 5. Questions; for parameters for right side-procedure-patient surgery 7th and 8th questions; for parameters for related infections 9th question; for parameters for right side-procedure-patient surgery 10th and 11th questions and last question related with whether or not they know that need to make security reporting notices (İskenderoğlu, 2019). Score evaluation of the answers given to the questionnaire; It was made between 1 and 3 scores. If the answer was completely correct, 3 scores were scored, 2 scores if partially correct or incomplete, and 1 point if incorrect answer was given. In this case, the maximum score of a health personnel answering the questionnaire is 36, at least 12. For example, there are 3 correct answers for questions 1 and 2. The respondent received 3 scores that answered all 3 questions correctly, 2 scores that answered 2 or 1, 1 point that left blank or gave the wrong answer. If a parameter which should not be in the patient description is checked, 1 point is also given. If all of the stages given for question 3 are marked, 3 scores are given, if some of them are marked, 2 scores are given and 1 point is given if none is checked. Since multiple choice questions had only one correct answer, they were given 3 scores if they were marked and 1 point if they were not. For the 9th question, the assessment was made by giving 3 scores if all 5 indication rules were written, 2 scores if more than 1 items were written, and 1 point if not answered at all.

3. FINDINGS In order to determine the general consciousness level of the IPSG, the scores of the answers given to the questions (12 questions) were collected and divided by the number of people. Obtained average was accepted as the limit of consciousness level and it was accepted that those who had a total score above the average had a higher level of consciousness about the IPSG and those who had a lower total score were considered low. Table 1 shows the average score (x̅), the number of participants (n), and 228 • Health Sciences Research Papers the total score of the personnel for the 12 questions in the questionnaire prepared by us regarding IPSG. Table 11: The General Consciousness Levels for IPSG Consciousness level n % x̄ s Low (Total scores < x̅) 61 48 31,38 3,326 High (Total scores ≥ x̅) 66 52

As can be seen from Table 1, the average level of general consciousness (x ̅) of health personnel related to IPSG was calculated as 31.38 ± 3,326. The average of the answers given to the questions was accepted as the level of consciousness. The total score of the answers they gave to the 12 questions in the questionnaire was lower than 31.38 and the 61 people who make up 48% of the health care workers had low level of consciousness towards IPSG. On the other hand, 52% (66 people) answered all of the 12 questions in the questionnaire and their scores were higher than 31.38. Therefore, 52% of the employees have a high level of consciousness towards IPSG.

3.1. Personnel Consciousness Levels for Verification of Patient Identity

Questions 1, 2, and 3 of the 12 questions in part 2 of the questionnaire relate to the process of patient identification. The distribution of employees' opinions about the parameters related to this process is given in Table 2. The average value, the level of consciousness, calculated by dividing the total scores of the answers given to these three questions by the number of participants. The total score of the answers to these three questions is higher than the average of the patients who have high level of consciousness about the process of identity verification; and those whose total score was below the average were considered as low-level employees regarding the process of verifying patient identity.(Table 2). During the verification of patient identity the identity of the patient is identified by name- surname, date of birth and protocol number while the patient in a coma is identified by gender, date of arrival, and protocol number. The conditions for verifying patient identity are given in the options of the questions. The cognitive status of the conscious patient Nuray GİRGİNER , Mine İSKENDEROĞLU • 229 identification parameters by healthcare workers is as follows: 99.21% of health care workers know the name-surname parameter, 93.70% know the protocol number parameter, and 16.54% know the date of birth parameter. The health workers who answered the room number which is a wrong way of identification is 2.36%. The parameters to identify the coma patient; 86,61% of the employees in the gender parameter, 77,95% in the day of arrival and 65,35% of the employees in the protocol number answered correctly. There is a 4.72% group of employees marking the room number. For cases where patient ID needs to be defined; 92.13% of health workers should be identified during hospitalization, 63.78% should be verified during vascular access, 88.98% should be authenticated when taking laboratory samples, 91.9% of employees should be verified during treatment. , 34, 89.76% of the patients need to be confirmed without giving medication and 89.76% of health care workers know that pre-operative verification should be done. Table 2: Distribution of answers to questions and consciousness levels towards patient identity verification

Marked Marked Empty Unmarked PARAMETERS ratio person Rate person

Name and surname 99,21 126 0,79 1

Protocol number 93,70 119 6,30 8

Date of Arriving 12,60 16 87,40 111

T. C. Identification number 14,96 19 85,04 108

Date of birth 16,54 21 83,46 106

Physician Name 8,66 11 91,34 116

The room number 2,36 3 97,64 124

Patient Diagnostic Parameters Diagnostic Patient Gender 18,90 24 81,10 103

Diagnosis Parameters of Coma 11,81 15 88,19 112 Patient

Protocol number 65,35 83 34,65 44

The room number 4,72 6 95,28 121

T.R. Identification number 3,15 4 96,85 123

Date of birth 7,09 9 92,91 118

Physician Name 5,51 7 94,49 120

Diagnosis Diagnosis Parameters of Coma Patient 230 • Health Sciences Research Papers

Date of Arriving 77,95 99 22,05 28

Gender 86,61 110 13,39 17

During Hospitalization 92,13 117 7,87 10

Vascular Path Opening 63,78 81 36,22 46

Laboratory Sample Collection 88,98 113 11,02 14

During Treatment 91,34 116 8,66 11

Authentication before giving the patient medication 89,76 114 10,24 13

Preoperative 89,76 114 10,24 13

Consciousness levels n % x̄ s Low (Total scores < x̅) 48 37,8 7,69 1,193 High (Total scores ≥ x̅) 79 62,2

In the International Patient Safety questionnaire 1, 2, 3. The average value, the level of consciousness, calculated by dividing the total scores of the answers given to these three questions by the number of participants. According to Table 2 the mean score of the questions asked about verifying the patient identity was 7.6 ± 1.19. The total score of the answers to these three questions is higher than the average of the patients who have high level of consciousness (62,20%) about the process of identity verification; and those whose total score was below the average were considered as low- level employees (37,80%) regarding the process of verifying patient identity.(see Table 2).

3.2. Personnel Consciousness Levels for Effective Communication Regarding effective communication; the 4th and 5th questions in the second part of the questionnaire were evaluated. The total scores of the answers given to the questions were divided by the number of participants and the mean value and the level of consciousness were accepted. Those whose answers to questions 4 and 5 were higher than the average score had higher level of consciousness about effective communication; and those whose total score is below the average are considered to have low level of consciousness about effective communication process. Distribution of answers to questions and consciousness levels towards effective communication given in Table 3. Nuray GİRGİNER , Mine İSKENDEROĞLU • 231

Table 3: Distribution of answers to questions and consciousness levels towards effective communication

Marked Marked Empty Unmarke PARAMETERS ratio person Rate d person Writing and registering notification 5,51 7 94,49 120 from the phone Writing the notification from the phone first and then verifying it by 69,29 88 30,71 39 reading Verification of repeated notification 25,20 32 74,80 95 Readback Application Readback by phone

Writing and registering notification 3,15 4 96,85 123 from the phone Writing the notification from the

Application phone first and then verifying it by 25,20 32 74,80 95 reading Repeat the notification from the phone and verification of the 71,65 91 28,35 36 Repeatback Repeatback notification Consciousness levels n % x̄ s Low (Total scores < x̅) 51 40,2 4,91 1,405 High (Total scores ≥ x̅) 76 59,8

Read back and repeat back applications were asked to the participants about the process of effective communication. Read back; the notification received by telephone is written before the recording and then back to the notification by reading the verification is done. Due to Table 4, 69.29% of healthcare workers answered this question correctly, while 25.20% confused it with repeat back. Repeat back; verification of the information received only by repetition and 71.65% of healthcare workers answered the question correctly. 25.20% of its employees confused read back and repeat back with each other. The average score (x ̅) of the answers given to the 4th and 5th questions of the International Patient Safety Questionnaire and the number of the respondents (n) related to the effective communication process and the answers given to the questions posed by the employees about the effective communication are summarized in Table 4. The average score of the questions about effective communication is 4.91 ± 1.19. 232 • Health Sciences Research Papers

When this score is accepted as limit; 59.8% of the healthcare workers were conscious about the process of effective communication because the total score of the answers they gave to the 4th and 5th questions was higher than 4.91, and 40.2% of the answers they gave to the 4th and 5th questions It was determined that the level of awareness about effective communication process was low due to the fact that the total scores were lower than 4.91.

3.3. Personnel Consciousness levels for Ensuring the Safety of High Risk Drugs The average score was obtained by dividing the total score of the answers given to a single question (Question 6) to employees in order to ensure drug safety. Those with a score higher than the average of the answer given to question 6 were considered to be conscious, while those with a lower than the average score of question 6 were considered to have incomplete or lacking knowledge of the subject. The data obtained for ensuring drug safety are given in Table 4. The question about the safety of high-risk drugs is related to the application of red labels. 99.21% of the employees know that the red label means high risk medicine0.79% replied that close to due date. Table 4: Distribution of answers to questions and consciousness levels towards Ensuring the Safety of High Risk Drugs

Marked Marked Empt Unmarked PARAMETERS ratio person y Rate person

Close to due date 0,79 1 99,21 126

It is defined as a similar drug in terms of 100,0 0,00 0 127 its reading, writing and packaging 0

Red Red Label High Uygulaması -risk medication 99,21 126 0,79 1 Consciousness levels n % x̄ s Low (Total scores < x̅) 1 0,8 2,98 0,177 High (Total scores ≥ x̅) 126 99,02

As can be seen in Table 4, the mean score of the questions asked about drug safety was 2.98 ± 0.177. This score was considered the level of consciousness limit. It was accepted that healthcare workers were conscious about providing drug safety for those whose total scores were higher than 2.98, and those who were lower than 2.98 had lower levels of consciousness. In this context, it was found that 99.02% of health workers Nuray GİRGİNER , Mine İSKENDEROĞLU • 233 were conscious about the process of effective communication. One person who gave the wrong answer to the sixth question is a nurse working in the institution for 2 years.

3.4. Personnel Consciousness levels Towards the Right Field, Right Procedure, Right Patient Surgery Regarding the right side, the right procedure, and the right patient surgery; Questions 7 and 8 of the International Patient Safety Objectives section of the questionnaire were evaluated. The total scores of the answers given to the questions were divided by the number of participants and the mean value and the level of consciousness were accepted. The total score of the answers to these two questions, the higher than the average level of awareness about the process of providing safe surgery; and those whose total score is below the average are considered to have low level of awareness about the process of providing safe surgery. Table 5 shows the distribution of consciousness levels for the purpose of ensuring safe surgery. Table 5: Distribution of answers to questions and consciousness levels towards Right Field, Right Procedure, Right Patient Surgery

Marked Marked Empty Unmarke PARAMETERS ratio person Rate d person

The letter ‘E’ 7,09 9 92,91 118 Round Sign 7,87 10 92,13 117 Cross Sign 7,09 9 92,91 118

Party Sign Party Letter ‘E’ in round 77,95 99 22,05 28 Break between surgery 23,62 30 76,38 97

The surgeon, surgical nurse and anesthesiologist confirm the identity and side of the patient and register them in 72,44 92 27,56 35 the Safe Surgery Checklist

Out Application Out - before surgery in the operating room. Time Pre-operative preparation of 3,94 5 96,06 122 the patient Consciousness levels n % x̄ Low (Total scores < 푥̅) 50 39,4 5,13 High (Total scores ≥ 푥̅) 77 60,06 One of the questions asked about the right area, the right procedure and the right patient surgery; is the symbol of the party sign identified and 234 • Health Sciences Research Papers standardized by the organization. The E standard mark within the round defined by the institution, 77.95% of the health workers know. There is an average distribution of 7% for the remaining three answers. The second question is the pre-time time-out application. 72.44% of employees dominate the process and these employees know that the surgeon, surgical nurse, and anesthesiologist should confirm the identity and side of the patient and register it on the safe surgical checklist just before the surgery begins. On the other hand, 23.62% of the employees know the time-out application as a break between operations. It is seen that the average score of the questions about the right area, the right procedure and the right patient surgery is 5.13 ± 1.21 from Table 5. When this score is accepted as the level of consciousness limit; the scores of the answers they gave to the 7th and 8th questions were found to be higher than the total of 5.13 (60.06%), the right field, the right procedure, the right patient surgery process to ensure that they are conscious, while the answers to questions 7 and 8, the total score of less than 5.13 health workers (39.4%), the level of consciousness was found to be low.

3.5. Personnel Consciousness Levels to Reducing Risks Due to Infection The total score of the answers to the 9th question related to the prevention of infections in the questionnaire was divided by the number of respondents and the average score was obtained. This mean value was accepted as the limit of consciousness for the prevention of infections. The scores of the answers to the 9th question, those below the average were low and those above were considered conscious about the infection prevention process. Distribution and awareness levels related to the level of awareness of the process of prevention of infections are given in Table 6.

Nuray GİRGİNER , Mine İSKENDEROĞLU • 235

Table 6: Distribution of answers to questions and consciousness levels towards Reducing Risks Due to Infection

Marked Marked Empt Unmarked PARAMETERS ratio person y Rate person Before Contacting Patient 61,42 78 38,58 49

After Contact with Patient 60,63 77 39,37 50 After Contact with Blood and Body Fluids 55,91 71 44,09 56 and Removing Gloves

After Contact with Surfaces Around the 57,48 73 42,52 54 5 Indication Rules 5 Indication Patient Before Aseptic Procedures 55,91 71 44,09 56 Consciousness levels n % x̄ Low (Total scores < 푥̅) 52 40,9 2,22 High (Total scores ≥ 푥̅) 75 59,1

The five indication rules for the prevention of infections were asked as open ended. The correct options for this question are: before contact with the patient, after contact with the patient, after contact with blood and body fluids and removing gloves, after contact with the surrounding surfaces of the patient and before aseptic procedures. According to Table 6 61.42% of healthcare workers before contact with the patient, 60.63% after contact with the patient, 55.91% after contact with blood and body fluids and removing gloves, 57.48% after touching the surfaces around the patient and 55.91% know that they should wash their hands before aseptic procedures. Approximately 40% of the employees did not know about the subject. According to Table 6, the mean score of the correct answers for the prevention of infection is 2.22 ± 0.95. This score is based on the level of consciousness; It was determined that 59.1% of the health care workers whose scores of answers to question 9 were higher than 2.22 had a high level of consciousness about the prevention of infections, whereas 40.9% had deficiencies in their knowledge about the prevention of infections. .

3.6. Personnel Consciousness Levels for the Prevention of Falls The 10th and 11th questions included in the questionnaire regarding the fall prevention process were evaluated. The total scores of the answers given by the health care workers to these two questions were accepted as the mean, 236 • Health Sciences Research Papers level of awareness obtained by dividing the number of participants. The total score of the answers to these two questions, the ones higher than the average level of awareness about the prevention of falls is high; and those whose total score is below the average are considered to be low level of awareness about the fall prevention process. Distribution and awareness levels related to fall prevention awareness levels are given in Table 7. Table 7: Distribution of answers to questions and consciousness levels towards Prevention of Falls

Marked Marked Empty Unmarked PARAMETERS ratio person Rate person

identity identifier 0,79 1 99,21 126

Falling risk 99,21 126 0,79 1

Infection risk 0,00 0 100,00 127

Bracelet

Green Color Color Green Specify gender 0,00 0 100,00 127

Consult the nurse before entering the 4,72 6 95,28 121 room Patient at risk for infection 6,30 8 93,70 119

leaf clover leaf

- Patient at risk of falling 88,98 113 11,02 14

Four Consciousness levels n % x̄ Low (Total scores < 푥̅) 16 12,6 5,76 High (Total scores ≥ 푥̅) 111 87,4

Two questions were asked about the measures taken to prevent falls: The first is the meaning of the green bracelets used, the other is the meaning of the 4-leaf clover image on the doors of the patient rooms. Both indicate that the patient is at risk of falling. According to Table 7, 99.21% of the employees know the purpose of the green bracelet and 88.98% know the meaning of the four-leaf clover image. 6.30% confused the four-leaf clover image with the infection-risk patient image. As can be seen from Table 7, the mean score of the questions asked about the fall prevention process is 5.76 ± 0.65. The scores of 87.4% of healthcare workers' answers to the 10th and 11th questions were higher than 5.76 and they were conscious about the fall prevention process and 12.6% of the answers they gave to the 10th and 11th questions It is seen that the level of consciousness about the fall prevention process is lower than 5.76. Nuray GİRGİNER , Mine İSKENDEROĞLU • 237

3.7. Personnel Consciousness Levels for Security Reporting The last question of the questionnaire was asked to see if the person who saw the incident should know whether to report security reporting when an incident occurred. The total score of the answers given to the question was divided by the number of participants in the survey and the average score was obtained. Those below the average score were deemed to lack knowledge about safety reporting notices, and those above the average were aware of the need to make safety reporting notices. The distribution of the need for security reporting notification is given in Table 8. In case of an incident within the scope of International Patient Safety, if necessary, 85,04% of the employees who know about the security reporting notification that should be made after medical intervention know that they should make security reporting notification and 11,02% need to forward it to the field manager. 3.94% of the employees answered that root cause analysis was performed. The root cause analysis takes place in the next step after the security reporting notification is made. Table 8: Distribution of answers to questions and consciousness levels towards Security Reporting

Marked Marked Empty Unmarked PARAMETERS ratio person Rate person

Inform the field manager 11,02 14 88,98 113 Security Reporting 85,04 108 14,96 19 Notification root cause analysis 3,94 5 96,06 122

GRS Notifications GRS Consciousness levels n % x̄ Low (Total scores < 푥̅) 19 15 2,70 High (Total scores ≥ 푥̅) 108 85

According to Table 8 the average score of the problem related to the use of security reporting system is 2.70 ± 0.71. Health workers are more conscious about the use of security reporting system than those whose total score is higher than 2.70, and those whose total score is less than 2.70, who have less than 2.70, levels were considered to be low. It was found that 85% of healthcare workers were conscious and 15% had low level of consciousness.

238 • Health Sciences Research Papers

4. Discussions and Conclusion In this study carried out in a private hospital which is accredited by JCI and adopted a simple philosophy, the awareness levels of the health personnel of the institution on IPSG were examined separately for each goal. According to findings: The patient identification: The best-known parameter for the patient identification process is the name-surname, and very few employees know the date of birth parameter. While the best known parameter for identifying the coma patient is gender, the lack of information is the protocol number. The order from the best to the least known for cases where patient identity needs to be verified; during hospitalization, during treatment, before giving medication to the patient and before surgery, before taking a laboratory sample, when opening a vascular access. Although the parameter of necessity of patient authentication when opening the vascular access is the least known parameter, it is one of the critical steps for the right treatment to the right patient. Therefore, at that stage, authentication should be done without neglecting. In line with these findings, the hospital managers can organize trainings and complete the information that is missing from the personnel. Ensuring effective communication: In terms of effective communication, 25% of employees confuse what readback and repeatback are. Considering this may be a confusion due to the fact that the words are in English; In order to solve the problem, transferring the training with video or visual support will be more memorable. Ensuring drug safety: Employees are well aware of the meaning of the red label to ensure drug safety.. Of the 127 health workers surveyed, only 1 person confused the green label with the red label used for near-term medication. In future studies, questions about drug safety can be expanded by adding various parameters to the scope of the assessment. Ensuring safe surgery: 77.95% of the employees are aware of the standard mark set by the institution regarding safe surgery. However, it is important that physicians know this sign correctly because of they are the physicians who will mark the surgical side of the patient in the safe surgical checklist. Nevertheless, knowing this sign by employees, reminding physician to files that they see as missing will be effective in reducing the errors or deficiencies. Similarly, although time-out is a process involving surgeons, surgical nurses and anesthesiologists rather than all healthcare Nuray GİRGİNER , Mine İSKENDEROĞLU • 239 workers, it is a process that all healthcare workers should know within the culture of patient safety. Although Time-out is known by 72% of health workers, this rate can be considered as a low rate in line with the objectives of the institution. If the time-out application performed in a surgery is made by video recording and watched in the trainings, with the effect that everyone knows people, the application will be more memorable. Prevention of infections: Hand hygiene is one of the basic rules for the prevention of infections. Health workers were also asked 5 indication rules in this direction. Sort from best known to least known; before contact with the patient, after contact with the patient, after contact with the patient, after contact with blood and body fluids and removing the glove, before aseptic procedures. The answers are distributed between 50% and 60%, and hand hygiene compliance monitoring performed by the infection nurse in the areas is a trigger for staff to be more careful, indicating that people have deficiencies. More attention should be paid to handwashing, and those responsible for the area should check their staff and support the infection nurse. Hand hygiene is not only important for the patient but also for the health of the individual. The need for security reporting notifications: 85.04% of the employees are sensitive to the issue regarding the necessity of making security reporting notifications. Awareness about making security reporting notifications in the organization is critical for data accuracy. The others employees should have access to hospital information management systems in order for other employees to gain the habit of reporting. It should be explained that the notifications are approached oriented process not human. Even the person who made the mistake should be asked the idea and the solution proposal should be evaluated. Within the scope of the compliance trainings to be given to every new employee in the institution, the training contents can be revised in such a way that the missing parts are supported by taking into account all these deficiencies. For each IPSG, it is not possible to rank on the average of the answers given to the questions because the number of questions is not equal. On the other hand, if an assessment is made based on the percentages of the conscious employees, the ranking from the highest consciousness level to the lowest goal; safety of high-risk drugs, prevention of falls, reporting safety reports, verification of patient identity, correct field, correct procedure, correct patient surgery and effective communication. 240 • Health Sciences Research Papers

Cultures related to patient safety culture or quality processes in hospitals are gained through adaptation trainings and in-service trainings after individuals start working in hospitals. The fact that the processes related to quality processes take place in university syllabuses and that people start to work with some awareness will be effective in increasing the quality of the service provided.

Nuray GİRGİNER , Mine İSKENDEROĞLU • 241

REFERENCES Aslan, Ö., & Ünal , Ç. (2005). "Cerrahi Yoğun Bakım Ünitesinde Parenteral İlaç Uygulama Hataları", Gülhane Tıp Dergisi, 47, 175-178. Aştı, T., Acaroğlu R. (2000). “Hemşirelikte Sık Karşılaşılan Hatalı Uygulamalar” , C.Ü.Hemşirelik Yüksek Okulu Dergisi, 4(2), 22-27. Bişkin, S., Cebeci, F. (2017). Acil Servislerde İlaç Uygulama Hataları, Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, 6(4), 180-185. Burke, J.P. (2003). “Infection Control – A Problem For Patient Safety”, The New Englans Journal of Medicine, 348(7), 651-656. Candaş , B., & Gürsoy , A. (2015). "Cerrahide Hasta Güvenliği: Güvenli Cerrahi Kontrol Listesi", Erciyes Üniversitesi Sağlık Bilimleri Fakültesi Dergisi , 3(1), 40-50. Cook R. I., Render M., Woods D. D. (2000). “ Gaps in the continuity of care and progress on patient safety”, BMJ , 320, 791-794, doi: 10.1136/bmj.3207237.791 Davis, E.R., Jacklin R., Sevdalis N., Vincent C.A. (2007). “Patient involvement in patient safety: what factors influence patient participation and engagement?” Health Expectations, 10(3), 259-267. Gaba, D. M. (2000). “Anaesthesiology As A Model for Patient Safety In Health Care”, BMJ, 320, 785-788. Hellings, J., Schrooten, W., Klazinga, N., Vleugels, A. (2007). "Challenging Patient Safety Culture: Survey Results", International Journal of Health Care Quality Assurance, 20 ( 7), 620-632. İntepeler, Ş., Dursun, M. (2012). "Tıbbi Hatalar ve Tıbbi Hata Bildirim Sistemleri", Anadolu Hemşirelik ve Sağlık Bilimleri Dergisi, 15(2), 129- 135. İskenderoğlu, M., Fidan , B., Kocatepe, B., Soydan, D., Yılmazlar, A., & Opsar, F. (2018). "Uluslararası Hasta Güvenliği Hedeflerinden Düşmelerden Kaynaklanan Hastaların Zarar Görme Riskinin Önlenmesi Süreci Sigma Seviyesi Analizi: Özel Medicabil Hastanesi Örneği", 2. Uluslararası 12. Ulusal Sağlık ve Hastane İdaresi Kongresi, Bodrum/Muğla: Muğla Sıtkı Koçman Üniversitesi, ss.122-131. İskenderoğlu, M. (2019), Uluslararası Hasta Güvenliği Hedefleri Sigma Seviyesi ve Personel Bİlinç Düzeyleri Analizi: Özel Hastane Örneği” ESOGÜ Sosyal Bİlimler Enstitüsü, yüksek lisans tezi, Eskişehir. 242 • Health Sciences Research Papers

Khuri, S. F., Henderson, W. G., Daley, J., Jonasson, O., Jones, R. S., Campbell, D. A., Fink, A. S., Mentzer, R. M, Steeger, J. E., “The Patient Safety in Surgery Study: Background, Study Design, and Patient Populations”, Journal of the American College of Surgeons, 204(6), 1089-1102. Korkmazer, F., Yıldız, A., & Ekingen, E. (2016). "Sağlık Personeli Hasta Güvenliği Kültürü Algılarının Değerlendirilmesine Yönelik Bir Araştırma", Muş Alparslan Üniversitesi Sosyal Bilimler Dergisi, 4(2), 141- 154. Leape, L.L., Berwick, D. M., Bates, D. W. (2002). “What Practices Will Most Improve Safety?Evidence-Based Medicine Meets Patient Safety”, JAMA, 288(4), 501-507. Longtin, Y., Sax, H., Leape, L.L., Sheridan, S.E., Donaldson, L., Pittet, D. (2010). “Patient Participation: Current Knowledge and Applicability to Patient Safety”. Mayo Clinic Proceedings, 85(1), 53-62. Marx, D. (2019). “Patient Safety and Just Culture”, Obstetric and Gynecology Clinics of North America, 46(2), 239-245, doi: 10.1016/j.ogc.2019.01.003. Menken Y. (2018). Günübirlik Anestezi Uygulamalarında Hasta Güvenliği Kültürünün Algılanmasının Değerlendirilmesi”, T.C. Sağlık Bilimleri Üniversitesi Okmeydanı Sağlık Uygulama Ve Araştırma Merkezi Anesteziyoloji Ve Reanimasyon Anabilim Dalı, Tıpta Uzmanlık Tezi, İstanbul, 2018. Özata, M., Aslan, Ş., & Mete, M. (2008). "Rasyonel İlaç Kullanımının Hasta Güvenliğine Etkileri Hekimlerin Rasyonel İlaç Kullanımına Etki Eden Faktörlerin Belirlenmesi", Selçuk Üniversitesi Sosyal Bilimler Enstitüsü Dergisi, 2008(20), 530-542. Rogers, A. E., Hwang, WT., Scott, L. D., Aiken, L. H., Dinges, D. F. (2004). “The Working Hours Of Hospital Staff Nurses And Patient Safety”, Health Affairs, 23(4),202-212. Stone, P. W., Mooney-Kane, C., Larson, E. L., Horan, T., Glance, L. G., Zwanziger, J., Dick, A. W. (2007). “Nurse Working Conditions and Patient Safety Outcomes”, Medical Care, 45(6), 571-578. Vincent C.A., Coulter A. (2002). “Patient Safety: What About the patient?”, BMJ Quality & Safety, 11, 76-80. Vries, E. N., Prins, H.A., Crolla, R.M.P.H., den Outer, A. J., van Andel, G., van Helden, S. H., Schlack, W. S., van Putten, M. A., Gouma, D. J., Dijkgraaf, M. G.W., Smorenburg, S. M., Boermeester, M. A. (2010). “Effect of a Comprehensive Surgical Safety System on Patient Outcomes”, Massachusetts Medical Society, 363, 1928-1937.

REVIEW

MONOSODIUM GLUTAMATE

CHAPTER 15

Nurhayat ATASOY1

1 Yuzuncu Yil University, Faculty of Science, Department of Chemistry/Biochemistry Section, Van/Turkey, [email protected]

Nurhayat ATASOY • 245

INTRODUCTION Developments and innovations in technology and nutrition eliminated some of the problems encountered in the past, while creating new ones. Healthy nutrition is very important particularly to maintain the quality of life in an aging population. As a result of new production techniques brought by developing technology and the diversity in food consumption of consumers, the use of food additives (FA) in the food industry has increased day by day. Sweetening agents are of vital importance in the production of delicious food. Many industrially prepared foods are attractive for the consumers particularly owing to their typical flavors. Therefore, it is not surprising that the manufacturer demonstrates great interest in the use of food or food components with typical umami taste and flavor enhancement systems. Providing the necessary attractiveness particularly for the foods that are not very tasty, sweeteners can play an important nutritional role. Food safety is an issue of priority for all health authorities, particularly for the World Health Organization. These authorities determined three main issues and conducted intensive work on them. These issues are non- standard food production, microbiological pollution, and chemical pollution in foods. There are two factors influencing the historical development of food additives. The first of these is the need for developing new food storage methods in parallel with the developing technology. The second factor is ensuring the customer to better perceive the current quality of the food. Considering the international food trade, the first factor demonstrates the necessity of these substances. The world market for food additives reached 10 billion dollars in the 1900s and today it is well above this number.

1.1.Definition of the Food Additives The definition made by the Ministry of Health in the food additives regulation is as follows; "The substances that, under normal circumstances, are not consumed alone or not used as a raw food, having or not having a nutritional value itself; whose residues or derivatives can exist within the finished products during the process or manufacture based on the selected technology, and the substances that are allowed to be used to protect and improve the taste, smell, appearance, structure and other qualities of foods during the production, sorting, processing, preparation, packaging, transportation, and storag. However, the counterfeit substances that 246 • Health Sciences Research Papers increase the nutritional value of foods or added into the counterfeit foods are not included in this group.

1.2.Points to Consider for Food Additives 1- It should not be harmful to human health and this should be determined by laws. 2- There should be a technological obligation in its use. 3- It should be used in permitted foods and in permitted quantities. 4- It should not reduce the nutritional value of the food. 5- Food additives should be used for the purpose of maintaining quality and not concealing the poor quality. 6- Food additives may be natural, nature identical, or artificial.

1.3.Classification of Food Additives We can classify food additives in 4 groups according to their intended use. 1. Those extending shelf life by maintaining quality (Preservatives) 2. Those improving the structure, preparation, and cooking feature 3. Those improving the aroma and color, 4. Those preserving and improving nutritional value (nutrients).

1.4.Functions of the Food Additives Acidity regulators, Anti-aggregation inhibitors, Antioxidants, Antimicrobial substances, Stabilizers, Gelling agents, Sweeteners, Colorants, Emulsifiers, Flavor (taste and smell) agents, and Flavor enhancers.

1.5.Flavor enhancers (Mono Sodium Glutamate, MSG-E621) They are added into the foods to make the aroma more attractive, to correct or preserve the natural aroma. MSG fits well with red meat, fish, chicken meat, numerous vegetables, sauces, soups, and marinades. In the early 20th century, Professor Ikeda from the University of Tokyo discovered in tomatoes, cheese, asparagus and meat that there was a taste different from sweet, bitter, sour, and salty. He named this fifth taste as Nurhayat ATASOY • 247

"Umami", which means delicious in Japanese. He then isolated the substance that gave this taste from a type of seaweed called kombu, which has been traditionally used in Japanese cuisine for 1,000 years. This substance was glutamic acid and sodium salt of glutamic acid (MSG) was prepared to ensure easy water solubility. Studies on taste receptor physiology proved the existence of the 5th basic taste. The most important feature of the substance in question is that it increases saliva secretion and reveals the properties of the taste profile. MSG is observed under different names in all kinds of chips, some fats, bouillons, instant soups, sauces, processed red meat, fish and chickens, mayonnaises, spice mixtures, colored yogurts, baby foods and many other consumer products (glutamic acid, glutamine). Glutamic acid is a chemical substance found abundantly in every living organism, including human beings, and is one of the 20 amino acids that make up the structure of human beings. It is especially vital for the functioning of brain. It is an amino acid that mediates most functions of the brain, including learning, focusing, comprehension, and memory recording. All protein-rich foods, such as meat, milk, fish and some vegetables, naturally contain high amounts of glutamate. For example, 100 grams of breast milk contains 229 milligrams of protein-bound glutamate and 22 milligrams of free glutamate. However, for many people, the fact that there is a debate that MSG may have harmful effects and that it has not been proven to be completely harmless is enough to cause reservations for its use. According to the Turkish Food Codex "Directive on Food Additives Other than Colorants and Sweeteners" updated on 22.05.2008, the limit for the use of glutamic acid or its salts in all foodstuffs was determined as 10 g/kg, and in its seasoning substances, it was determined as QS (Unspecified amount, Quantum Satis). In the United States, it was obliged to specify its amounts on the label of foods including high levels of free total glutamate. All food additives can be used with the approval of international and national health and food authorities. International organizations responsible for the safety of food additives are the World Health Organization (WHO) functioning under the United Nations, the Codex Alimentarius Commission established by the Food and Agriculture Organization (FAO), and the "FAO/WHO Joint Expert Committee for Food Additives" named as the JECFA. Additionally, the US-FDA (United States Food and Drug Administration) and the EFSA (European Food Safety Authority) and many other national 248 • Health Sciences Research Papers food safety authorities are responsible for the safety and control of food additives. Monosodium glutamate is considered safe for human health by all the health/food authorities and scientific bodies mentioned above. Consequently, although no direct relationship could be detected, it has been stated in several studies that MSG may cause allergic reactions in some sensitive groups. When used as a flavorer, it is known to cause chest pain, headache, redness on the face, difficulty in breathing, edema and sweating, which is referred to as Chinese Restaurant Syndrome.

DISCUSSION Monosodium glutamate (MSG) is one of the salt forms of glutamic acid, a nonessential amino acid, and is widely used as a taste enhancer food additive. Today, studies evaluating the possible effects of MSG on human health are still ongoing. The reported effects are usually attributed to the direct acts of MSG in the brain, which would affect food intake, body weight and lipid metabolism. MSG has also been interested to the glycolytic process, especially when it is swallowed with carbohydrates, but its effects on glucose metabolism are bad characterized. In a study conducted by Thomas et al., rats were orally given 8 mg/g dose of MSG for 20 consecutive days. Significant increases were observed in the serum alanine amino transferase (ALT) and aspartate amino transferase (AST) activities. Akanya et al., conducted a study in 2010 on the evaluation of changes in some liver functions and hematologic parameters in rats given MSG. As a result of the study, they found that MSG, which was given at a high dose to rats, increased the activity of alkaline phosphatase (ALP) and alanine aminotransferase (ALT). MSG intake in rodents was determined to increase the risk of insulin resistance and obesity and concentrations exceeding the daily human diet were stated to have potential damage to the hypothalamic region that regulates appetite. Recent research studies have demonstrated that MSG exposure produces metabolic changes that can result in serious disorders in animals and humans. Many experimental studies have shown that MSG is toxic to various organs such as liver, brain, thymus and kidneys. In a study named as the Effects of Monosodium Glutamate on the Liver Tissues of Wistar Albino Rats conducted by Shrestha et al., in 2018, it was determined that there was an increase in the liver enzyme alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transferase serum in the MSG-administered experimental group compared to the control group. As the conclusion, it was observed Nurhayat ATASOY • 249 that the MSG influenced the liver histology and liver function of the given rats.In addition to data demonstrating that its excessive use in the neonatal period has harmful effects on nervous system, retina, and kidneys, it is suggested that it causes disorders in learning and memory mechanism, as well as obesity, infertility, growth disorder, Alzheimer, neurodegenerative diseases such as Parkinson and epilepsy with the increasing age.

CONCLUSION As with all additives, the dose is the critical factor for the MSG as well. Certain substances added to the foods for benefit under normal conditions may also cause adverse effects in some cases. This usually emerges as a result of excessive or improper use of the additive, adding it in a wrong step of production or storage, or the poor quality or purity of the additive. Therefore, it is recommended to consume products that have been approved and subjected to inspections by the Ministry of Agriculture, and whose additives, intermediate step products, and end products known to have properly checked in terms of quality and reliability. Only through this way will it be possible to rely on the necessary controls in the appropriateness of additives, as in all other subjects. Otherwise, any other substance administered without any control may also cause adverse health effects. When talking about the safety of any substance, what should be taken into account is that it cannot be considered independently of the environmental conditions, its quantity, exposure time and interaction with other substances consumed with it. Consequently, the purpose of this review is to create awareness about the harmful effects of the MSG in order to encourage higher numbers of scientific studies and raise awareness about this issue.

250 • Health Sciences Research Papers

REFERENCES Altuğ T. (2001): Gıda Katkı Maddeleri. Meta Basım, İzmir. Bağcı T. (1995): "Gıda Katkı Maddeleri ve Gıda Kontrolü"; Halk Sağlığı Temel Bilgiler. Güneş Kitapevi Ltd. Ankara. Di Sebastiano K., Bell K. E., Barnes T., Weeraratne A., Premji T., et al. (2013): Glutamate supplementation is associated with improved glucose metabolism following carbohydrate ingestion in healthy males. British Journal of Nutrition, 110: 2165-2172. Diniz Y. S., Fernandes A. A., Campos K. E., Mani F., Ribas B.O., Novelli E.L. (2004): Toxicity of hypercaloric diet and monosodium glutamate: oxidative stress and metabolic shifting in hepatic tissue. Food and Chemical Toxicology, 42: 313-319. Diniz Y. S., Faine L. A., Galhardi C. M., Rodrigue H. G., Ebai G. X., Burneik R. C., Cicogna AC, Novelli E. L. (2005): Monosodium glutamate in standard and high-fiber diets: metabolic syndrome and oxidative stress in rats. Nutrition, 21: 749-755. Farombi E., Onyem O. O. (2006): Monosodium glutamate-induced oxidative damage and genotoxicity in the rat: modulatory role of vitamin C, vitamin E and quercetin. Human and Experimental Toxicology, 25: 251-259. Hermanussen M, Tresguerre JA 2003. Does high glutamate intake cause obesity? Journal of Pediatric Endocrinology and Metabolism, 16: 965-968. IFIC. Review on Monosodium Glutamate: Examining the Myths. (1994). Ikeda K. (2002) New Seasonings. Chem Senses, 27: 847-849. T., Selmi C., Cha'on U., Pethlert S., Yongvanit P., Areejitranusorn P., Boonsiri P., Khampitak T., Tangrassameeprasert R., Pinitsoontorn C., Prasongwattana V., Gershwin M. E., Hammock B. D. (2012): Monosodium glutamate (MSG) intake is associated with the prevalence of metabolic syndrome in a rural Thai population. Nutrition and Metabolism (Lond), 9: 50. Loliger J. (2000): Function and importance of glutamate for savory foods. Journal of Nutrition, 130: 915-920. Macho L., Fickova M., Zorad S. (2000): Late effects of postnatal administration of Monosodium glutamate on insulin action in adult rats. Physiological Research, 49:79- 85. Olguin M. C., Posadas M. D., Revelant G. C., Marinozzi D., Labourdette V., Venezia M. R. Nurhayat ATASOY • 251

(2018): Monosodium Glutamate Affects Metabolic Syndrome Risk Factors on Obese Adult Rats: A Preliminary Study. Journal of Obesity and Weight- Loss Medication, 4:1- 5 Olney J.W. (1969). Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 164: 719-721. Panel on Food Additives and Nutrient Sources added to Food (2017). Re- evaluation of glutamic acid (E 620), sodium glutamate (E 621), potassium glutamate (E 622), calcium glutamate (E 623), ammonium glutamate (E 624) and magnesium glutamate (E 625) as food additives. EFSA Journal, 15: 4910. Sasaki Y., Suzuki W., Shimada T., Iizuka S., Nakamura S., Nagata M., Fujimoto M., Tsuneyama K., Hokao R., Miyamoto K., Aburada M. (2009). Dose dependent development of diabetes mellitus and non-alcoholic steatohepatitis in monosodium glutamate-induced obese mice. Life Sciences, 85: 490-498. Shrestha S., Jha C. B., Lal Das B. K., Yadav P. (2018). Effects of Monosodium Glutamate on Liver Tissue of Wistar Albino Rats-A Histological and Biochemical Study. International Journal of Therapeutic Applications, Volume 35. Schiffman S.S. (2000). Intensification of sensory properties of foods for the elderly. Journal of Nutrition, 130: 927-930. Rotimi O.A., Olayiwola I.O., Ademuyiwa O, Balogun E.A. (2012). Effects of fibre-enriched diets on tissue lipid profiles of MSG obese rats. Food and Chemical Toxicology, 50 (11): 4062-4067. Thomas M, Sujatha KS, George S 2009. Protective effect of Piper longum Linn. on monosodium glutamate induced oxidative stress in rats. Indian Journal of Experimental Biology, 47: 186-92. Türk Gıda Kodeksi Yönetmeliği. T.C. Resmi Gazete. Sayı: 23172 16. Kasım (1997). Janssen MMT. Food Additives. deVries J(Ed). Food Safety and Toxicology. CRC Press. USA, 1997. Walker, R., Lupien, J.R. (2000). The safety evaluation of monosodium glutamate. Journal of Nutrition, 130: 1049-1052.

LASERS IN DENTISTRY

CHAPTER 16

Özge PARLAR ÖZ1, Mahmut ERÇİL2

1 Dr. Öğr. Üyesi, Gaziantep Üniversitesi, Diş Hekimliği Fakültesi, Protetik Diş Tedavisi Anabilim Dalı, Gaziantep, TÜRKİYE, [email protected] 2 Araş. Gör., Gaziantep Üniversitesi, Diş Hekimliği Fakültesi, Protetik Diş Tedavisi Anabilim Dalı, Gaziantep, TÜRKİYE,

Özge PARLAR ÖZ, Mahmut ERÇİL • 255

INTRODUCTION Light has been used therapeutically for centuries. Using light to treat various pathologies is called “phototherapy” (Daniell,1991). The term “LASER” consists of the first letters of the words “Light Amplification by Stimulated Emission of Radiation”. The word “laser” was first used by Goldon Gould. Lasers emerged when Albert Einstein introduced the theory of “stimulated emission” in 1917, and since then it has changed and developed considerably. The first laser beam was produced experimentally in a laboratory in 1960 by Thedore Harold Mainman, using a synthetic ruby bar made of aluminum oxide coated with chromium oxide. Laser technology was first used in medicine in 1963. The first dentistry-specific lasers were introduced in 1989 (Coluzzi,2008; George,2009).

Since other laser types such as CO2, Nd:YAG, Erbium (Er), Argon, Holmium (Ho) were only experimental, the Ruby laser, which was the only laser in use in those years, was used in studies, and it was found that the energy level required to cut the enamel may harm the dental pulp (Coluzzi,2008). The first devices introduced for intraoral applications were

CO2 lasers. These devices have been used by otorhinolaryngologists and surgeons throughout the 1970s and 80s. The first device designed specifically for dentistry was the Nd:YAG laser, which was FDA approved for soft tissue procedures in 1990. For dental laser devices, FDA approval was also obtained for composite polymerization in 1991, teeth whitening in 1995, subgingival curettage, cavity cleaning and cavity preparation in 1997, and for selective ablation of caries in 1999 (Gimbel,2000; Sulewski,2000).

CHARACTERISTICS OF LASER BEAMS

Monochromaticity The laser light has a special color. This color is called monochromatism. Since the beam has a single wavelength, it has a single color, and this color varies in each laser along the wavelength (Bass et al,1995; Baxter,1994).

Coherence The mixing of the beams in two different phases is called coherence. Coherence in laser beams provides longitudinal and transverse beams (Bass et al,1995; Baxter,1994). 256 • Health Sciences Research Papers

Parallelism and diffraction Unlike other types of light, a laser beam shows a high degree of parallelism. In lasers produced for commercial purposes, the amount of diffraction in laser beams is important, not their degree of parallelism. The diffraction angles of the laser beam vary between 3-10°. Lasers showing high degrees of parallelism have a small diameter and high efficiency. Laser beams with high diffraction rates have less effect since target diameter increases, and they are generally used in low intensity laser therapy (Bass et al,1995; Baxter,1994). The light generated by the laser energy induces four different interactions in the targeted tissue. The first interaction is reflection. Reflection is when the beam bounces off the surface without affecting the tissue. If it contacts another unwanted tissue, it may be harmful to that tissue. The second effect is the ability of the targeted zones to absorb the laser energy. This effect is the primary desired effect obtained from the laser and in particular, it depends on the amount of water in the tissue and the wavelength. The third interaction is transmission. In contrast to absorption, this is when the laser energy moves through the tissue without affecting it. Another interaction of laser light is dissemination. This interaction weakens the laser energy and prevents it from producing a beneficial biological effect. It may have a harmful effect if it comes into contact with an undesirable tissue. However, this effect produces a beneficial effect for polymerized composite resins (Sulewski,2000; Coluzzi, 2000). Factors determining the application areas of laser devices, i.e. the amount of absorption by tissue, are wavelength and waveform. Wavelength: This is the primary feature that determines the absorption of light by the target material. Depending on the tissue, some lasers have a deeper penetration than others, while others have a more shallow penetration, and can only affect the tissue superficially (Dederich,1993; Miserandino et al,1995). Waveform: Laser energy may be in the form of two waves that cause different tissue effects; continuous waveform lasers deliver a high amount of energy to the tissue in a constant and continuous manner with low and medium intensity. Pulsed or threshold waveform lasers deliver small amounts of energy to the tissue in intermittent pulses and generally, in higher intensity (Dederich,1993; Miserandino et al,1995). Özge PARLAR ÖZ, Mahmut ERÇİL • 257

Factors in Laser Tissue Interaction • Wavelength of laser used • Technical application options • Type of tissue applied Photobiological effects of lasers The laser light functions by demonstrating the following effects in tissues; 1. Photochemical, 2. Photothermal, 3. Photomechanical and photoelectric.

Photochemical Effects of Lasers: Photochemical effects are the changes caused by the laser light on a surface and the applied target tissue without any thermal effect. Molecular bonds may be disrupted by the high photon energy, or chemical reactions may be triggered (Vogel et al, 2003). Photothermal Effects of Lasers: Laser energy transforms into thermal effect and heats the tissue. This causes events such as coagulation, protein denaturation (above 60°C), obstruction of small blood vessels, increased lymphatic hemostasis, sterilization of wound sites, and tissue bonding (between 70-90°C) (Dederich,1993; Convissar,2004).

Photomechanical and Photoelectric Effects of Lasers: Photoablation: Fast thermal expansion, mechanical shock waves. Photodistruption: Optical refractions occurring as a result of mechanical shock waves (Harris,1995; Vogel et al, 2003, Moritz 2006).

LASER CLASSIFICATION Lasers may be classified according to the active agent, movement of light, wavelength, energy, or mode of application (Blahnik,2003; Harris,1995; Wintner,2006). Laser classification according to an active agent

➢ Solid-State Lasers: 258 • Health Sciences Research Papers

Nd:YAG,Ho:YAG,Er:YAG,Ruby, Alexandrite, Er,Cr:YSGG

➢ Gas Lasers: CO2, Ar/Krypton, Excimer (Excited Dimer), Ultraviolet (UV), He-Ne

➢ Liquid Lasers: Dye (various) VIS

➢ Electronic Lasers: Semiconductors, Diode Lasers (infrared-IR) Classification according to the movement of laser light

➢ Continuous lasers

➢ Pulse lasers

➢ Chop lasers Classification according the wavelength of laser light

➢ Ultraviolet-UV spectrum (140400 nm)

➢ Visual-VIS spectrum (400-700 nm)

➢ Infrared (IR) spectrum (700 nm and above) Classification according to the energy of laser light

➢ Soft laser

➢ Mid laser

➢ Hard laser Classification according to the application of laser light

➢ Contact

➢ Noncontact

TYPES OF LASERS USED IN DENTISTRY

1- Argon Lasers (487-514 nm) The ion laser is the most commonly used, and these lasers have two visible emission wavelengths. The first has a wavelength of 488 nm and is blue in color. The second has a wavelength of 514 nm and is blue-green Özge PARLAR ÖZ, Mahmut ERÇİL • 259 color. The lasers with a wavelength of 514 nm are highly absorbed in soft tissue. The lasers with a wavelength of 488 nm are the wavelengths needed for camphorquinone. This reduces polymerization time and allows composite resins to absorb less liquid. If there are tooth cavities, they become dark orange and red when the laser comes in contact with the tooth, which makes it easy to find the cavity. It provides effective hemostasis by having a wavelength that is absorbed by hemoglobin. It is not absorbed much in hard dental tissues. It is used in resin polymerization, soft tissue incision, removal of pigmented lesions, removal of vascular anomalies, teeth whitening, diagnosis of dental caries, increasing of resistance to caries, and excessive dentin desensitization (Baxter,1994; Dederich,1993; Sulewski,2000; Fleming,1999; Bader,2000). Studies suggest that argon laser can be used to prevent enamel decalcification by changing the crystal structure of the enamel (Oho T,1990). There are studies suggesting that an argon laser demonstrates its effect not only by increasing the resistance of enamel to demineralization, but also by increasing the intake of minerals from saliva (Flaitz,1996;Westerman,1996). This theory is supported by the results of various scanning electron microscopy studies (Gwinnett,1979). These studies have shown that the argon laser changes the surface properties of the enamel by creating micro-cavities that allow the ions to be captured rather than letting them leak during an acid attack. Free ions form spherical precipitates that occlude micro-cavities and provide a protective effect by including calcium, phosphate, and fluoride ions in the saliva of the enamel surface (McCormack,1995).

2- Nd:YAG Lasers (Neodymium: Yttrium-Aluminum-Garnet Lasers) (1064 nm) This is a crystal laser with good hemostatic properties. This laser is highly absorbed in pigmented tissue, cuts quickly, but it is slower in lighter-colored tissues. In dentistry, it is absorbed by the pigments of bacteria (brown or similar pigments) that often cause infections, and therefore, a rapid healing effect can be observed. As it has the highest penetration depth of all laser systems used in dental surgery as tissues under the surgical surface are also exposed to the laser energy. This causes undesired environmental damage. If the light is directed to the crown or root, the pulp is directly affected. This effect is manifested by reduced pulpal functions such as sensitivity. In fact, decreased sensitivity is 260 • Health Sciences Research Papers preferred by the patient and physician, but it cannot be sure whether this loss of function will require an endodontic treatment. This laser is used in gingival procedures, treatment of oral ulcers, frenectomy, soft tissue surgery, periodontal treatment, increasing of resistance against caries, elimination of dentin hypersensitivity, etching enamel and dentin, sterilization of cavity and root canal, and pulp capping (Miserandino,1995; Bader,2000; Parker,2007). Moritz, et al. investigated the antibacterial efficacy of Nd:YAG laser in their in vivo study. Then, they applied 1.5 W and 30 mj Nd:YAG laser to the root canals of these teeth and examined them by taking samples from the root canals. As a result of the study, they reported that an energy level of 1.5 W and 30 mj is sufficient for the disinfection of root canal, and that laser energy can be used to disinfect root canals (Moritz,1997).

3-Erbium Lasers (Er: YAG and Er, Cr: YSGG) Erbium-Doped: Yttrium-Aluminium-Garnet laser (Er:YAG 2940 nm), and Erbium, Chromium: Yttrium-Scandium-Gallium-Garnet (Er, Cr: YSGG 2780 nm) are often used in dentistry as lasers. Taking into account the mechanism by which lasers create a more acid- resistant surface, there are two accepted theories for crystallographic changes and decomposition of the organic matrix. Although enamel has less than 1% organic content; organic matrix may have great potential to control the diffusion path. Therefore, enamel plays an important role in the prevention of demineralization. Liu and Hsu observed that the band density of the organic matrix decreased by about 30% after laser treatment in a study conducted with Er:YAG laser (Liu Y,2007). The organic matrix can be denatured such that enamel diffusion can be blocked by laser treatment. Liu et al. (2013) reported that hydroxyapatite turns into fluoride hydroxyapatite after sub-ablative low-energy Er:YAG laser treatment following the fluoride treatment, and thus an increase in acid resistance of enamel was observed (Liu Y et al. 2013). The Er, Cr:YSGG laser operates at a 2780 nm wavelength, and although it is often used in cavity preparation due to the ablation mechanism, it was also investigated for protective applications. However, in order to prevent caries, it is important that the laser does not cause ablation on the applied tooth surface, and that it only changes the tissue morphologically or chemically. Consequently, in order to provide an anti-caries effect, studies Özge PARLAR ÖZ, Mahmut ERÇİL • 261 were performed with low energy densities (sub-ablative parameters), and without water cooling (Apel et al. 2004). An application of Er, Cr: YSGG laser at an energy density of 8J/cm2 has been shown to provide an adequate temperature increase to transform the chemical structure of the enamel surface into a less soluble structure (De Freitas et al. 2010). However, higher energy densities may lead to enamel ablation and a greater loss of minerals under acidic conditions (Hossain et al. 2001).

4- Carbon Dioxide (CO2) Lasers (10600 nm) The active substance is gas, it is a soft tissue laser and is the most absorbed laser by hydroxyapatite. It is highly controlled, almost operating by removing cellular layers and conducting the process without contacting the transmitter tip surface. A necrotic zone occurs at a depth of 0.5 mm around the evaporation area, and coagulation occurs in the capillary vessels. The bleeding tendency is very low (Miserandino,1995; Dederich,2004). As the cutting process is not performed with mechanical equipment, the physician cannot detect the tissue’s resistance, and controlling the laser head becomes difficult. This may result in unwanted tissue injuries when working on soft tissue. Wound healing may be delayed for a few days. Tissues have a black- brown appearance due to carbon residues. This dissipates after washing a few times over a few days. The color of the treated area becomes normal and healthy in 10-14 days. This laser is used in soft tissue incision and erosion, gingival procedures, treatment of oral ulcers, frenectomy, preprosthetic surgery, treatment of submucous abscess, hard and soft tissue surgery, periodontal treatment, etching enamel and dentin, increasing resistance against caries, cavity preparation, elimination of dentin hypersensitivity, sterilization of cavity and root canal, shaping of root canal, pulp capping, and teeth whitening (Miserandino,1995; Dederich,2004; Pick et al,1993). 5- Excimer Lasers (193-308 nm) This laser active substance contains inert gas (xenon) and halogen gas (chlorine). The beams of the excimer laser may separate the material from the main mass without burning it, thus causing material loss in hard tissues, despite its low energy. The laser is extremely expensive and has no potential to reduce its cost or size. The short UV wavelength (248 nm) has 262 • Health Sciences Research Papers a carcinogenic potential as it can break chromosomes within the cell nucleus. This laser is used for the sterilization of cavity and root canal, etching enamel or dentin (Sulewski,2000; Vogel,2003).

6- Helium Neon Lasers (632 nm) This was one of the precursors of lasers theoretically presented for the first time and then put into practice in 1961. Only the spot irradiation applications of this laser are known in dentistry. It was used in the removal of pigmented lesions, elimination of dentin hypersensitivity, sterilization of cavity (Baxter,1994; Azevedo,2012).

7- Ruby And Alexandrite Lasers (720-780 nm) Historically, ruby was the first laser and works on the basis of synthetic ruby crystal sticks. In dentistry, red wavelengths allow for a variety of applications. It is used to remove plaque and debris from the root surface (Baxter,1994; Azevedo,2012).

8- Titanium Sapphire Lasers They are the most widely used, adjustable solid-state lasers today. In addition to its high thermal conductivity, the main crystal sapphire has an unusual chemical structure and mechanical hardness (Baxter,1994; Azevedo,2012).

9- KTP Lasers (Potassium Titanyl Phosphate Lasers) (532 nm) It was originally the Nd:YAG laser, with the wavelength changed to have a filter in front of the crystal. The mechanism of action is almost identical to that of the Nd:YAG. Whitening using green light with KTP laser is much more effective than halogen light whitening with blue light. This is used in teeth whitening (Baxter,1994; Azevedo,2012).

10- Low-Level Lasers Lasers used in low-level laser therapy (LLLT) are commonly referred to as “therapeutic lasers”. For this type of non-invasive lasers; names such as soft laser, medical laser, biostimulator laser, soft laser, cold laser are also used. The most important purpose of LLLT is to accelerate the healing of wounds in the tissues. It also has effects that reduce edema, Özge PARLAR ÖZ, Mahmut ERÇİL • 263 inflammation and pain. LLLT has been used as a medical treatment for more than 40 years (Sun G,2004). Laser Types Used in Low-Level Laser Therapy:

➢ Helium-Neon (He-Ne) Laser These laser systems take their name from helium gas and neon atoms present in their active environment. He-Ne laser beams are obtained at a wavelength of 633 nm by stimulating the active environment with electrical energy (Baxter,1995). He-Ne lasers are the most common low- cost systems. Although there are many different types of He-Ne lasers, the ones most commonly used are pen-sized and battery-powered (Baxter,1995; Greguss,1984). He-Ne lasers induce epithelial growth and healing of peripheral nervous system injuries. It can be applied easily with or without contacting the applied surface (Greguss,1984).

➢ Semiconductor Diode Lasers (Gallium-Aluminum-Arsenide, Gallium-Arsenide) Diode lasers contain solid elements such as aluminum, indium, gallium, and arsenide that have semiconductor properties in their inactive environments. Wavelengths of semiconductor diode lasers vary between 800-980 nm depending on the elements used. These wavelengths correspond to the start of the infrared electromagnetic color spectrum. The resulting laser beams can be transmitted in continuous or pulsed modes by means of fiber optic cables. (Coluzzi,2004). The semiconductor diode laser systems allow the beam parameters to be adjusted with their control panel (Baxter,1995).

264 • Health Sciences Research Papers

REFERENCES

Apel C, Birker L, Meister J, et al. (2004). The caries-preventive potential of subablative Er:YAG and Er:YSGG laser radiation in an intraoral model: a pilot study. Photomed Laser Surg, 22(4), 312-7. Azevedo DT, Faraoni-Romano JJ, Derceli JR, Palma-Dibb RG. (2012). Effect of Nd: YAG laser combined with fluoride on the prevention of primary tooth enamel demineralization. Braz Dent J, 23(2), 104-9. Bader HI. (2000). Use of lasers in periodontics. Dent Clin North Am, 44(4), 779- 91. Bass LS, Treat MR. (1995). Laser tissue welding: a comprehensive review of current and future clinical applications. Lasers Surg Med, 17(4),315-49. Baxter CD, Waylonis GW. (1995). Therapeutic lasers: theory and practice. Am J Phys Med Rehabil, 74(4), 327. Baxter GD. (1994). Therapeutic Lasers; Theory and Practice. 1sted. London: Churcill Livingstone;. p.5-20. Blahnik JA, Ringde DW. (2003). Laser Therapy: A Clinical Manual. 1sted. Melbourne, FL: Healing Light Seminars Inc;. p.52-9. Coluzzi DJ. (2000). An overview of laser wavelengths used in dentistry. Dent Clin N Am, 44, 753-765. Coluzzi DJ. (2004). Fundamentals of dental lasers: science and instruments. Dent Clin North Am, 48(4), 751-70. Coluzzi JD. (2008). Fundamentals of lasers in dentistry: basic science, tissue interaction and instrumentation. J Laser Dent, 16, 4-10. Convissar RA. (2004). The biologic rationale for the use of lasers in dentistry. Dent Clin North Am, 48(4), 771-94. Daniell MD, Hill JS. (1991). A history of photodynamic therapy. Aust NZJ Surgery, 61(5), 340-8. De Freitas PM, Rapozo-Hilo M, Eduardo Cde P, et al. (2010). In vitro evaluation of erbium, chro mium:yttrium-scandium-gallium-garnet laser-treated enamel demineralization. Lasers Med Sci, 25(2), 165-70. Dederich DN, Bushick RD, (2004). Lasers in dentistry: separating science from hype. J Am Dent Assoc, 135(2), 204-12. Dederich DN. (1993). Laser/tissue interaction: what happens to laser light when it strikes tissue? J Am Dent Assoc, 124(2), 57-61. Özge PARLAR ÖZ, Mahmut ERÇİL • 265

Flaitz CM, Hicks MJ, Westerman GH, et al. (1995). Argon laser irradiation and acidulated phosphate fluoride treatment in caries-like lesion formation in enamel: an in vitro study. Pediatr Dent, 17(1), 31-5. Fleming MG, Maillet WA. (1999). Photopolymerization of composite resin using the argon laser. J Can Dent Assoc, 65(8), 447-50. George R. (2009). Laser in dentistry-Review. Int Dent J, 1: 13-9. Gimbel CB. (2000). Hard tissue laser procedures. Dent Clin N Am, 44, 931-953. Greguss P. (1984). Low-level laser therapy-reality or myth? Optics & Laser Technology, 16(2), 81-5. Gwinnett AJ, Ceen RF. (1979). Plaque distribution on bonded brackets: a scanning microscope study. Am J Orthod, 75(6), 667-77. Harris DM, Pick RM. (1995). Laser physics. In: Misserandino LJ, Pick RM, eds. Lasers in Dentistry. 1st ed. Hanover Park, IL: Quintessence Publishing Co Inc.. p.27-38. Hossain M, Kimura Y, Nakamura Y, et al. (2001). A study on acquired acid resistance of enamel and dentin irradiated by Er,Cr:YSGG laser. J Clin Laser Med Surg, 19(3), 159-63. Liu Y, Hsu CY, Teo CM, et al. (2013). Potential mechanism for the laser-fluoride effect on enamel demineralization. J Dent Res, 92(1), 71-5. Liu Y, Hsu CY. (2007). Laser-induced compositional changes on enamel: a FT- Raman study. J Dent, 35(3), 226-30. McCormack SM, Fried D, Featherstone JD, et al. (1995). Scanning electron microscope observations of CO2 laser effects on dental enamel. J Dent Res, 74(10), 1702-8. Miserandino LJ, Levy G, Miserandino CA. Laser interaction with biologic tissues. In: Miserandino LJ, Pick RM, eds. (1995) Moritz A, Doertbudak O, Gutknecht N, Goharkhay K, Schoop U, Sperr W. (1997). Nd:YAG laser irradiation of infected root canals in combination with microbiological examinations. J Am Dent Assoc, 128(11), 1525-30. Moritz A, Schoop U, Strassl M, Wintner E. (2006). Cavity preparation. In: Moritz A, ed. Oral Laser Application. 1st ed. Berlin: Quintessenz Verlags;. p.75- 138. Oho T, Morioka T. (1990). A possible mechanism of acquired acid resistance of human dental enamel by laser irradiation. Caries Res, 24(2), 86-92. Parker S. (2007). Surgical laser use in implantology and endodontics. Br Dent J, 202, 377-386. 266 • Health Sciences Research Papers

Parker S. (2007). Verifiable CPD paper: introduction, history of lasers and laser light production. Br Dent J, 202(1), 21-31. Pick RM, Colvard MD. (1993). Current status of lasers in soft tissue dental surgery. J Periodontol, 64(7), 589-602. Sulewski JG. (2000). Historical Survey of Laser Dentistry. Dent Clin Nort Am; 44: 717-752. Sun G, Tuner J. (2004). Low-level laser therapy in dentistry. Dent Clin North Am, 48(4), 1061-76. Vogel A, Venugopalan V. (2003). Mechanisms of pulsed laser ablation of biological tissues. Chem Rev, 103(2), 577-644. Westerman GH, Hicks MJ, Flaitz CM, et al. (1996). Surface morphology of sound enamel after argon laser irradiation: an in vitro scanning electron microscopic study. J Clin Pediatr Dent, 21(1), 55-9. Wintner E, Strassl M. (2006). Basic information on lasers. In: Moritz A, ed. Oral Laser Applications. 1st ed. Berlin: Quintessenz Verlags;. p.1-55.

SPECIAL USAGE AREAS OF LASERS IN DENTISTRY

CHAPTER 17

Özge PARLAR ÖZ1, Mahmut ERÇİL2

1 Dr. Öğr. Üyesi, Gaziantep Üniversitesi, Diş Hekimliği Fakültesi, Protetik Diş Tedavisi Anabilim Dalı, Gaziantep, TÜRKİYE, [email protected] 2 Araş. Gör., Gaziantep Üniversitesi, Diş Hekimliği Fakültesi, Protetik Diş Tedavisi Anabilim Dalı, Gaziantep, TÜRKİYE,

Özge PARLAR ÖZ, Mahmut ERÇİL • 269

INTRODUCTION Using lasers for different purposes in many areas of dentistry is becoming increasingly popular. This is mostly due to many factors such as faster healing of the tissue, shorter application time, and greater patient satisfaction etc. However, if the laser is used incorrectly, it may also cause complications. Therefore, in-vivo and in-vitro studies related to lasers used for dental purposes are required to guide and inform clinicians more accurately. This chapter provides information about the special usage areas of lasers in dentistry, which are listed below: 1. Low-level Laser therapy 2. Protective applications 3. Treatment of dentin sensitivity 4. Caries diagnosis 5. Restorative treatments 6. Endodontic treatments 7. Surgical treatment 8. Surface etching (laser etching) 9. Laser bleaching 10. Teeth scaling in periodontics

1- LOW LEVEL LASER THERAPY (LLLT) LLLT therapy is becoming popular in today's dentistry. As a general rule, in oral applications, doses of 2 to 4 J are applied by means of special oral apparatus to an area of 1 cm2; in extra-oral applications, doses of 4 to 10 J should be applied to an area of 1 cm2. LLLT therapy is used in dentistry for many purposes. These are; - Treatment of inferior alveolar and lingual nerve paresthesia, - Treatment of peri-implantitis, - Treatment of TMJ disorders, - Acceleration of bone healing (especially after dental implant applications), 270 • Health Sciences Research Papers

- Dentin desensitization, - Treatment of aphthous lesions, - Local analgesic and anti-inflammatory, - Supportive treatment for dental infections, - After rapid maxillary expansion, - Supportive after all surgical procedures of soft and hard tissue (Prazeres et al,2013; Nencheva-Sveshtarova et al,2015). No side effects were observed in dental applications. However, in the presence of suspected malignancy, treatment should not be performed except by specialists. Although LLLT therapy does not have a definite contraindication, patients with coagulation disorder should be paid attention to. It has also been reported that patients who were given LLLT therapy, fatigue was increased for a short time and the pain was prolonged temporarily. However, the literature on this subject is limited (Zecha et al,2016).

2- PROTECTIVE APPLICATIONS In addition to anti-caries, remineralization stimulants, and the anti- bacterial effects of fluoride, lasers are known to prevent enamel demineralization and reduce enamel permeability. Researches have shown that the effectiveness of the combined use of laser and fluoride is greater than the effect of laser or fluoride alone. It was determined that fluoride uptake on the enamel surface increased when used with laser (Apel et al. 2004). The efficacy mechanism of the use of laser and fluoride together has not been completely clarified. However, there are some assumptions on this issue. In their study, Liu et al. (2013) stated that the combination of laser and fluoride strengthens the hydroxyapatite structure, organic laser therapy reduces the diffusion from enamel by reducing the organic structure by the photothermal effect, the application of the laser increases the storage of fluoride on the enamel surface leading to a more stable structure, and the use of laser increases the formation of fluoroapatite. In their study published in 2011, Banda et al. (2011) investigated the effects of the use of Nd:YAG lasers with topical fluorides on the surface morphology and microhardness of the enamel of the primary teeth. They stated that the combination of a Nd:YAG laser and acidulated phosphate Özge PARLAR ÖZ, Mahmut ERÇİL • 271 fluoride (APF) gel increases enamel surface hardness, and therefore, this method can provide protection against cariogenic acid attacks. Vitale et al. (2011) investigated how the fluoride uptake of teeth is affected by a diode laser application. They reported that laser application may be significantly effective on the fluoride uptake of teeth. In their study investigating the preventive effect of combined use of CO2 laser and fluoride against dental caries, Esteves-Esteves-Oliveira et al. (2011) found that the combined use of CO2 laser and fluoride reduces the loss of calcium and phosphate on the dentin surface, and therefore may be successful in caries prevention. Moslemi et al.(2009) investigated the effect of the combined use of Er, CR:YSGG laser and APF gel on enamel demineralization. According to the results, the combined use of Er, Cr:YSGG laser and APF gel was found to be more successful in preventing enamel demineralization than using gel or laser alone.

3- TREATMENT OF DENTIN SENSITIVITY Dentin sensitivity is characterized by an acute, sudden and sharp pain that occurs as a result of the tooth being exposed to thermal, chemical, mechanical, or osmotic stimuli. Although there are many theories about the mechanism of dentin sensitivity, the hydrodynamic theory is the most accepted theory. This theory is based on the principle that stimuli that cause the movement of fluid in dentine tubules causes dentin sensitivity. There are many methods used to treat dentin sensitivity. The most commonly used agents in these methods include protein precipitates, tubule-occluding agents, and tubule blockers (Yılmaz et al,2011). The use of lasers in the treatment of dentin sensitivity has brought a new dimension to this field. There are various theories about the mechanism of laser application eliminating the dentin sensitivity. The effects of these lasers may be stated as follows; causing melting and recrystallization in dentin, providing dentinal fluid evaporation, providing analgesia by depressing nerve conduction, and obliterating the tubules by stimulating tertiary dentin formation. Helium-neon (He-Ne) laser, Nd:YAG laser, Er:YAG laser and CO2 lasers are known to have desensitizing effects. Gallium aluminum-arsenide (GaAlAs) lasers and Er,Cr:YSGG lasers are suggested to be effective (Yılmaz et al,2011). 272 • Health Sciences Research Papers

Yılmaz et al (2011) evaluated the effect of GaAlAs laser and Er, Cr:YSGG laser on dentin sensitivity in a randomized controlled clinical trial. Accordingly, it was stated that both lasers have a dentin desensitizing effect, and there was no significant difference between the two laser types. Gholami et al.(2011) evaluated dentin’s desensitizing effects of Nd:YAG, Er, Cr:YSGG, CO2 and diode lasers by comparing them. Accordingly, it was stated that Nd:YAG, Er, Cr:YSGG, and CO2 lasers swift melting and recrystallization of dentin, causes a decrease in dentin sensitivity; however, diode laser does not have a significant effect. Blatz (2012) investigated the efficacy of lasers in treating dentin sensitivity by comparing it with the efficacy of topical desensitizing agents. Accordingly, although the studies included in the collection contain contradictions from time to time, lasers are more effective than topical desensitizing agents.

4- CARIES DIAGNOSIS Caries diagnosis is traditionally done by visual inspection and radiographical methods. However, these methods are subjective; therefore their repeatability is low. As a result of searching for less researcher- dependent and subjective methods of caries diagnosis, laser fluorescence (LF) devices called “diagnodent” (Kavo, Biberach, Germany) and “diagnodent-pen” (Kavo, Biberach, Germany) were developed and introduced to the market (Celiberti et al,2010). The efficacy of the LF device in detecting occlusal caries in primary teeth has been evaluated in many studies in vivo and in vitro, and its performance has been found to be high, such as visual inspection and radiographic measurements. These devices were found to be more effective in determining occlusal dentin caries in primary teeth than in determining caries in enamel. Bitewing radiographs were found to be particularly effective in detecting approximal caries (Neuhaus et al,2011). In their study, Çınar et al. (2013) compared the efficacy of conventional methods and LF devices to determine occlusal caries in primary teeth. Accordingly, they stated that all methods produced similar results, and that the LF pen was found to be more effective than LF. It was determined that visual inspection and LF pen devices can be sufficient for the detection of occlusal caries. Özge PARLAR ÖZ, Mahmut ERÇİL • 273

In the study conducted by Jabloski-Momeni et al. (2011), after measurement of extracted teeth with occlusal caries with the LF device and ICDAS-II method, an evaluation was done after histological sectioning. Accordingly, it has been reported that the combined use of the LF device and the ICDAS system in the diagnosis of occlusal caries will allow for objective evaluation.

5- RESTORATIVE TREATMENTS  Cavity preparation The noise, vibration and pain caused by various dental instruments are effective in the occurrence of dental fear. It is stated that the use of lasers for cavity preparation may prevent some of these disadvantages. Although cavity preparation with a laser produces an unpleasant odor and takes longer time than conventional methods, it has been reported to be preferred by the majority of patients (Hjertton et al,2012). In their studies published in 2012, Bohari et al. (2012) evaluated conventional, chemomechanical, and laser techniques for the removal of caries from primary teeth. Accordingly, they stated that the use of laser and chemomechanical techniques may be suitable for clinical use as they are less painful.  Adhesion Composite resins are used in pediatric dentistry for anterior, posterior direct and indirect restorations, and for bonding of brackets. Acid etching also contributes to the satisfactory bonding of composite resins to dental tissues. Disadvantages that may occur during acid etching can be stated as follows: loss of enamel tissue, differences in the depth of etching, etched surface being easily contaminated, improper washing and drying adversely affecting the bonding strength (Goswami and Sing ,2011). In their study published in 2011, Goswami and Sing (2011) compared the bonding strengths of tooth surfaces etched with acid and the Nd:YAG laser with composite resin. They then examined the tooth sections obtained by scanning electron microscopy (SEM). According to the results, the bonding between Nd:YAG laser-etched enamel surface and composite resin was found to be weaker than the bonding between acid-etched enamel and composite resin. When the SEM images were examined, it was observed that the acid-etched enamel exhibited a typical honeycomb 274 • Health Sciences Research Papers image, while the laser-applied enamel surface exhibited irregular, bubble- like superficial formations that were not sufficient for bonding. In their study published in 2011, Scatena et al. (2011) evaluated the bonding strength achieved by applying Er:YAG laser with acid etching to the dentin of primary teeth. Accordingly, the control group with only a 37% phosphoric acid application, and groups with laser applied at different distances after acid-etching were compared. It has been reported that as the application distance increases, the bonding force increases, and the laser application increases the bonding force. In their study published in 2013, Khogli et al.(2013) investigated the effect of the application of a hydrophilic fissure sealant and conventional fissure sealants after different surface treatments on microleakage and penetration depths. For this purpose, only acid-etching, diamond bur preparation with acid-etching and Er:YAG laser with acid-etching groups were used. Accordingly, it was stated that the least microleakage occurs in combination of acid etching+laser+conventional fissure sealant.

 Polymerization The polymerization process includes the steps of activating the molecules of camphorquinone with blue light, converting the monomers into a polymer network, thus resulting in polymerization. During the conversion of the monomers to the polymer network, the Van der Waals interactions between the monomers are broken, and covalent bonds forming the polymer network are formed. Polymerization shrinkage inevitably occurs in the meantime. Residual monomers that cannot be incorporated into the polymer network may cause gaps between the restorative material and the tooth wall. The light source used is very important in that it initiates polymerization. Conventionally, argon lasers, quartz-tungsten-halogen light sources, plasma arc light sources, light- emitting-diode (LED) light sources are used for polymerization. Recently, diode-pumped solid state (DPSS) lasers have been used for polymerization (Shin et al,2011). In their study published in 2008, Ramos et al. (2008) compared an argon laser and a halogen light source in terms of their effect on the physical properties of composite resins. Flex resistance and microleakage parameters were used for the evaluation. Accordingly, it was concluded that no light source prevents microleakage. Argon lasers have been Özge PARLAR ÖZ, Mahmut ERÇİL • 275 reported to produce similar results to the halogen light source, and they have been reported as an alternative to polymerization. In their study, Jang et al. (2009) compared DPSS lasers by evaluating the physical properties of composite resins polymerized with other light sources. Accordingly, they stated that DPSS lasers showed satisfactory results, and they could be an important alternative to the light sources used in polymerization.

6- ENDODONTIC TREATMENTS With the emergence of fiber optic transmission systems, the possibilities of using lasers in a narrow space such as a root canal have been started to be investigated. Lasers studied for endodontic use include; CO2, XeCl, Argon, Nd:YAG, Er:YAG, Er:YSGG, Ho:YAG, Nd:YAG, and diode lasers. Procedures such as cleaning and disinfection of the canal, pulpotomy and pulpectomy, apex occlusion, polymerization of the canal fillers, and trauma can be performed with lasers (Todea,2004; Parker,2007).

7- SURGICAL TREATMENT Many benign pathologies and oral anomalies that may occur in the soft tissues of pediatric patients can be treated by dentists. Conventional treatment methods of these pathologies include knife incision, electrocauterization and cryosurgery. Laser treatment can be considered as an alternative to these methods (Boj et al,2011). Oral pathologies that can be treated using laser can be stated as follows; frenectomy, vascular lesions, gingival hyperplasia, mucositis, eruption cysts, abscesses, cysts mucocele and ranula lesions, gingival melanin pigmentation, lesions caused by papilloma virus, premalignant lesions, and other soft tissue lesions (Boj et al,2011). The advantages of using laser treatment in soft tissue are as follows; it reduces the need for anesthesia compared to conventional treatments, greatly assists in bleeding control, eliminates the need for sutures, facilitates healing, has anti-inflammatory properties, antibacterial and disinfectant activity, and reduces the need for postoperative care (Boj et al,2011).

276 • Health Sciences Research Papers

8- SURFACE ETCHING (LASER ETCHING): After laser application, the morphology of the enamel surface takes an irregular and etched shape due to microbursts. The etching characteristics of this resulting surface is different from that of acid etching. This conventional method creates a homogeneous structure in which prisms and boundaries are monitored, while surface structures of different sizes and depths occur after laser application (Hobson et al,2002). The etching amount of the surface exposed to the laser increases. There are studies suggesting that the values for etching is lower than the conventional etching method, as well as the studies showing that the values for etching is higher (Ying et al,2004; Nakamura et al,2006). It has been suggested that morphological changes on the surface, resulting irregular sized and shaped structures, increase retention between the tooth surface and restorative material and that Erbium lasers can be used for etching purposes (Usumez et al,2003; Başaran et al,2007). However, there are also studies in the literature indicating that excessive irregularity and fissure on the surface obtained by Erbium laser will affect micro-mechanical retention and are disadvantageous in terms of adhesion (Martínez-Insua; 2000). Also some researchers reported that the highest bonding strength values were achieved after acid etching following the laser application (Svizero,2007; Sasaki;2008).

9- LASER BLEACHING: The purpose of laser bleaching is to provide an effectively powered bleaching process using the most suitable energy source which does not cause any side effects. In the bleaching application, Argon, CO2, and GaAlAs diode and Nd:YAG lasers can be used, and all discolorations, including tetracycline related ones, can be removed (Sasaki;2008).

10- TEETH SCALING: In addition to facilitating conventional scaling with laser application, the process can also be carried out specifically with the laser itself. For this purpose, among the studied Nd:YAG, Er:YAG, CO2, XeCl and Alexandrite lasers. Alexandrite laser, which can selectively remove dental calculus, was found to be the most effective. This laser does not damage tooth surfaces during the procedure (Todea,2004 ;Coluzzi,2000).

Özge PARLAR ÖZ, Mahmut ERÇİL • 277

REFERENCES Apel C, Birker L, Meister J, et al. (2004). The caries-preventive potential of subablative Er:YAG and Er:YSGG laser radiation in an intraoral model: a pilot study. Photomed Laser Surg, 22(4), 312-7. Banda NR, Reddy VG, Shasikiran ND. (2011). Evaluation of primary tooth surface morphology and microhardness after Nd: YAG laser irradiation and APF gel treatment-An in-vitro study. J Clin Pediatr Dent, 35, 377-82. Başaran G, Özer T, Berk N, Hamamcı O. (2007). Etching enamel for orthodontics with an Erbium,Chromium:YttriumScandium-Gallium-Garnet Laser System. Angle Ort, 77, 117-123. Blatz MB. (2012). Laser therapy may be better than topical desensitizing agents for treating dentinal hypersensitivity. J Evid Based Dent Pract, 12, 69-70. Bohari MR, Chunawalla YK, Ahmed BM. (2012). Clinical evaluation of caries removal in primary teeth using conventional, chemomechanical and laser technique: an in vivo study. J Contemp Dent Pract, 13, 40-7. Boj JR, Poirier C, Hernandez M, Espasa E, Espanya A. (2011). Review: laser soft tissue treatments for pediatric dental patients. Eur Arch Pediatr Dent, 12, 100-5. Celiberti P, Leamari VM, Imparato JCP, Braga MM, Mendez FM. (2010). In-vitro ability of a laser fluorescence device in quantifying aproximal caries lesions in primary molars. J Dent, 38, 666-70. Coluzzi DJ. (2000). An overview of laser wavelengths used in dentistry. Dent Clin N Am, 44, 753-765. Çınar Ç, Atabek D, Odabaş ME, Ölmez A. (2013). Comparison of laser fluorescence devices for detection of caries in primary molars. Int Dent J, 63, 97-102. Esteves-Oliveira M, Zezell DM, Ana PA, Yekta SS, Lampert F, Eduardo CP. (2011). Dentine caries inhibition through CO2 laser (10,6μm) irradiation and fluoride application, in vitro. Arch Oral Biol, 56, 533-9. Gholami GA, Fekrazad R, Esmaiel-Nejad A, Kalhori KA. (2011). An evaluation of the occluding effects of Er;Cr:YSGG, Nd:YAG, CO2 and diode lasers on dentinal tubules: a scanning electron microscope in vitro study. Photomed Laser Surg, 29, 115-21. Goswami M, Singh A. (2011). Comperative evaluation of shear bond strength of composite resin bonded to acid etch or Nd:YAG lased enamel. J Indian Soc Pedod Prev Dent, 29, 140-3. 278 • Health Sciences Research Papers

Hjertton PM, Bagesund M. (2013). Er:YAG laser or high-speed bur for cavity preperation in adolescents. Acta Odontol Scand, 71, 610-15. Hobson RS, Rugg-Gunn AJ, Booth TA. (2002). Acid etch patterns on the buccal surface of human permanent teeth. Arch Oral Biol, 47, 407-412. Jabloski-Momeni A, Rickets DNJ, Rolfsen S, Stoll R, Heinzel-Gutenbrunner M, Stachniss V, Pieper K. (2011). Performance of laser fluorescence on tooth surface and histological section. Lasers Med Sci, 26, 171-8. Jang CM, Seol HJ, Kim HI, Kwon YH. (2009). Effect of different blue light-curing systems on the polymerization of nanocomposite resins. Photomed Laser Surg, 27, 871-6. Khogli AE, Cauwels R, Vercruysse C, Verbeeck R, Martens L. (2013). Mikroleakage and penetration of a hydrophilic sealant and a conventional resin based sealant as a function of preperation techniques: a laboratory study. Int J Pediatr Dent, 23, 13-22. Liu Y, Hsu CY, Teo CM, et al. (2013). Potential mechanism for the laser-fluoride effect on enamel demineralization. J Dent Res, 92(1), 71-5. Martínez-Insua A, Da Silva Dominguez L, Rivera FG, Santana-Penín UA. (2000). Differences in bonding to acidetched or Er:YAG-laser-treated enamel and dentin surfaces. J Prosthet Dent, 84, 280-8. Moslemi M, Fekrazad R, Tadayon N, Ghorbani M, Torabzadeh H, Shadkar MM. (2009). Effects of Er,Cr:YSGG laser irradiation and fluoride treatment on acid resistance of the enamel. Pediatr Dent, 31, 409-13. Nakamura Y, Hossain M, Yamada Y, Masuda YM, Jayawardena JA, Matsumoto K. (2006). Basic study of morphological changes and surface roughness of cavities prepared by TEA CO2 laser irradiation. Photomed Laser Surg, 24, 503-507. Nencheva-Sveshtarova S, Sveshtarov V, Uzunov TS, Prodanova K. (2015). Effectiveness of gaalas phototherapy according to diagnostic criteria for tемрoromandibular disorders. Acta Medica Bulgarica, 42(2), 36-41. Neuhaus JW, Rodriguez JA, Hug I, Stich H, Lussi A. (2011). Performance of laser fluorescence devices, visual and radiographic examination for the detection of occlusal caries in primary molars. Clin Oral Invest, 15, 635-41. Parker S. (2007). Surgical laser use in implantology and endodontics. Br Dent J, 202, 377-386. Parker S. (2007). Verifiable CPD paper: introduction, history of lasers and laser light production. Br Dent J, 202(1), 21-31. Özge PARLAR ÖZ, Mahmut ERÇİL • 279

Prazeres LD, Muniz YV, Barros KM, Gerbi ME, Laureano Filho JR. (2013). Effect of infrared laser in the prevention and treatment of paresthesia in orthognathic surgery. J Craniofac Surg, 24(3), 708-11. Ramos-Lloret P, Lacalle Turbino M, Kawano Y, Sanchez Aguilera F, Osorio R, Toledano M. (2008). Flexural properties, microleakage, and degree of conversion of a resin polymerized with conventional light and argon laser. Quintessence Int, 39, 581-6. Sasaki LH, Lobo PDC, Moriyama Y, Watanabe LS, Villaverde AB, Tanaka CS, Moriyama EH, Brugnera A. (2008). Tensile bond strength and SEM analysis of enamel etched with Er:YAG laser and phosphoric acid: A comparative study in vitro. Braz Dent J, 19, 57-61. Scatena C, Torres CP, Gomes-Silva JM, Contente MMMG, Pecora JD, PalmaDibb RG, Borsatto MC. (2011). Shear strength of the bond to primary dentin: influence of Er:YAG laser irradiation distance. Lasers Med Sci, 26, 293-7. Shin DH, Yun DI, Park MG, Ko CC, Garcia-Godoy F, Kim HI Kwon YH. (2011). Influence of DPSS Laser on Polymerization Shrinkage and Mass Change of Resin Composites. Photomed Laser Surg, 29, 545–50. Svizero NR, Carvalho RS, Domingues LA, Pegoraro CN, Francischone CE, Rocha MCA. (2007). Shear bond strength of resin composite to enamel treated with Er:YAG laser and phosphoric acid. Cienc Odontol Bras, 10, 13-18. Todea CDM. (2004). Laser applications in conservative dentistry. TMJ, 54, 392- 405. Usumez A, Aykent F. (2003). Bond strengths of porcelain laminate veneers to tooth surfaces prepared with acid and Er,Cr:YSGG laser etching. J Prosthet Dent, 90, 24-30. Vitale MC, Zaffe D, Botticell AR, Caprioglio C. (2011). Diode laser irradiation and fluoride uptake in human teeth. Eur Arch Paediatr Dent, 12, 90-2. Yılmaz HG, Yılmaz SK, Cengiz E, Bayındır H, Aykaç Y. (2011). Clinical evaluation of Er,Cr:YSGG and GaAlAs laser therapy for treating dentine hypersensitivity: A randomized controlled clinical trial. J Dent, 39, 249- 54. Ying D, Chuah GK, Hsu CY. (2004). Effect of Er:YAG laser and organic matrix on porosity changes in human enamel. J Dent, 32, 41-46. Zecha JA, Raber-Durlacher JE, Nair RG, Epstein JB, Sonis ST, Elad S, et al. (2016). Low level laser therapy/photobiomodulation in the management of side effects of chemoradiation therapy in head and neck cancer: part 1: mechanisms of action, dosimetric, and safety considerations. Support Care Cancer, 24(6), 2781-92.

HERBAL THERAPY FOR LEISHMANIASIS: ROLE OF NATURAL PRODUCTS

CHAPTER 18

Rahsan ILIKCI SAGKAN1, Sercin OZLEM CALISKAN2

1 Usak University, Faculty of Medicine, Department of Medical Biology, Usak, Turkey 2 Usak University, Faculty of Medicine, Department of Biophysics, Usak, Turkey

Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 283

INTRODUCTION Leishmaniasis is a disease caused by Leishmania species and transmitted to humans through the bite of infected Phlebotomus. There are three clinical forms of the disease: cutaneous, mucocutaneous and visceral. According to World Health Organization, approximately 12 million people have been diagnosed with leishmanias in 98 countries around the world and 350 million people at risk. It has been reported that about 2 million new cases every year, and approximately three-quarters of these cases are Cutaneous Leishmaniasis (CL). Leishmaniasis is an important health problem in Turkey. Thirteen of Leishmania species are dermotropic in the world. Leishmania tropica is the main agent of Cutaneous Leishmaniasis in our country. Leishmania species are important in treatment. Because the treatment methods used differ according to the type of Leishmania. Each Leishmania species has spesific biochemical and molecular properties. These differences in Leishmania species are also reflected in the drug susceptibility. Drug resistance has increased with Leishmaniasis in recent years. Therefore, there has been a need to investigate new therapeutic agents and alternative treatment methods in the treatment of Leishmaniasis caused by different species of Leishmania.

Leishmaniasis Leishmaniasis, which are reported to be endemic in 98 countries worldwide, infect humans, as well as wild and domestic carnivores and mammals, and are transmitted by Phlebotomus. It is documented that 350 million people are at risk and 2 million new cases are seen each year. Leishmaniasis is listed to be ninth of the most serious infectious diseases according to the World Health Organization (Hotez et al., 2007; Leishmaniasis & Organization, 2010). The infection varies according to the Leishmania species and the individual's immune resistance. If the infection is left untreated, visceral involvement, which can be fatal, may develop, or it may lead to different clinical manifestations of the skin that are self-healing. Visceral (VL) and cutaneous leishmaniasis (CL) is observed in Turkey. There are 13 dermotropic in the skin. L. tropica, L. major, L. aethiopica and L. infantum are the agents of CL seen in the Old World. Old World CL is a skin disease characterized by lesions that may involve the skin and sometimes mucous membranes and usually heal by leaving a permanent scar and immunity. It is characterized by a painless crusted skin lesion that starts with a papule up to the number of bites and 284 • Health Sciences Research Papers gradually ulcers in 3-6 months after 1-12 weeks after being infected by blood sucking by infected phelebotomus. Lesions are usually seen in exposed parts of the body and often in the face, hands, arms and legs, and the number of lesions (1-20). It is reported that most of CL cases are seen in countries such as Afghanistan, Iran, Syria, Saudi Arabia, Brazil and Peru. In addition, the disease can be seen in people who move to endemic areas in North Europe and North America for different purposes (tourists, soldiers, workers, etc.). Therefore, the disease can threaten all populations (Torres-Guerrero et al., 2017). The disease is one of the notifiable group A diseases in our country and the treatment is applied at the Şark Çıbanı Diagnosis and Treatment Centers established by the Ministry of Health. The Ministry of Health has initiated studies to eradicate the disease from our country. The majority of cases are reported from southeastern Anatolia in Turkey over 2000 cases with KL each year. (Ok et al., 2002; Ozbel et al., 1995). Treatment in CL is performed with the aim of accelerating healing, especially in cosmetically unwanted scar development, especially in mucosal cases, reducing the dissemination of the agent, preventing relapses and preventing the source of the case (Murray and Saravia 2005). Depending on the type of leishmania, the location, severity, number and immune status of the lesion, intralesional therapy, topical therapy, systemic drug therapy or any one or more of the physical methods are applied together in the treatment of CL (Hepburn, 2000). Other important factors of CL treatment are related to the characteristics of the lesion. It has been reported that systemic drug treatment should be preferred in multiple, diffuse, resistant, lymphangitic lesions or mucosal involvement (Alrajhi et al., 2002). For topical treatment of CL, imidazole compounds have been tried and shown to be less effective than other methods (Hepburn, 2000). Among the physical methods, the most commonly used are curettage, surgical excision, cryotherapy, heat application, radiotherapy and laser. It is reported that relapses occur after treatment with these methods (Alkhawajah, 1998). Sodium Stibogluconate (Pentostam ™) and Meglumine Antimonate (Glucantime ™), pentavalent antimony compounds, have been used as the first choice in intralesional therapy for the last 50 years. When these drugs need to be administered systemically, they need to be administered parenterally, as they show toxic effects and their effects can be variable. In addition, the increase in resistance to these drugs is a persistent issue (Croft & Yardley, 2002). Pentavalent antimony Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 285 compounds are applied free of charge by the Ministry of Health as the first choice in our country. It has been reported that various drugs (topical paromomycin, oral miltefosin, ketoconasal, rifampin and zinc) have been tried in CL treatment, especially in endemic areas, and response to treatment at different rates (Berman, 2003).

Plant Extracts in Leishmaniasis Treatment Plants have been used for medicinal purpose since ancient times. Natural extracts of plant have been used as drug for hundreds of years. World Health Organization and many researchers suggested that primarily used in the local treatment of the plants should be researched, so that the synthetics of some natural compounds can be made against the Leishmaniasis (Bhadra, 1993; Carvalho et al., 2000; Weniger et al., 2001). All over the world, studies on this subject have begun, are continuing rapidly and successful results are obtained. A plant screening program for Leishmaniasis was initiated in 1984 in French Guiana based on the traditional medical knowledge of the local population. Singha et al. found that the extracts obtained from five plants in the Madras region of India (Alstonia scholaris, Swertia chirata, Tibouchina semidecandra, Tinospora cordifolia and Nyctanthes arbortristis) showed that the anti-leishmanial activity in the hamster leishmaniasis model (Singha et al., 1992). The anti- leishmanial activity of various plant extracts was tested in vitro on the amastigote stages of Leishmania amazonensis and evaluated in vivo using cutaneous L. amazonensis lesions in mice. As a result, Faramea guianensis plant has been reported that good anti-leishmanial activity (Sauvain et al., 1994). Bocconia integrifolia and B. Pearcei from five plants in the colonial region of Bolivia have been shown to be effective in vitro in anti- lesihmanial effect and Ampelocera edentula, Galipea longiflora and Pera benensis in L. amazonensis-infected mice (Fournet et al., 1994). In Sudan, Azadirachta indica, Maytenus senegalensis and Eucalyptus globulus plants have been reported to have anti-leishmanial effects in vitro on L. major promastigotes (Tahir et al., 1998). Inula montana, Bupleurum rigidum and Scrophularia scorodonia collected from different regions of Spain showed efficacy in terms of antiparasitic activity against L. infantum (Martin et al., 1998). Ethanolic extracts of Yucca filamentosa showed strong activity against L. amazonensis (Plock et al., 2001). Other plants such as Khaya senegalensis and Antigenema senegalense have been reported to have significant effects against L. donovani (Abreu et al., 1999). Studies 286 • Health Sciences Research Papers conducted with Annona muricata against L. braziliensis and L. panamenis in Colombia have shown that its activity is greater than meglumine antimoniate (Glucantime) (Jaramillo et al., 2000). In a study conducted in Mexico; Aphelandra scabra, Byrsonima bucidaefolia, Byrsonima crassifolia, Clusia flava, Cupania dentata, Diphysa carthagenensis, Dorstenia contrajerva, Milleria quinqueflora, Tridax procumbens and Vitex gaumeri species against L. mexicana was highly effective. (2007). In a study conducted in Brazil, it was found that Portulaca werdermannii and P. hirsutissima against L.amazonensis contain active substance with anti- leishmanial effect (J. F. O. Costa et al., 2007). In the screening of plant species used in traditional treatment in the public in Mali, Sarcocephalus latifolius, Zanthoxylum zanthoxyloides, Entada africana, Bobgunnia madagascarensis, Pseudocedrela kotschyi and Psorospermum guineense species were found to contain very strong anti-leishmanial active molecules (2007). Ostan et al. reported that Haplophyllum myrtifolium extract has both in vivo and in vitro activity on L.tropica (Ostan et al., 2007). Ethanol extracts of Tinospora sinensis showed a strong anti- leishmanial effect on Leishmania donovani promastigotes (Singh et al., 2008). Al-Jubouri and colleagues in an in vitro study found that the extracts of M. azedarach and N. oleander fruits showed 97% and 81.5% inhibition of L. tropica promastigotes, respectively (Al-Jubouri et al., 2009). In a study conducted by Kıvcak et al. In 2009, ethanol, water and n-hexane extracts obtained from the leaves of Arbutus unedo were tested in vitro against L. tropica promastigotes and the ethanol extract of A. unedo leaves was found to be more effective than other extracts (Kivçak et al., 2009) ). When anti-leishmanial effects of Tunisian endemic plants, including P. halepensis, against L.major and L.infantum were investigated, anti- leishmanial activities of some essential oils with cytotoxic effect were found to be low. Based on this finding, cytotoxic effect and anti- leishmanial activity are generally not correlated. L. infantum was reported to be more sensitive than L. major in all tested plant extracts (Ahmed et al., 2011). Ozbilge and colleagues reported that antileishmanial effect of some endemic plant extracts on L. tropica in 2014. (Ozbilge et al., 2014). In their study with Veaman et al. L.amanozensis and L.chagasi promastigotes, they showed that Virola surinamensis leaf extract which they solved in hexane was effective (Veiga et al., 2017). Domeneggeti et al. reported that the anti-leishmanial and anti-inflammatory effect of C. brasiliense may have potential as an alternative treatment for Leishmaniasis in a study conducted with L.amanozensis promastigotes in 2018 (Domeneghetti et al., Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 287

2018). In 2018, Ohashi et al. found that nine leaf extracts, the most effective of which were Annona senegalensis and Cassia alata leaf extract, showed anti-leishmanial effect (Ohashi et al., 2018). In addition, Baccharis (Asteraceae) (Tempone et al., 2008), Annona (Annonaceae) (EV Costa et al., 2006), Calophyllum (Clusiaceae) (Brenzan et al., 2007), Copaifera some other plant extracts and purified molecules, ( Fabaceae (Santos et al., 2008), Cassia (Fabaceae) (Sartorelli et al., 2007), Lippia (Verbenaceae) (Tempone et al., 2008), Croton (Euphorbiaceae) (do Socorro S Rosa et al., 2003), Jacaranda (Bignoniaceae) (Passero et al., 2007), Piper (Piperaceae) (Nakamura et al., 2006), Stachytarpheta (Verbenaceae) (Moreira et al., 2007), Guatteria (Annonaceae) (Correa et al., 2006), Plectranthus (Lamiaceae) (Tempone et al., 2008) and Pourouma (Moraceae) (Torres ‐ Santos et al., 2003). Some of the studies on the anti-leishmanial effect of plant extracts have been mentioned above. The various criteria are considered to evaluate the anti-leishmanial or leishmanicidal activity of a compound including the MIC, IC50, and selectivity index. MIC is defined as the minimum inhibitory concentration of a compound or drug against interference. IC50 is defined as the concentration of the drug that causes 50% growth inhibition of the amastigote or promastigote forms of Leishmania spp. (Monzote et al., 2014). The IC50 is also used for host macrophages. CC50 is the concentration of the cytotoxicity of the drug, which usually causes 50% of macrophage death from BALB/c mice (Islamuddin et al., 2012). An anti- leishmanial drug or natural compound is safe when the selectivity index is greater than 10 (Mahmoudvand et al., 2014). It is well documented that various medicinal plants and plant derived compounds such as crude extracts and essential oils administered to in vitro and in vivo animal models with CL and VL caused by different species of Leishmania are summarized in Table 1 and Table 2.

288 • Health Sciences Research Papers

Table 1. Plant-derived compounds against CL and VL in vitro

Table 1. Plant-derived compounds against CL and VL in vitro Guimaraes et al. 2010 80% inhibition, significant reduction in the number of amastigotes and morphological changes in promastigotes of L. amazonensis without any toxicity treatment with Croton pullei var. glabrior De Medeiros et al. 2011 Decreased amastigote and promastigote of L. amazonensis survival without any cytotoxic effect treatment with Lippia sidoides Cham. (Alecrim pimenta) Ghosh et al. 2011 Methanol and chloroform extracts of Valeriana wallichii lead to apoptotic cell death on L. donavani and L.major promastigotes and amastigotes. Iqbal et al. 2012 The significant effect of T. aphylla on parasite mobility and anti-promastigote activity against L.tropica promastigotes. Machado et al. 2012 The inhibition of the growth of L. infantum, L. major and L. tropica promastigotes after the treatment of Cymbopogon citratus and citra.

Colares et al. 2013 Anti-leishmanial activity of Vanillosmopsis arborea and α- bisabolol in L. amazonensis promastigotes

Garcia et al. 2013 Treatment of Piper regnellii var. pallescens lead to programmend cell death, G0/G1 arrest and ROS production against L. amazonensis

Lage et al. 2013 Strychnos pseudoquina prepared with Quercetin, ethyl acetate and strychnobilavoneth solvents in L. amazonensis promastigotes. Ethyl acetate extract and two purified flavonoid, antileishmanial potential without significant toxicity. Mansour et al.2013 Reduction of L.infantum promastigote number after Vitis vinifera treatment Nogueira et al. 2013 A promising anti-leishmanial effect without reduction of parasitic number and cytotoxicity after administration of Physalis angulata on L. amazonensis and L. braziliensis promastigotes Ogeto et al .2013 Inhibition of L.major promastigotes growth by treatment of Aloe Secundiflora Vila-Nova et al. 2013 L. donovani and L. major were more sensitive than L. mexicana as a result of the treatment of Annona muricata acetogenins on L. donovani, L. mexicana and L. major promastigotes. Albuquerque Melo et al. Significant leishmanicidal activity without toxic effect as a 2014 result of treatment of Senna spectabilis to L.major promastigotes Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 289

Machado et al. 2014 Thymus Capitellatus Hoffmanns treatment led to induction of apoptosis without any toxicity on L.infantum, L.major and L.tropica promastigotes. Mahmoudvand et al.2014 Inhibition of promastigote in both strains as a result of Berberis vulgaris and B. barbering treatment in L.major and L.tropica promastigotes Monzote et al. 2014(a) As a result of the treatment of Chenopodium ambrosioides prepared with with ascaridole, carvacrol and caryophyllene solvents had better activity on L. amazonensis amastigote and promastigotes: Great selectivity index in all three oils Rodrigues et al. 2014 Inhibition of peptidase activity of L. amazonensis and L. infantum promastigotes treatment with raw hexanic extract of Arrabidaea chica

Table 2. Plant-derived compounds in vivo studies against CL and VL Calla-Magarinos et al. Reduction of parasitic load and footpath thickness in 2013 Leishmaniasis model as a result of treatment of Galipea longiflora Tiuman et al. 2012 Shrunken skin lesions and anti-leishmanial activity of Calophyllum brasiliense similar to the reference drug Glucantime® in a CL model Monzote et al. 2014 (b) Inhibition of disease progression shown against L.amazonensis amastigote as a result of Bixa orellana treatment in mice Rabito et al. 2014 Decrease in parasite growth and size of mouse lesions in L.amazonensis amastigote and promastigotes treatment with Tanacetum parthenium (L.) treatment Salehabadi et al. 2014 90% and 55% improvement in L. major-induced mouse lesions treated with 20% and 10% root extract Berberis vulgaris L., respectively Yamamoto et al. 2014 Reduction in lesion size after Baccharis uncinella treatment in experimental Leishmania model of L. braziliensis and L. amazonensis mice. Sachdeva et al. 2014(a) L. donavani-induced experimental Leishmania model of Tinospora cordifolia combined with cisplatin resulted in proliferation and differentiation of lymphocytes, Th1 polarisation (induction of IFN-γ and IL-2, decreased IL-4 and IL-2 levels). Sachdeva et al. 2014(b) An increase in both cellular and humoral immunity as a result of treatment of Asparagus racemosus (shatavari) combined with cisplatin in an experimental Leishmania model with L. donovani 290 • Health Sciences Research Papers

Conclusions Although the epidemiology of leishmaniasis is well known, it is still uncontrolled. Scientists from all over the world should devote more attention and time to leishmaniasis, a protozone disease that is of great importance in public health but is neglected. In general, identifying the most effective control strategies to reduce mortality and morbidity, appropriate approaches to control vectors, identifying and combating risk factors, developing safe, non-invasive and inexpensive treatment modalities are among the approaches to the complete elimination of leishmaniasis from developing countries. This review demonstrates that a wide variety of plant extracts exhibiting antileishmanial properties in in vitro and in vivo experimental studies have great potential as drug candidates for the treatment of leishamaniasis. Many compounds extracted from plants can replace synthetic antileishmanial agents to overcome cytotoxicity problems. This section emphasizes the use of natural products as a potential source of new and selective agents that contribute significantly to primary health care. Plant extracts are thought to be promising therapeutic therapeutic agents in place of chemicals for the treatment of possible protozoan diseases, particularly leishmaniasis, and bring the need for the search for alternative new plant extracts.

Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 291

REFERENCES Abreu, P. M., Martins, E. S., Kayser, O., Bindseil, K. U., Siems, K., Seemann, A., & Frevert, J. (1999). Antimicrobial, antitumor and antileishmania screening of medicinal plants from Guinea-Bissau. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 6(3), 187- 195. Ahmed, S. B. H., Sghaier, R. M., Guesmi, F., Kaabi, B., Mejri, M., Attia, H., … Smaali, I. (2011). Evaluation of antileishmanial, cytotoxic and antioxidant activities of essential oils extracted from plants issued from the leishmaniasis-endemic region of Sned (Tunisia). Natural Product Research, 25(12), 1195-1201. Ahua, K. M., Ioset, J.-R., Ioset, K. N., Diallo, D., Mauël, J., & Hostettmann, K. (2007). Antileishmanial activities associated with plants used in the Malian traditional medicine. Journal of Ethnopharmacology, 110(1), 99-104. Alkhawajah, A. (1998). Recent trends in the treatment of cutaneous leishmaniasis. Annals of Saudi Medicine, 18(5), 412-416. Berman, J. (2003). Current treatment approaches to leishmaniasis. Current Opinion in Infectious Diseases, 16(5), 397-401. Bhadra, R. (1993). Antileishmanial agents. Drugs of the Future, 18(5), 451-463. Calla-Magarinos, J; Quispe, T; Gimenez, A; Freysdottir, J; Troye-Blomberg, M and Fernandez, C (2013). Quinolinic alkaloids from Galipea longiflora Krause suppress production of proinflammatory cytokines in vitro and control inflammation in vivo upon Leishmania infection in mice. Scand. J. Immunol., 77: 30-38. Carvalho, P. B. de, Arribas, M. A. da G., & Ferreira, E. I. (2000). Leishmaniasis. What do we know about its chemotherapy? Revista Brasileira de Ciências Farmacêuticas, 36(1), 69-96. Colares, AV; Almeida-Souza, F; Taniwaki, NN; Souza Cda, S; da Costa, JG; Calabrese Kda, S and Abreu-Silva, AL (2013). In vitro antileishmanial activity of essential oil of Vanillosmopsis arborea (Asteraceae) baker. Evid. Based. Complement. Alternat. Med., 2013: 727042. doi: 10.1155/2013/727042. Correa, J. E., Ríos, C. H., del Rosario Castillo, A., Romero, L. I., Ortega-Barría, E., Coley, P. D.,Rios, L. C. (2006). Minor alkaloids from Guatteria dumetorum with antileishmanial activity. Planta Medica, 72(3), 270–272.

292 • Health Sciences Research Papers

Costa, E. V., Pinheiro, M. L. B., Xavier, C. M., Silva, J. R. A., Amaral, A. C. F., Souza, A. D. L.,Leon, L. L. P. (2006). A Pyrimidine-β-carboline and Other Alkaloids from Annona foetida with Antileishmanial Activity. Journal of Natural Products, 69(2), 292-294. Costa, J. F. O., Kiperstok, A. C., David, J. P. de L., David, J. M., Giulietti, A. M., de Queiroz, L. P., … Soares, M. B. P. (2007). Anti-leishmanial and immunomodulatory activities of extracts from Portulaca hirsutissima and Portulaca werdermannii. Fitoterapia, 78(7–8), 510-514. Croft, S. L., & Yardley, V. (2002). Chemotherapy of leishmaniasis. Current Pharmaceutical Design, 8(4), 319-342. Channon JY, Blackwell JM. (1985), “A study of the sensitivity of Leishmania donovani promastigotes and amastigotes to hydrogen peroxide. I. Differences in sensitivity correlate with parasite-mediated removal of hydrogen peroxide.” Parasitology, 91(2):197-206. de Albuquerque Melo, GM; Silva, MC; Guimaraes, TP; Pinheiro, KM; da Matta, CB; de Queiroz, AC; Pivatto, M; Bolzani Vda, S; Alexandre-Moreira, MS and Viegas, CJr (2014). Leishmanicidal activity of the crude extract, fractions and major piperidine alkaloids from the flowers of Senna spectabilis. Phytomedicine. 21: 277-281. de Medeiros, Md; da Silva, AC; Cito, AM; Borges, AR; de Lima, SG; Lopes, JA and Figueiredo, RC (2011). In vitro antileishmanial activity and cytotoxicity of essential oil from Lippia sidoides Cham. Parasitol. Int., 60: 237-241. do Socorro S Rosa, M. do S. S., Mendonça-Filho, R. R., Bizzo, H. R., de Almeida Rodrigues, I., Soares, R. M. A., Souto-Padrón, T., … Lopes, A. H. C. S. (2003). Antileishmanial activity of a linalool-rich essential oil from Croton cajucara. Antimicrobial Agents and Chemotherapy, 47(6), 1895-1901. Domeneghetti, L., Demarchi, I. G., Caitano, J. Z., Pedroso, R. B., Silveira, T. G. V., & Lonardoni, M. V. C. (2018). Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death. Mediators of Inflammation, 2018, 6148351. Fournet, A., Barrios, A. A., & Muñoz, V. (1994). Leishmanicidal and trypanocidal activities of Bolivian medicinal plants. Journal of Ethnopharmacology, 41(1–2), 19-37. Hepburn, N. C. (2000). Cutaneous leishmaniasis. Clinical and Experimental Dermatology, 25(5), 363-370.

Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 293

Hotez, P. J., Molyneux, D. H., Fenwick, A., Kumaresan, J., Sachs, S. E., Sachs, J. D., & Savioli, L. (2007). Control of Neglected Tropical Diseases. New England Journal of Medicine, 357(10), 1018-1027. Gantt KR, Goldman TL, McCormick ML, Miller MA, Jeronimo SM, Nascimento ET, (2001), “Oxidative responses of human and murine macrophages during phagocytosis of leishmania chagasi,” Journal of immunology,167(2):893-901. Jaramillo, M. C., Arango, G. J., González, M. C., Robledo, S. M., & Velez, I. D. (2000). Cytotoxicity and antileishmanial activity of Annona muricata pericarp. Fitoterapia, 71(2), 183–186. Garcia, FP; Lazarin-Bidoia, D; Ueda-Nakamura, T; Silva Sde, O and Nakamura, CV (2013). Eupomatenoid-5 isolated from leaves of Piper regnellii induces apoptosis in Leishmania amazonensis. Evid. Based. Complement. Alternat. Med., 2013: 940531. doi: 10.1155/2013/940531. Ghosh, S; Debnath, S; Hazra, S; Hartung, A; Thomale, K; Schultheis, M; Kapkova, P; Schurigt, U; Moll, H; Holzgrabe, U and Hazra, B (2011). Valeriana wallichii root extracts and fractions with activity against Leishmania spp. Parasitol. Res., 108: 861-871. Guimaraes, LR; Rodrigues, AP; Marinho, PS; Muller, AH; Guilhon, GM; Santos, LS; do Nascimento, JL and Silva, EO (2010). Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis. Parasitol. Res., 107: 1075-1081. Iqbal, H; Khattak, B; Ayaz, S; Rehman, A; Ishfaq, M; Naseer Abbas, M; Rehman, H; Waheed, S and Wahab, A (2012). Comparative efficacy of Aloe vera and Tamarix aphylla against cutaneous leishmaniasis. IJBMSP., 2: 42-45. Jaramillo, M. C., Arango, G. J., González, M. C., Robledo, S. M., & Velez, I. D. (2000). Cytotoxicity and antileishmanial activity of Annona muricata pericarp. Fitoterapia, 71(2), 183-186. Kivçak, B., Mert, T., Ertabaklar, H., Balcioğlu, I. C., & Ozensoy Töz, S. (2009). In vitro activity of Arbutus unedo against Leishmania tropica promastigotes. Turkiye Parazitolojii Dergisi, 33(2), 114-115. Lage, PS; de Andrade, PH; Lopes Ade, S; Chavez Fumagalli, MA; Valadares, DG; Duarte, MC; Pagliara Lage, D; Costa, LE; Martins, VT; Ribeiro, TG; Filho, JD; Tavares, CA; de Padua, RM; Leite, JP and Coelho, EA (2013). Strychnos pseudoquina and its purified compounds present an effective in vitro antileishmanial activity. Evid. Based. Complement. Alternat. Med., 2013: 304354. doi: 10.1155/2013/304354.

294 • Health Sciences Research Papers

Leishmaniases, W. E. C. on the C. of the, & Organization, W. H. (2010). Control of the leishmaniases: report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases, Geneva, 22-26 March 2010. Machado, M; Pires, P; Dinis, AM; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C and Sousa, MC (2012). Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major. Exp. Parasitol., 130: 223-231. Machado, M; Dinis, AM; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C and Sousa, MC (2014). Activity of Thymus capitellatus volatile extract, 1,8-cineole and borneol against Leishmania species. Vet. Parasitol.,200: 39-49. Mahmoudvand, H; Ayatollahi-Mousavi, SA; Sepahvand, A; Sharififar, F; Ezatpour, B; Gorohi, F; Saedi Dezaki, E and Jahanbakhsh, S (2014). Antifungal, antileishmanial, and cytotoxicity activities of various extracts of Berberis vulgaris (Berberidaceae) and its active principle berberine. ISRN. Pharmacol., 2014: 602436. doi: 10.1155/2014/ 602436. Mansour, R; Haouas, N; Ben Kahla-Nakbi, A; Hammami, S; Mighri, Z; Mhenni, F and Babba, H (2013). The effect of Vitis vinifera L. leaves extract on Leishmania infantum. Iran. J. Pharm. Res., 12: 349-355. Martin, T., Villaescusa, L., Gasquet, M., Delmas, F., Bartolome, C., Diaz-Lanza, A. M., … Balansard, G. (1998). Screening for protozoocidal activity of Spanish plants. Pharmaceutical Biology. Monzote, L; Garcia, M; Pastor, J; Gil, L; Scull, R; Maes, L; Cos, P and Gille, L (2014a). Essential oil from Chenopodium ambrosioides and main components: activity against Leishmania, their mitochondria and other microorganisms. Exp. Parasitol., 136: 20-26. Monzote, L; Garcia, M; Scull, R; Cuellar, A and Setzer, WN (2014b). Antileishmanial activity of the essential oil from Bixa orellana. Phytother. Res., 28: 753-758. Moreira, R. C. R., Costa, G. C., Lopes, T. C., Bezerra, J. L., Guerra, R. N. M., Rebêlo, J. M. M., … Costa, J. M. L. (2007). In vitro leishmanicidal effect of Stachytarpheta cayennensis (Rich.) Vahl (Verbenaceae). Revista Brasileira de Farmacognosia, 17(1), 59–63. Murray, H. W., Berman, J. D., Davies, C. R., & Saravia, N. G. (2005). Advances in leishmaniasis. Lancet (London, England), 366(9496), 1561-1577.

Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 295

Murray HW, (1981), “Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages,” The Journal of Experiental Medicine, 153(5):1302-15. Murray HW, Berman JD, Davies CR, Saravia NG, (2005), “Advances in leishmaniasis,” Lancet, 4;366 (9496):1561-77. Nakamura, C. V., Santos, A. O., Vendrametto, M. C., Luize, P. S., Filho, D., P, B., … Ueda-Nakamura, T. (2006). Antileishmanial activity of hydroalcoholic extract and fractions obtained from leaves of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.). Revista Brasileira de Farmacognosia, 16(1), 61-66. Nogueiraa R.C, Fabiana V.P.C.R, Nonatoa R, Tomassinib T.C.B, Ribeirob I.M, dos Santosa R.R, Soaresa M.B.P.(2013) Genotoxicity and antileishmanial activity evaluation of Physalis angulata concentrated ethanolic extract. Environmental Toxicology and Pharmacology,36:(3),1304-1311. Ogeto, TK; Odhiambo, RA; Shivairo, RS; Muleke, CI; Osero, BO; Anjili, C; Ingonga, JM and Osuga, IM (2013). Antileishmanial activity of Aloe secundiflora plant extracts against Leishmania major. Adv. Life. Sci. Tech., 13: 9-18. Ohashi, M., Amoa-Bosompem, M., Kwofie, K. D., Agyapong, J., Adegle, R., Sakyiamah, M. M., … Ohta, N. (y.y.). In vitro antiprotozoan activity and mechanisms of action of selected Ghanaian medicinal plants against Trypanosoma, Leishmania, and Plasmodium parasites. Phytotherapy Research, 0(0). Ok, U. Z., Balcioğlu, I. C., Taylan Ozkan, A., Ozensoy, S., & Ozbel, Y. (2002). Leishmaniasis in Turkey. Acta Tropica, 84(1), 43-48. Ostan, I., Saglam, H., Limoncu, M. E., Ertabaklar, H., Toz, S. O., Ozbel, Y., & Ozbilgin, A. (2007). In vitro and in vivo activities of Haplophyllum myrtifolium against Leishmania tropica. The New Microbiologica, 30(4), 439-445. Ozbilgin, A., Durmuskahya, C., Kayalar, H., Ertabaklar, H., Gunduz, C., Ural, I. O., … Ozbel, Y. (2014). Antileishmanial Activity of Selected Turkish Medicinal Plants. Tropical Journal of Pharmaceutical Research, 13(12), 2047-2055. Passero, L. F. D., Castro, A. A., Tomokane, T. Y., Kato, M. J., Paulinetti, T. F., Corbett, C. E. P., & Laurenti, M. D. (2007). Anti-leishmania activity of semi-purified fraction of Jacaranda puberula leaves. Parasitology Research, 101(3), 677–680.

296 • Health Sciences Research Papers

Peraza-Sánchez, S. R., Cen-Pacheco, F., Noh-Chimal, A., May-Pat, F., Simá- Polanco, P., Dumonteil, E., … Mut-Martín, M. (2007). Leishmanicidal evaluation of extracts from native plants of the Yucatan peninsula. Fitoterapia, 78(4), 315-318. Plock, A., Sokolowska-Köhler, W., & Presber, W. (2001). Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp. Experimental Parasitology, 97(3), 141-153. Rabito, MF; Britta, EA; Pelegrini, BL; Scariot, DB; Almeida, MB; Nixdorf, SL; Nakamura, CV and Ferreira, IC (2014). In vitro and in vivo antileishmanial activity of sesquiterpene lactone-rich dichloromethane fraction obtained from Tanacetum parthenium (L.) Schultz- Bip. Exp. Parasitol., 143: 18-23. Rodrigues, IA; Azevedo, MM; Chaves, FC; Alviano, CS; Alviano, DS and Vermelho, AB (2014). Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania spp. Biomed. Res. Int., 2014: 985171. doi: 10.1155/2014/985171. Sachdeva, H; Sehgal, R and Kaur, S (2014a). Tinospora cordifolia as a protective and immunomodulatory agent in combination with cisplatin against murine visceral leishmaniasis. Exp. Parasitol., 137: 53-65. Sachdeva, H; Sehgal, R and Kaur, S (2014b). Asparagus racemosus ameliorates cisplatin induced toxicities and augments its antileishmanial activity by immunomodulation in vivo. Parasitol. Int., 63: 21-30. Salehabadi, A; Karamian, M; Farzad, MH and Namaei, MH (2014). Effect of root bark extract of Berberis vulgaris L. on Leishmania major on BALB/c mice. Parasitol. Res., 113: 953-957. Santos, A. O., Ueda-Nakamura, T., Dias Filho, B. P., Veiga Junior, V. F., Pinto, A. C., & Nakamura, C. V. (2008). Effect of Brazilian copaiba oils on Leishmania amazonensis. Journal of Ethnopharmacology, 120(2), 204-208. Sartorelli, P., Andrade, S. P., Melhem, M. S. C., Prado, F. O., & Tempone, A. G. (2007). Isolation of antileishmanial sterol from the fruits of Cassia fistula using bioguided fractionation. Phytotherapy Research: PTR, 21(7), 644- 647. Sauvain, M., Dedet, J.-P., Kunesch, N., & Poisson, J. (1994). Isolation of Flavans from the Amazonian Shrub Faramea guianensis. Journal of Natural Products, 57(3), 403-406. Singh, N., Kumar, A., Gupta, P., Chand, K., Samant, M., Maurya, R., & Dube, A. (2008). Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis. Parasitology Research, 102(3), 561-565. Rahsan ILIKCI SAGKAN, Sercin OZLEM CALISKAN • 297

Singha, U. K., Guru, P. Y., Sen, A. B., & Tandon, J. S. (1992). Antileishmanial Activity of Traditional Plants against Leishmania donovani in Golden Hamsters. International Journal of Pharmacognosy, 30(4), 289-295. Tahir, A. E., Ibrahim, A. M., Satti, G. M. H., Theander, T. G., Kharazmi, A., & Khalid, S. A. (1998). The potential antileishmanial activity of some Sudanese medicinal plants. Phytotherapy Research, 12(8), 576-579. Tempone, A. G., Sartorelli, P., Teixeira, D., Prado, F. O., Calixto, I. A., Lorenzi, H., & Melhem, M. S. (2008). Brazilian flora extracts as source of novel antileishmanial and antifungal compounds. Memórias do Instituto Oswaldo Cruz, 103(5), 443-449. Tiuman, TS; Brenzan, MA; Ueda-Nakamura, T; Filho, BP; Cortez, DA and Nakamura, CV (2012). Intramuscular and topical treatment of cutaneous leishmaniasis lesions in mice infected with Leishmania amazonensis using coumarin (-) mammea A/BB. Phytomedicine. 19: 1196- 1199. Torres-Guerrero, E., Quintanilla-Cedillo, M. R., Ruiz-Esmenjaud, J., & Arenas, R. (2017). Leishmaniasis: a review. F1000Research, 6. Torres‐Santos, E. C., Silva, S. A. G. D., Costa, S. S., Santos, A. P. P. T., Almeida, A. P., & Rossi‐Bergmann, B. (2003). Toxicological analysis and effectiveness of oral Kalanchoe pinnata on a human case of cutaneous leishmaniasis. Phytotherapy Research, 17(7), 801-803. Vila-Nova, NS; de Morais, SM; Falcao, MJ; Alcantara, TT; Ferreira, PA; Cavalcanti, ES; Vieira, IG; Campello, CC and Wilson, M (2013). Different susceptibilities of Leishmania spp. promastigotes to the Annona muricate acetogenins annonacinone and corossolone, and the Platymiscium floribundum coumarin scoparone. Exp. Parasitol., 133: 334-338. Veiga, A., Albuquerque, K., Corrêa, M. E., Brigido, H., Silva E Silva, J., Campos, M., … Dolabela, M. (2017). Leishmania amazonensis and Leishmania chagasi: In vitro leishmanicide activity of Virola surinamensis (rol.) warb. Experimental Parasitology, 175, 68-73. Weniger, B., Robledo, S., Arango, G. J., Deharo, E., Aragón, R., Muñoz, V., … Anton, R. (2001). Antiprotozoal activities of Colombian plants. Journal of Ethnopharmacology, 78(2-3), 193-200. Yamamoto, ES; Campos, BL; Laurenti, MD; Lago, JH; Grecco Sdos, S; Corbett, CE and Passero, LF (2014). Treatment with triterpenic fraction purified from Baccharis uncinella leaves inhibits Leishmania amazonensis spreading and improves Th1 immune response in infected mice. Parasitol. Res., 113: 333-339.

FOOD SUPPLEMENTS FOR MOTHERS-BABIES AND RELATED ANALYSIS

CHAPTER 19

Esra KIZAR, Serap SAĞLIK ASLAN1

1 Istanbul University Faculty of Pharmacy Department of Analytical Chemistry 34116 Beyazit Istanbul Turkey [email protected] [email protected]

Esra KIZAR, Serap SAĞLIK ASLAN • 301

INTRODUCTION Nowadays, the need for supplementary foods has increased, as people's diets change, their meals skip, and their tendency to fast food increases. In the past, people who fed regularly did not need such things. But in a fast lifestyle, our bodies are deprived of some essential and essential nutrients. They can also be stored in a box and taken in daily doses by means of supplements. The Sen Dietary Supplement Health and Education Act - (DSHEA), adopted by the US Senate in 1994, provides one or more nutritional supplements (minerals, vitamins, amino acids, and plant drugs) used to support diet. tablets, capsules and liquid form (Halsted, 2003; Tek and Pekcan, 2008; McWhorter, 2009; Geller et al., 2015; Rautiainen et al., 2016). DSHEA; categorize food supplements under the general name of alar foods çok rather than medication (McWhorter, 2009). In addition, it is stated that food supplements are used for performance enhancement, cosmetic or balanced nutrition, strengthening the immune system and improving some diseases (Petroczi et al., 2011; Soare et al., 2014; Rautiainen et al., 2016). The prevalence and frequency of use of food supplements has been increasing in recent years. In a study conducted in 2001, 61.2% of 376 adults randomly selected used food supplements; on the advice of some of these patients from their doctor; most of them are reported to use food supplements via family, friends or the internet (Harnack et al., 2001). According to the study conducted by Durante et al. (2001); In a study of 118 patients, the use of vitamin, mineral or herbal supplements was 73% in women; It is reported that 44% of males and 70% of users under 50 years of age, 26% of those over 50 years of age use food supplements without the knowledge of their doctors. In particular, it was found that young patients found food supplements safer and more effective than using medications recommended by the doctor. The use of food supplements is known to become widespread and popular in recent years, especially through sales strategies, advertisements and the Internet (Halsted, 2003). Nutritional needs of individuals vary according to age, gender, pregnancy. Pregnancy is a period in which nutrition is very important both for maternal and child health. There are some micronutrients that we should take in addition to our normal diet during pregnancy. In this study, 302 • Health Sciences Research Papers nutritional supplements and analysis required for mothers and infants were examined.

FOOD SUPPLEMENT Food supplement; nutrients such as vitamins, minerals, proteins, carbohydrates, fiber, fatty acids, amino acids, and / or concentrates or extracts of plant, plant and animal origin substances, bioactive substances and the like, which have nutritive or physiological effects. refers to the product for which the daily dose of administration is determined by itself or in mixtures thereof in capsule, tablet, lozenge, disposable powder package, liquid ampoule, dropper bottle and other similar liquid or powder forms.

SPECIAL NURITION NEEDS DURING PREGNANCY Deficiency in pregnancy causes congenital malformations such as neural tube defects, orfacial clefts, cardiac anomalies, miscarriages, anemia and preeclampsia. Adequate intake before and during pregnancy is important because it is protective against neural tube defect. It is recommended that pregnant women consume 600 µg of diet folate equivalent of all foods daily. All women, including adolescents, are advised to take 400 µg of enriched nutrients and / or supplements daily in addition to folate from food sources. It is recommended that mothers with a neural tube defect should receive support from health institutions for the use of 4,000 g folic acid during the first trimester of pregnancy (Procter and Campbell, 2014). Calcium deficiency during pregnancy is associated with preeclampsia and restriction of intrauterine growth. The use of calcium supplementation during pregnancy reduces preeclampsia (Barger, 2010). When sufficient calcium is not taken during pregnancy, the requirement is met by withdrawing calcium from the bones. Frequent births, lack of movement and inadequate use of sunlight cause bone softening and tooth decay. Vitamin D should also be taken sufficiently to facilitate the absorption of calcium. Increased calcium intake during pregnancy is important for the health of the mother and the baby. Calcium supplementation should be used if dietary sources are not sufficient. The United States Medical Institute's recommendation of the Institute of Medicine is 1000 mg / day for pregnant and lactating women aged 19-50 years and 1300 mg / day for 14-18 years (Uzdil and Özenoğlu, 2015). Esra KIZAR, Serap SAĞLIK ASLAN • 303

Iron deficiency is the most common micronutrient deficiency that affects pregnant and lactating women and young children and results in anemia. Iron deficiency in the first two trimesters of pregnancy leads to increased preterm birth, low birth weight and increased infant mortality. Increased plasma volume in pregnant women, increased iron requirement of the fetus, high consumption of phytate-containing cereals that prevent iron absorption, intestinal parasites, frequent births, excessive tea and coffee consumption adversely affect iron absorption, and this leads to the development of iron deficiency anemia. anemia in eastern Turkey, 4.5% of pregnant women in research in 2010 (Hb <11 g / dL) in 40.3% iron deficiency (ferritin <15 microg / L) in 10.9% and vitamin B12 (<200 pg / ml) and iron deficiency was also detected (Karabulut et al., 2011). Iodine is essential for normal brain development and growth. Severe iodine deficiencies are seen as infertility, miscarriages, stillbirths, cognitive dysfunction, and an increase in the frequency of low-weight births. Medium iodine deficiency, defined as the detection of 50-150 µg / day iodine in urine, increases the risk of developing goiter in the mother and fetus. In the seminar on kullanım Use of iodine and folic acid supplementation in pregnancy and lactation In Spain, iodine salt, iodine content of milk and dairy products should be at a level that meets the needs of pregnant and lactating women. (Donnay et al., 2014). Accordingly, although the use of supplements such as potassium iodinate is not deemed necessary, the use of iodine supplements pharmacologically during pregnancy and lactation should be used selectively in cases of high-risk iodine deficiency or decreased thyroid functions. Adequate intake of vitamin A is required for visual function, fetal growth, immunity and epithelial tissue integrity. Estimated Average Requirement for Pregnant Women 14-18 years old (530) RAg RAE / day and the recommended dietary intake (RDA) is 770 µg / day. However, the upper limit for tolerable vitamin A precursor (UL = Upper Level) is 2800- 3000 µg / day. The recommended upper limit for retinol support is 3000 IU / day. High retinol supplementation of 8000-10000 IU / day does not carry an increased risk of malformation. It is known that vitamin A supplement increases the birth weight and growth of the child born from an HIV-infected woman, but it should be avoided. (Hovdenak and Haram, 2012) 304 • Health Sciences Research Papers

Approximately 50% of biochemical thiamine deficiency develops during pregnancy, serum levels fall but remain within normal limits in many women. Thiamine is required for insulin production and cell growth from pancreatic beta cells for glucose oxidation. Thiamine administration increases glucose tolerance in pregnant women, provides intrauterine growth and prevents low birth weight (Bakker et al., 2000) Thiamine deficiency is also present in newborns born in 19% of 77 pregnant women with low birth weight and vitamin deficiencies. also available (Baker et al., 2000). Thiamine concentration of all newborns is higher than pregnant women. At the same time, thiamine concentration in cord blood is higher in mothers receiving insulin therapy for gestational diabetes (GDM). Significant weight reduction is observed in babies born to mothers receiving GDM treatment. It may cause healthy pregnant women to have macrosomic babies, but only macrosomic babies treated with diabetes have a marked decrease in thiamine concentration in blood. Riboflavin acts as a coenzyme in tissues that produce energy through respiration in oxidation and reduction reactions. It is also associated with protein and energy metabolism and erythropoiesis. Agte et al. (1998), growth parameters in the average weight gained during pregnancy on the 21st day of average fetus weight and hemoglobin percentage of riboflavin supplementation, reported that significant improvements in mice. Riboflavin has the capacity to make complex. Thus, it increases the absorption of iron and zinc by increasing cellular transport. Therefore, riboflavin has direct and indirect effects on growth. The precise mechanism of riboflavin transport from the placenta has not been well studied. It was not stated that pantothenic acid should be confirmed in pregnant and non-pregnant subjects. When pantothenic acid supplementation is performed to pregnant women, the level increases. If pantothenic acid deficiency is present in rats with cerebral disorders (Moghissi KS., 1981), there is no evidence that this causes congenital malformation in humans. Niacin is an important component of two enzymes in glucolysis and tissue respiration. Many enzyme reactions involving NAD and NADP contain nicotinamide components. During pregnancy, niacin is reduced in serum and niacin metabolites are increased in urine (Moghissi, 1981). Generally, niacin deficiency is rare and it has not been reported that any effect of niacin is low or high in pregnant women. Esra KIZAR, Serap SAĞLIK ASLAN • 305

Vitamin B6 helps the development of the central nervous system. Although vitamin B6 levels decrease during pregnancy, it is unclear whether pregnancy is a process leading to vitamin B6 deficiency and whether this deficiency is associated with an important clinical picture. Pyridoxine is also of interest due to the protection of the placental vascular bed. It is also protective against tooth decay. Hillman et al. (1962) investigated the effect of two types of pyridoxine treatment on dental caries. Local and systemic pyridoxine treatment has been shown to prevent tooth decay compared to placebo. Swartwout et al. (1960) investigated the histological effect of antenatally administered pyridoxine and placebo on placental arterioles. Vitamin B12 is a coenzyme essential for erythropoiesis and lipid, protein, carbohydrate metabolism. It is synthesized in the liver and called extrinsic factor. Although it acts on all cells, bone marrow plays a major role in the gastrointestinal system (GIS) and central nervous system (CNS). It acts as cofactor in bone marrow and DNA synthesis. Due to insufficient DNA synthesis, erythroblasts are indivisible and erythroblasts are given to the blood as megaloblast and megaloblastic anemia occurs. It is not clear whether folate deficiency or vitamin B12 deficiency contributes to this anemia. The exact effect of vitamin D function during pregnancy has not been defined for the mother and the fetus. However, although it is recommended as a prophylaxis against some problems during pregnancy such as low birth weight, its necessity, safety and efficacy of vitamin D supplementation are controversial. In addition to bone health, glucose regulation is also important for immune function and uterine contraction at birth. The United States Medical Institute (Institute of Medicine) recommends 600 IU of vitamin D to meet the daily needs of pregnant women of any age (Erata and Güçlü, 2003). The main physiological effect that binds to vitamin E is that it is an antioxidant. The maternal level of vitamin E increases during pregnancy, peaks at around 37 weeks and returns to pre-pregnancy levels immediately after birth. The etiology of the increase in vitamin E during pregnancy may be attributed to the increased need for fetal growth, but probably reflects the normal hyperlipidemic condition associated with pregnancy. The mechanism of vitamin E transport from the maternal circulation to the fetal circulation is not clear, but the serum tocopherol level of the cord blood is 306 • Health Sciences Research Papers approximately one third of the levels in the mother. Vitamin E deficiency in animals is associated with gonadal atrophy, degeneration of the placenta, possible resorption of the embryo, and increased hemolysis in erythrocytes. Studies in Japan found that vitamin E deficiency in human pregnancies was associated with a low threat, premature rupture of membranes, intrauterine growth retardation and neonatal hemolytic anemia. Reactive oxygen species, which emerge as the body's response to many harmful conditions, such as infection, cigarette smoke, bleeding, or cocaine use, can activate collagenolytic enzymes and disrupt fetal membrane integrity. Vitamin K functions as a cofactor for the production of prothrombin, factors 7, 9 and 10 in the liver. The only clinical practice of vitamin K in the perinatal period is based on the fact that premature babies develop vitamin K deficiency more frequently than term babies. This develops due to decreased tissue stores and impaired intestinal absorption. Therefore, premature babies show coagulation disorder more frequently as a sign of vitamin K deficiency. Pregnant women receiving coumadine, a vitamin K antagonist, during pregnancy are at risk of intrauterine bleeding with their fetuses. The exact mechanism of vitamin K passage through the placenta is not understood. Studies in rats have suggested a facilitated diffusion pathway. The increase in total fats, saturated fats and linoleic acid associated with modern nutrition has led to the formation of a pro-inflammatory environment. Inflammation is the cause of many pregnancy problems; Pregnancy and postpartum depression, clinical studies in 23 countries, the presence of sufficient concentration of DHA in breast milk has been reported to reduce the incidence of postpartum depression. Pregnancy and preeclampsia In a study, 7.6 times more preeclamptic diagnosis was found in patients with insufficient omega 3 fatty acids. It has been shown that the consumption of fish 3 times a week shows a protective effect in premature labor if it starts 23 weeks ago. (Mark et al., 2011) Probiotics are living living organisms. They mostly contain Bifidobacterium and Lactobacillus. Probiotics are contained in nutrients or added to the diet as a nutritional supplement. In the study of probiotic supplementation and placebo in pregnant women who started 2 months before birth and continued for 2 months following breastfeeding, Lactobacillus rhamnosus + Bifidobacterium longum and Lactobacillus paracasei + Bifidobacterium longum supplementation were observed for Esra KIZAR, Serap SAĞLIK ASLAN • 307

24 months after birth. When the effects of probiotic use in pregnancy were investigated, positive effects on the digestive system were observed, while possible positive effects on Group B Streptococcus colonization were observed (Uzdil and Ozenoglu, 2015).

FOOD SUPPLEMENTS IN INFANTS The micronutrient content of breast milk varies according to maternal nutrition and storage (such as vitamins A, B1, B2, B6, B12, D and iodine). Since maternal diet is not always optimal, it is recommended that the mother be given multivitamin supplementation during lactation. Since the amount of vitamin K in milk is very low regardless of maternal feeding, 1 mg of vitamin K i.m. required. Vitamin D is also low in breast milk, especially when sunlight is not sufficiently absorbed; therefore, early support is recommended. Iron deficiency anemia is rare in the first 6-8 months in infants who are fed exclusively with breast milk and whose umbilical cord is not attached early, because the iron content of breast milk is low but its absorption is very high.

REVIEW OF ANALYTICAL METHODS Adolfo et al. have developed a method based on direct solid sample analysis and high resolution continuity-based graphite furnace atomic absorption spectrometry for the side-by-side determination of iron and nickel as contaminants in multimineral and multivitamin supplements. Measurements were made with secondary lines of Fe (352.604 nm) and Ni (352.454 nm) to prevent spectral interference. The best temperatures for the pyrolysis and atomization of Fe and Ni elements are 1000 and 2700 C., respectively. For calibration, aqueous standard solutions were used. The limit of detection is 0.517 (g (-1) for Fe and 0.011 gg g (-1) for Ni. Precision for Fe and Ni ranges from 4.3% to 17% and 4.4-20%, respectively. The method was confirmed by statistical comparison of the results obtained with solid sampling and the results of sample acid digestion. The proposed methodology has been successfully applied to identify both metals in different multimineral and multivitamin supplements (Adolfo et al., 2019). Amayreh et al. have developed a method for the determination of iron in dietary supplements based on voltammetric analysis of iodine-coated polycrystalline platinum electrodes. The method developed is based on screening the iodine-coated polycrystalline platinum electrode potential between the hydrogen evolution limit (-0.25 V) and a potential lower than 308 • Health Sciences Research Papers the iodine desorption threshold at the platinum surface (about +0.80 V). An anodic peak of about 0.56 V is assigned to the oxidation of Fe (II) to Fe (III). The anodic peak current (i (p)) showed linearity with Fe2 + concentration in the Fe concentration range of 4500 ppm (R-2 = 0.995). Anodic peak current also showed a linear relationship with the square root of the scanning rate (R-2 = 0.999). The detection limit (according to S / N = 3) for the developed method is 2.34 ppm, while the detection limit (according to S / N = 10) is 7.8 ppm. The analysis of real samples and the application of statistical tests showed no systematic error in the results and the validity of the null hypothesis. There is no significant difference between the nominal values reported by the manufacturers and the results at p = 0.05 (Amayreh, et al., 2018). Araujo et al. have developed a method based on high performance liquid chromatography (HPLC-ICP-MS) coupled to inductively coupled plasma mass spectrometry for the side-by-side determination and specification analysis of iron supplements used in the treatment of iron deficiency anemia. For specification analysis, water was used as extraction solvent at 90 ° C for 30 minutes. Chromatographic separation was performed using a mobile phase containing 1.0 mM tetrabutylammonium hydroxide (TBAH), 0.7 mM ethylenediaminetetraacetic acid (EDTA) and 5% methanol at pH 7.2. Under optimized conditions, separation and detection of As (III), As (V), Cr (III) and Cr (VI) can be performed within 5 minutes. The assay limits obtained were 0.008, 0.010, 0.5 and 0.14 gg g (-1) for As (III), As (V), Cr (III) and Cr (VI), respectively. The accuracy of the method was evaluated and confirmed by recovery tests. The recoveries obtained ranged from 81% to 110%. The content of arsenic is 0.01 to 1.3 gg g (-1) and chromium is 0.4 to 61.2 gg g (-1). Between (Araujo et al., 2017). Garvey et al. developed a statistical comparison method in the analytical laboratory to determine calcium content in dietary supplements. A laboratory experiment has been described in which students compare two methods to determine the calcium content of dietary drug tablets. In a two-week sequence, the sample tablets are first analyzed by complexometric titration with ethylenediaminetetraacetic acid followed by ion exchange of the calcium ion present for hydronium ion by acid base titration with sodium hydroxide. Upon completion of the laboratory study, the students collect the data and perform a statistical analysis to determine whether the average values of the calcium content obtained by the two Esra KIZAR, Serap SAĞLIK ASLAN • 309 methods are equivalent. Taken together with analysis time, accuracy, simplicity and ease of use, sensitivity to interference and waste generation, these results enable students to evaluate the relative value of calcium determination in two approaches. In doing so, they introduce an important aspect of the "real world" chemical analysis, namely the selection of the most appropriate method for the determination of a particular analyte in a given sample (Garvey et al., 2015). Ellis, et al. have developed a method based on optical density and ion chromatography at 595 nm (OD595) to investigate the levels of Oxalobacter formigens and Oxalo capsules in probiotic supplements and to measure the ability of these preparations to degrade oxalate in vitro. In the polymerase chain reaction (PCR), specific and degenerate Lactobacillus primers of Oxalobacter formigens to oxalate decarboxylase gene (oxc) were used. It has been concluded that probiotic supplements sold online do not contain identifiable Oxalobacter formigens or viable oxalate-disrupting organisms, and are unlikely to benefit calcium oxalate kidney stone patients (Ellis et al., 2015). Lu et al. developed a gas chromatography-mass spectrometer (GC-MS) method for the simultaneous analysis of the vitamin E isomer and alpha- tocopherol acetate in functional foods and nutritional supplements. The isomers of vitamin E were extracted directly without saponification with solvents of methanol and hexane (7: 3, v / v). Chromatographic analyzes were performed within 13 minutes using a VF-5MS column (30 m x 0.25 mm, 0.25 mm). Correlation coefficients are greater than 0.997. The method is linear in the range of 0.1 to 40 mLg mL (-1). The detection limits are from 0.09 ng mL (-1) to 0.46 ng mL (-1). The quantity limits are from 0.29 ng mL (-1) to 1.52 ng mL (-1). Relative standard deviations (RSDs) within the day and between days (for 1 mLg mL (-1) of standard solution) range from 4.9% to 8.0% and 2.1 to 4.9%, respectively. The mean recovery value of the method ranged from 83.2% to 107%, with RSDs ranging from 1.1% to 8.4%. The method was applied successfully. Figure 1 shows the total ion chromatogram of the standard (Lu et al., 2015). 310 • Health Sciences Research Papers

Figure 1: Total ion chromatogram of the standard (I.) Peak definitions: (1) d- tocopherol; (2) b-tocopherol; (3) g-tocopherol; (4) d-tocotrienol; (5) α-tocopherol; (6) b-tocotrienol; (7) g-tocotrienol; (8) α-tocopherol acetate; (9) dibenzanthracene; (10) α-tocotrienol (Lu et al., 2015). Struminska evaluated the determination of Po-210 in calcium supplements and the possible dose for consumers. The aim of this pioneering study was to investigate the most popular calcium supplements as a potential supplementary source of polonium Po-210 in the human diet. The calcium pharmaceutical agents analyzed include organic or inorganic calcium compounds; these are some natural sources such as scallop shells, fish extracts or sedimentary rocks. The objectives of this research are; to investigate the naturally occurring concentrations of Po-210 activity in calcium supplements, to find correlations between Po-210 concentration in drug and calcium chemical form, and to calculate the effective dose of radiation analyzed based on calcium supplement consumption. As the results show, Po-210 concentrations in calcium supplements of natural origin (especially sedimentary rocks) are higher than other analyzes. In addition, the results of the Po-210 analysis for inorganic calcium supplementation forms are higher. The highest Po-210 activity concentrations were determined in mineral tablets made from sedimentary rocks: -3.88 +/- 0.22 and 3.36 +/- 0.10 mBq g (-1) in dolomite and chalk, respectively. Organic calcium compounds have the lowest calcium level. Calcium lactate and calcium gluconate were 0.07 +/- 0.02 and 0.17 +/- 0.01 Esra KIZAR, Serap SAĞLIK ASLAN • 311 mBq g (-1), respectively. In addition, annual effective radiation doses from supplementation intake were calculated. The highest annual radiation dose from Po-210 taken with 1 tablet of calcium supplementation per day was linked to the sample made with chalk. - 2.5 +/- 0.07 mu Sv year (-1), the highest annual radiation dose from Po-210 was attributed to dolomite with 1 g of pure calcium per day.- 12.7 ± 0.70 mu Sv year (-1) (Struminska- Parulska, 2015). Brunetti and Desimoni have developed a simple and rapid voltammetric method for the determination of vitamin B-6 in food samples and food supplements. The effect of pH on voltammetric response was analyzed. The method is linear in the range (2.0 x 10 (-6) / 7.2 x 10 (-5) M). Calibration function, detection limit (1.5 x 10 (-6) M) and repeatability were determined. The proposed analytical system has been successfully applied for the determination of pyridoxine in multivitamin supplements, energy drinks and cereals using the standard addition method (Brunetti and Desimoni, 2014). Bekhit et al. have developed a method based on vibrational spectroscopy and chemometric method for the determination of omega-3 fatty acids in fish oil supplements. To estimate concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), FT-IR, NIR and Raman spectroscopic techniques of Fourier transformation were combined to investigate total omega-3 fatty acids (n-3 FAs) in fish oil supplements. FT-IR spectroscopy showed EPA (coefficient of determination 0.994 (R-2), standard estimation error 2.49% (SEP), standard cross-validation error 2.90% (SECV) and DHA (R-2 = 0.983, SECV = 2.89% and A simplified two-factor PLS model for total n-3 FA (R-2 = 0.985, SECV = 2.73% and SEP. = 2.75%) was obtained with a factor of six to seven PLS. selected regions gave good performing models and EPA (R-2 = 0.979, SECV = 2.43% and SEP = 3.11%) and DHA (R-2 = 0.972, SECV = 2%, with factor of four to six PLS, NIR regions yielded simple models (two PLS factors) with similar results (R-2 = 0.997, SECV = 2.18% and SEP = 1.60%) for total n-3 FAs. All of the Raman spectrum and selected regions are EPA (R-2 = 0.977, SECV = 3.18% and SEP = 2.73%) and DHA (R-2 = 0.966, SECV =) with a factor of 7-8 PLS giving comparable results. 3.31% and SEP = 2.56%) whereas a simpler model (three PLS factors) was obtained for R-2 = 0.993, SECV = 2.82% and SEP = 3.27% for total n-3FA. The results showed that FT-IR, NIR and Raman spectroscopy combined with PLSR can be used as simple and rapid 312 • Health Sciences Research Papers methods for the quantitative analysis of EPA, DHA and total n-3 FAs. In particular, FT-IR and NIR spectroscopy has the potential to be applied in process industries during the production of fish oil supplements (Bekhit et al., 2014). Suh et al. developed a simple HPLC-DAD method to determine the vitamin B content of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxine and folic acid in the finished food products. Chromatographic analyzes using a gradient of acetonitrile elution and 20 mM phosphate buffer (pH 3.0), on a column of Develosil RPAQUEOUS C (30) (4.6 mm x 250 mm, 5 pm particle size) at 275 nm [except pantothenic acid (205 nm)]. and 1.0 mL / min flow rate. Correlation coefficients (r (2)) are higher than 0.995. The calibration curves for all six vitamins are linear. The developed method was validated in terms of linearity, intraday and inter- day accuracy and sensitivity, limit of determination (LOQ), recovery and stability. The method showed good sensitivity and accuracy within and between analyzes with coefficients of variability at all concentrations below 15%. Recovery was carried out according to the standard addition procedure. The yield ranged from 89.8 to 104.4%. This method has been successfully applied in the determination of vitamin B groups in food supplements (Suh et al., 2011). Halvorsen and Blomhoff determined lipid oxidation products in vegetable oils and marine omega-3 supplements. There is convincing evidence that replacing dietary saturated fats with polyunsaturated fats (PUFA) reduces the risk of cardiovascular disease. PUFA-rich foods such as vegetable oils, oily fish and marine omega-3 supplements are therefore recommended. However, PUFA can be easily oxidized and there are concerns about the possible adverse health effects caused by ingestion of oxidized lipids. Little is known about the degree of lipid oxidation in such products. The aim is to evaluate the content of lipid oxidation products in a wide range of vegetable oils and marine omega-3 supplements found in Norway. The content of lipid oxidation products was measured as peroxide value and alkenal concentration. Twelve different vegetable oils were heated at a temperature (225 degrees C) and time (25 minutes) similar to the conditions typically used during cooking. Peroxide values were between 1.04-10.38 meq / kg for omega-3 supplements and 0.60-5.33 meq / kg for fresh vegetable oils. The concentration range of alkenes is 158.23- 932.19 nmol / mL for omega-3 supplements and 33.24-119.04 nmol / mL for vegetable oils. After heating, a 2.9-11.2-fold increase in alkenal Esra KIZAR, Serap SAĞLIK ASLAN • 313 concentration was observed in vegetable oils. It was concluded that the content of hydroperoxides and alkenals in omega-3 supplements was higher than vegetable oils. After heating of vegetable oils, it was observed that there was a large increase in alkenal concentration (Halvorsen and Blomhoff, 2011). Jochum et al. developed different methods for the determination of n3- polyunsaturated fatty acids (n3-PUFA) in food supplement samples and compared their applicability to starch or high fat containing samples. In the presence of starch, a hydrolysis step was needed due to lipid-starch complexes. In terms of stability and reproducibility, the method of application of ethanol and sulfuric acid is more advantageous than the Weibull-Stoldt method. Analysis of ethanol-sulfuric acid extracts can be carried out by GC or SFC. The recovery of bread with n3-PUFA added by this analysis method is about 65%. 30% of the losses are due to adsorption of n3-PUFA on the hydrolyzed bread matrix, degradation during sample preparation, and failure to completely remove unsaturated fatty acid ethyl esters from the bread matrix. In addition, small losses were observed due to the volatility of ethyl esters in water vapor and deterioration during cooking. They are estimated to be less than 5%. The determination of polyunsaturated fatty acids in food samples containing large amounts of fat should be examined in two cases. If the sample itself is bound to polyunsaturated fatty acids, the content of triglycerides and fortified fatty acid ethyl esters should be determined, one of the samples using soxhlet extraction. These extracts contain triglycerides and must be detected by SFC. If the entire content of N3-PUFA needs to be determined, the method using ethanol and sulfuric acid should be followed. The quantification can then be performed by GC or SFC. The recovery is about 100% by soxhlet extraction and about 95% by application of ethanol and sulfuric acid (Jochum et al., 2001).

314 • Health Sciences Research Papers

CONCLUSION Adequate, balanced nutrition and appropriate weight gain during pregnancy is important to prevent short and long term complications related to pregnancy. The need for energy and nutrients increases during pregnancy. Adequate intake of nutrients during this period has important effects on the health of both the mother and the developing fetus. The amount of nutrients consumed should not be less than the recommended amounts. Diversity of vitamins and minarels should be provided by consuming foods from different food groups. Pregnancy maternal obesity, excessive energy intake and excessive weight gain may cause health problems such as gestational diabetes, preeclampsia, macrosomia, and the risk of malformations, preterm birth and low birth weight as a result of vitamin and mineral deficiencies. In addition, insufficient intake of vitamins and minerals in the maternal period increases the risk of permanent problems in the baby's health. It is stated that maternal vitamin deficiencies may affect cognitive development negatively in children. Although there is no general nutritional support of vitamins other than folic acid, which prevents the risk of neural tube defects during pregnancy, studies on vitamin supplements during pregnancy are being conducted. It is also stated that the effects of folic acid along with vitamin B12 to decrease homocysteine levels may be protective against fetal growth failure and low birth weight risk. Iron is also one of the nutrients that need increasing during pregnancy. Increasing the blood volume of the mother, the use of iron from the mother's stores by the fetus, iron deficiency due to errors in nutrition should be prevented with iron supplementation. Calcium and vitamin D are important in protecting the bone development of the fetus and protecting the mother's calcium stores and preventing tooth decay. When sufficient calcium is not obtained with food, it should be used as an additional supplement to the diet. It is also reported that vitamin D can protect against risk of diabetes, preeclampsia, inflammation and infection. Many food supplements are now on the market and the analytical methods used are insufficient. For specific multivitamin preparations, more specific, rapid and economical side-by-side determination methods need to be developed. Thus, different applications can be made in the direction of the use of food supplements in the later stages of pregnancy. Esra KIZAR, Serap SAĞLIK ASLAN • 315

REFERENCES Adolfo FR, Cicero do Nascimento P, Leal CL, Bohrer D, Viana C, Machado de Carvalho L, Colim AN, 2019, Simultaneous determination of iron and nickel as contaminants in multimineral and multivitamin supplements by solid sampling HR-CS GF AAS, Talanta, 195, 745-751. Agte V, Paknikar KM, Chiplonkar SA, 1998, Effect of riboflavin supplementation on zinc and iron absorption and growth performance in mice, Biol Trace Elem Res, 65, 109. Amayreh M, Hourani MK, 2018, Determination of iron in dietary supplements by voltammetric analysis at an iodine coated polycyrstalline platinum electrode, Int J Electrochem, 13, 975-983. Antonangeli L, Maccherini D, Cavaliere R, 2002, Comparison of two different doses of iodide in the prevention of gestational goiter in marginal iodine deficiency: a longitudinal study, Eur J Endocrinol, 147(1), 29-34. Araujo Barbosa U, Peña-Vazquez E, Barciela MC, Costa Ferreira SL, Bermejo- Barrera P, 2017, Simultaneous determination and speciation analysis of arsenic and chromium in iron supplements used for iron-deficiency anemia treatment by HPLC-ICP-MS, Talanta, 170, 1, 523-529. Baker H, Hockstein S, DeAngelis B, Holland BK, 2000, Thiamin status of gravidas treated for gestational diabetes mellitus compared to their neonates at parturition, Int J Vitam Nutr Res, 70(6), 317-320. Bakker SJ, Maaten JC, Gans RO, 2000, Thiamine supplementation to prevent induction of low birth weight by conventional therapy for gestational diabetes mellitus, Med Hypotheses, 55 (1), 88-90. Bekhit MY, Grung B, Mjos SA, 2014, Determination of omega-3 fatty acids in fish oil supplements using vibrational spectroscopy and chemometric methods, Appl Spectrosc, 68(10), 1190-200. Brunetti B, Desimoni E, 2014, Voltammetric determination of vitamin B-6 in food samples and dietary supplements, Journal of Food Composition and Analysis, 33(2), 155-160. Donnay S, Arena J, Lucas A, Velasco I, Ares S, 2014, Iodine supplementation during pregnancy and lactation, Endocrinol Nutr, 61, 27-34. Durante KM, Whitmore B, Jones CA, Campbell NR, 2001, Use of vitamins, minerals and herbs: a survey of patients attending family practice clinics, Clin Invest Med, 24, 242-249. Ellis, ML, Shaw KJ, Jackson SB, Daniel SL, Knight J, 2015, Analysis of commercial kidney stone probiotic supplements, Urology, 85(3), 517-521. 316 • Health Sciences Research Papers

Erata YE, Güçlü S, 2003, Gebelikte vitamin desteği, Perinatoloji Dergisi, 11(1), 15-18. Garvey SL, Shahmohammadi G, McLain DR, Dietz ML, 2015, Determination of calcium in dietary supplements: Statistical Comparison of Methods in the Analytical Laboratory, J Chemical Ed, 92 (1), 167-169. Geller AI, Shehab N, Weidle NJ, Lovegrove MC, Wolpert BJ, Timbo BB, Mozersky RP, Budnitz DS, 2015, Emergency department visits for adverse events related to dietary supplements, New Eng J Med, 373(16), 1531- 1540. Halsted CH, 2003, Dietary supplements and functional foods: 2 sides of a coin, Am J Clin Nutr, 77, 1001-1007. Halvorsen BL, Blomhoff R, 2011, Determination of lipid oxidation products in vegetable oils and marine omega-3 supplements, Food Nutr Res, 55, 5792. Harnack LJ, Rydell SA, Stang J, 2001, Prevalence of use of herbal products by adults in the Minneapolis/St Paul, Minn, Metropolitan Area, Mayo Clinic Proceedings, 76, 688-694. Hillman RW, Cabaud RW, Schenone RA, 1962, The effects of pyridoxine supplements on the dental caries experience of pregnant women, Am J Clin Nutr, 10 (5), 512-515. Hovdenak N, Haram K, 2012, Influence of mineral and vitamin supplements on pregnancy outcome, Eur J Obst Gyne Reproduct Biol, 164(2), 127-132. Jochum M, Pieper A, Engelhardt H, 2001, Bestimmung von Omega-3-Fettsäuren in supplementierten Lebensmitteln, Deutsche Lebensmittel-Rundschau, 97, 285-295. Karabulut A, Şevket O, Acun A, 2011, Iron, folate and vitamin B12 levels in first trimester pregnancies in the Southwest region of Turkey, J Turk Ger Gynecol Assoc, 12(3), 153-156. Lu D, Yang Y, Wu X, Zeng L, Li, YX, Sun CJ, 2015, Simultaneous determination of eight vitamin E isomers and alpha-tocopherol acetate in functional foods and nutritional supplements by chromatography - mass spectrometry, Analytical Methods, 7(8), 3353-3362. Mark A, Klebanoff, MD, Margaret MPH, Harper MD, 2011, Fish consumption, erythrocyte fatty acids, and preterm birth, Obstet Gynecol, 117(5), 1071- 1077. McWhorter LS, 2009, Dietary supplements for diabetes: an evaluation of commonly used products, Diabetes Spectrum, 22(4), 206-213. Esra KIZAR, Serap SAĞLIK ASLAN • 317

Menger H, Lin AE, Toriello HV, Bernert G, Spranger JW, 1997, Vitamin K deficiency embryopathy: a phenocopy of the warfarin embryopathy due to a disorder of embryonic vitamin K metabolism, Am J Med Genet, 72 (2), 129. Moghissi KS, 1981, Risks and benefits of nutritional supplements during pregnancy, Obstet Gynecol, 58 (5), 68S-78S. Petroczi A, Taylor G, Naughton DP, 2011, Mission Impossible Regulatory and enforcement issues to ensure safety of dietary supplements, Food Chem Toxicol, 49, 393-402. Procter SB, Campbell CG, 2014, Position of the Academy of Nutrition and Dietetics: Nutrition and lifestyle for a healthy pregnancy outcome, Journal of The Academy of Nutrition and Dietetics, 114,1099-1103. Rautiainen S, Manson JE, Lichtenstein AH, Sesso HD, 2016, Dietary supplements and disease prevention a global overview, Nature Reviewes, 12, 407-420. Soare A, Weiss, EP, Holloszy, JO, Fontana, L, 2014, Multiple dietary supplements do not affect metabolic and cardiovascular health, Aging, 6(2), 149-157. Struminska-Parulska DI, 2015, Determination of Po-210 in calcium supplements and the possible related dose assessment to the consumers, J Environ Rad, 150, 121-125. Suh JH, Yang DH, Lee BK, Eom HY, Kim, U, Kim, J, Lee H, Han SB, 2011, Simultaneous determination of B group vitamins in supplemented food products by high performance liquid chromatography-diode array detection, Bull Korean Chem Soc, 32(8), 2648-2656. Swartwout JR, Ungalub WG, Smith RC, 1960, Vitamin B6, serum lipids and placental arteriolar lesions in human pregnancy, A preliminary report, Am J Clin Nutr, 434-444. Tek NA, Pekcan G, 2008, Besin destekleri kullanılmalı mı?, Klasmat Matbaacılık, 32s, 978-985. Uzdil Z, Özenoğlu A, 2015, Gebelikte çeşitli besin öğeleri tüketiminin bebek sağlığı üzerine etkileri, Balıkesir Sağlık Bilimleri Dergisi, 4 (3), 119.

THE ROLE OF IMMUNE SYSTEM IN PHOTODYNAMIC THERAPY

CHAPTER 20

Sercin OZLEM CALISKAN1, Rahsan ILIKCI SAGKAN2

1 Usak University, Faculty of Medicine, Department of Biophysics, Usak, Turkey 2 Usak University, Faculty of Medicine, Department of Medical Biology, Usak, Turkey

Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 321

INTRODUCTION

Photodynamic Therapy Light has been used as a treatment more than three thousand years. The use of light in the treatment of various diseases have been reported in Ancient Egypt, Indian and Chinese civilizations such as psoriasis, rickets, vitiligo and skin cancer (Spikes et al., 1985). Photodynamic therapy, a photosensitization mechanism used in medicine, is a method of treatment based on the excitation of a photosensitizer by a suitable light corresponding to the absorption band of wavelength light. Photodynamic therapy has become an accepted treatment for several types of cancer and a number of diseases that have been approved for use in many countries. The number of articles about PDT published in a year is increasing both at clinical and experimental level.

History The first studies on photodynamic therapy were performed in the late 19th century. It began when Oscar Raab's acridine dye and light used together killed the single-celled organism, Paramecium caudatum. Tappaneiner and Jesionek showed the first known application of photodynamic therapy in 1903 by activating the topical eosin dye with sunlight in 3 patients with skin cancer. However, the application of photodynamic therapy has not been popular until the 1960s. Hematoporphyrin derivative (HPD) in recurrent breast cancer patients who use for therapeutic purposes in 1966 published the 9th International cancer congress by Lipson et al. In 1978, Dougherty et al. successfully cured malignant tumors with red light application after HPD injection for 24-72 hours exposure time and called this process Photodynamic Therapy (PDT) (Macdonald IJ et al., 2001). After the purification studies, the first PDT drug Photofrin (PF) was developed. Starting with the arc lamp, the use of laser, LED or fiber optic systems provided sufficient irridation dose for the target tissues. The Canadian Department of National Health and Welfare approved the use of PDT in bladder cancer using PF in 1993. After this first official approval of PF-PDT, the Food and Drug Administration in the United States approved PF as a PDT drug for the treatment of palliative esophageal cancer in December 1995 (Dougherty TJ et al., 1998). 322 • Health Sciences Research Papers

In the following years, PF-PDT has been approved for one or more pathological conditions such as esophageal malignancies, gastric cancer, early and late stage lung cancers, bladder cancer, gynecological malignancies by the relevant health institutions in the USA, Canada, France, Japan, the Netherlands and Italy (Dolmans DE et al 2003). Since PF-PDT has become official, clinical PDT applications are increasing in the treatment of non-malignant diseases such as removal of various cancers and atherosclerotic plaques, macular degeneration and actinic keratosis with many new photosensitizer (Fayter D et al 2010). The efficacy of the treatment approach in skin diseases was first demonstrated by Kennedy et al. In non-melanoma skin cancers in 1990, and the first FDA approval was granted in 1999 for the use of aminolevulinic acid (ALA) in actinic keratoses (Kennedy JC et al., 1990; Ormrod D and Jarvis B 2000). One of the most important developments was the fact that the more lipophilic methyl ester form of ALA, mALA, was approved in 2001 for actinic keratoses and basal cell carcinomas and used in clinical level.

Mechanism of Photodynamic Therapy PDT is based on the principle that a photosensitive agent is exposed to visible light at a suitable wavelength that matches the absorption spectrum in a site-directed manner. The mechanism of photodynamic therapy is: (1) Following the uptake of the PDT agent, photosensitizer is exposed to irradiation in the target tissue and this agent goes into triplet energy state. 1 (2) Formation of singlet molecular oxygen ( O2) as a result of transferring electron excitation energy of the PDT agent in triplet state to the nearby 1 molecular oxygen, (3) Reacts with O2target tissue to initiate events that will eliminate the pathological condition (cancer, etc.) in the target tissue 1 (Bilgin, 1999). The production of O2 generated by the PDT agent in vitro is an important indicator of PDT activity. As a result, it causes reactions in cells by direct and indirect mechanisms leading to death.

Photosensitizers Photosensitizer are classified as porphyrin and non-porphyrin. Porphyrin-derived photosensitizers divided into first and second generation photosensitizers. In the early studies with PDT, hematoporphyrin derivative (HPD) which is the first example of first generation light- sensitive drugs was used. Then, Porfimer sodium is the agent that enables the progression of photodynamic therapy and has led to Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 323 the development of second generation light sensitive drugs (Nayak, 2005). The second generation of photosensitizers (metallaporphrines, chlorines, porphycins, phthalocyanines, protoporphrines, purpurines and chlorophyll derivatives) have a lower half-life and less side effects on the body and especially on the skin. These agents have begun to be produced to provide a safer and more effective PDT. Although the studies have focused on porphyrin-derived agents, there are also photosensitizers (cyanines, phenothiazine dyes, azadipyromethene, psorelans, anthracyclines) that are not porphyrin-derived (O’Conner et al., 2009).

Light Sources In photodynamic therapy, monochromatic wavelength argon ion pump laser, solid phase diode laser, gold vapor laser and similar laser light sources, polychromatic arc lamps, halogen and incandescent lamps such as halogen lamps and LED (Light Emmiting Diodes) can be used. Visible light wavelength range (400-700nm) is suitable for PDT, but in practice the light range wavelength of 600-900 nm is used. The light sources used in practice have a penetration depth of approximately 3 mm. As the wavelength increases, the ability of light to enter the tissue increases (Häder and Jori, 2003).

PDT and Immune Response in Cancer The relationship between mechanisms of death and immune response in cancer cells has been investigated in several studies, both in vitro and in vivo. Do apoptotic pathways in tumor cells or treatment options targeting necrosis increase the immune response? The answer to the question has been examined by various research groups (Magner et al., 2005; Bartholomae et al., 2004; Scheffer et al., 2003; Shaif-Muthana et al., 2000). In necrosis, the inflammatory response increases with the spread of cytosolic components outside the cell as a result of damage to the plasma membrane. Castano et al. (Castano et al., 2006). As a result, the cause of tissue damage is eliminated, necrotic cells are cleaned and tissue repair takes place. Immunogenic DAMPs are released after PDT administration and can thus be detected by innate immune cells programmed to detect microbial invasion (Korbelik et al., 2006). These stress signals are recognized by pattern recognition receptors. These receptors are mainly expressed by myeloid cells such as monocytes, macrophages, neutrophils and dendritic 324 • Health Sciences Research Papers cells, but can also be expressed in lymphocytes, fibroblasts and epithelial cells. For these reasons, innate immune system cells, such as monocytes or macrophages, neutrophils and dendritic cells, aggregate into the treated site to develop an immune response to tumor cells (Krosl et al., 1995). Primary function of inflammatory cells; To neutralize DAMPs by swallowing up cellular debris. This process promotes local healing by restoration of normal tissue function. On the other hand, at the onset of PDT-induced inflammation, tumor vasculature also undergoes significant changes and becomes adhesive to intracellular proteins and inflammatory cells by overexpression of adhesion molecules (ICAM-1, VCAM-1, selectins). Thus, migration of immune system cells into tumor tissue is supported (Korbelik et al., 2006). One of the most important events induced by PDT is the extracellular heat shock protein 70 (HSP70), one of the most critical cellular molecules released from necrotic tumor cells. It is known that the expression of heat shock proteins is increased under stress conditions, thereby preventing cell death to protect normal cells under stress. For example, the expression of HSP70 increased after PDT administration and apoptosis of tumor cells was stimulated. It was shown in 2005 by Yenari et al., 2005. Intracellular HSP70 stimulates not only the tumor to go into apoptosis but also complexes with cytoplasmic tumor antigens. Once expressed on the cell surface, these antigens interact with antigen-presenting cells (APCs) to stimulate the anti-tumor immune response. Extracellular HSP70 binds to receptors on the surface of APCs with high affinity. This leads to activation and maturation of dendritic cells, the most important group of APCs. The dendritic cell receiving the foreign antigen migrates to the nearest lymph node through the lymph system and presents the foreign antigen complex to the naive T lymphocytes in the lymph node. As a result, HSP70 and peptide antigen complex are introduced by CD8+ cytotoxic T lymphocytes (CTL) by dentritic cells and CTLs are activated (Melcher et al., 1999). Thus, T lymphocytes, which become effector, migrate to the tumor site, destroying tumor cells either with the secreting cytotoxic molecules such as perforin and granzyme, or activating other immune system cells, such as cytokines, chemokines.

Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 325

PDT and Inflammation Inflammation is one of the body's main defense mechanisms and develops in response to tissue damage. It is a process that can be induced by biological factors such as bacteria, viruses, fungi and allergens, as well as by chemical agents such as acids, bases and by physical factors such as radiation, heat and trauma. As a result of inflammation, the immune system is activated. Inflammation is a reaction to protect the cell that is functionally damaged for any reason, but it involves a complex mechanism including molecular, immunological and physiological processes. If pathogens or endogenous stress signals are detected by pattern recognition receptors on the host immune system cells, inflammation is induced. Inflammation basically progresses with cellular and plasma mediator molecules. The release of pro-inflammatory cytokines and chemokines as a result of cellular stimulation of inflammation triggers inflammatory processes. Cytokines activate endothelial cells to enhance vascular permeability and facilitate entry of immune cells into tissues at the site of infection, but may also lead to capillary leakage, vasodilatation, and hypotension. The main function of chemokines is the collection of immune cells at the site of infection, including neutrophils, which play a vital role in phagocytosis and killing of pathogens (Dinarello et al., 1996; Dinarello et al., 2007; Bonecchi et al., 2009). Among the proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1) have autocrine and paracrine effects, leading to local activation of interleukin-6 (IL-6) macrophages and neutrophils. PDT-induced oxidative stress is known to stimulate the release or expression of various proinflammatory mediators TNF-α, IL-6, IL-1, complement proteins, arachidonic acid metabolites and HSPs (Gollnick et al., 2003). Acute inflammation begins after PDT administration. Inflammatory cytokines and chemokines such as IL-6 and macrophage inflammatory protein 1 (MIP1) and MIP2 have been detected in the serum of mice undergoing PDT directed to a subcutaneous tumor or a normal skin site (Gollnick et al., 2003). The mechanisms that block the inflammatory response are crucial for the return to homeostasis. There are many mechanisms that initiate inflammation as well as various inhibitory mechanisms that prevent inflammation. IL-10 suppresses the production of pro-inflammatory cytokines and is produced mainly from regulatory T cells (Tregs) (Ouyang et al., 2011). Increased levels of IL1, IL6, IL8 and IL10 from interleukins were detected after PDT application in mesothelioma (Yom et al., 2003). The presence of both pro-inflammatory 326 • Health Sciences Research Papers and anti-inflammatory cytokines was determined. Inflammatory mediators may be caused by malignant cells themselves, but may also be released from various immune system cells, such as leukocytes infiltrating tumor tissue. The release of cytokines from activated monocytes and neutrophils in the bloodstream stimulates the release of prostaglandin, one of the derivatives of arachidonic acid that acts on the hypothalamus and mediates disease symptoms and symptoms (insomnia, fatigue, fever) (Shattuck et al., 2015). Prostaglandins released from fibroblasts are known as inflammatory mediators (Handerson et al., 1989; Handerson et al., 1992). Furthermore, the release of thromboxane from endothelial cells after PDT administration is responsible for vasoconstriction (Fingar et al., 1990). The onset of acute inflammation is important in triggering the immune response. Tumor environment is defined as a chronic inflammation state as opposed to acute inflammation. Nuclear factor κB (NFκB) and activator protein 1 (AP1) are the major transcription factors in expression of acute inflammation after PDT. Both signaling molecules activated by cellular oxidative stress regulate the transcription of pro-inflammatory proteins and the immune system (Sun et al., 2003; Baeuerle et al., 1994; Haefner et al., 2002). These studies have shown a significant association between PDT and NFκB signaling pathway and acute inflammation. NFκB activation is one of the most important mediator molecules of acute inflammation, and the demonstration of induction after PDT in vitro confirms that PDT induces acute inflammation in vivo. NFκB regulates the expression of the cyclooxygenase 2 (COX2) enzyme and produces inflammatory mediators known as eicosanoids (prostaglandin E2 (PGE2) and leukotrienes). In a study of tumors treated with PDT, a correlation between increased PGE2 synthesis and long-term expression of COX2 has been demonstrated (Ferrario et al., 2002). Inhibitors of the enzyme COX2, one of the target molecules of inflammation, do not sensitize cancer cells by themselves, but can be used to enhance the anti-tumor activity of PDT when administered after irradiation. This effect is due to inhibition of angiogenesis (Makowski et al., 2003). Further studies are needed to understand the precise role of COX2 and eicosanoids in PDT-induced immune response to tumors. Kick et al. compared the level of IL-6 expression, one of the pro- inflammatory cytokines after PDT or UVB treatment; IL-6 levels in PDT- induced samples were higher and earlier than UVB-treated samples. FD1- induced IL-6 expression increase was found to be mediated by AP1 as a transcription factor (Kick et al., 1995). Gollnick et al. demonstrated that in Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 327 the BALB / c mouse model, PDT caused significant changes in the expression of IL-6 and IL-10 after administration to normal and tumor tissue, but did not detect changes in the expression of TNF-α (Gollnick et al., 1997). On the other hand, pro-inflammatory cytokines have recently been tested for combination therapy with PDT. Studies have been conducted by Bellnier on the application of cytokines, such as TNF-α, which have been shown to enhance the Photofrin®-mediated PDT of murine SMT-F adenocarcinoma (Bellnier et al., 1991). Have reported that one of the photosensitizers, 2- [1-oxoxyethyl] -2-devinyl pyropheoporbide-α (HPPH) mediated a temporary and local increase in the expression of MIP2 from chemokines, resulting in neutrophil migration to the tumor site (Gollnick et al. (2003). Blocking the function of various adhesion molecules may affect the effectiveness of PDT (Gollnick et al., 2003; Sun et al., 2002). Luiter et al. firstly observed that neutrophils adhere to the microvascular wall in vivo after PDT, but that PDT does not induce expression of β-selectin (one of the major adhesion molecules that bind leukocytes) by endothelial cells. This finding was supported by a finding that the expression levels of the adhesion molecules in intracellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) were downregulated in endothelial cells after PDT (Volanti et al., 2004). In this study, an increase in the peripheral blood neutrophil count was detected 4 hours after PDT treatment and this increase was reported to last for 24 hours. In some tumor models, the reasons why neutrophils are so important in producing an effective response to PDT are still unclear. Krosl et al. demonstrated a 200-fold increase in neutrophil count in 5 minutes of PDT in a Photofrin®-mediated PDT-treated murine squamous cell carcinoma VII (SCCVII) model, followed by an increase in mast cells levels. In another study, monocyte / macrophages, another type of myeloid cells, were shown to infiltrate the tumor for 2 hours after PDT (Krosl et al., 1995). Cecic et al. reported that neutrophil populations increased rapidly after Photofrin® or mTHPC-mediated PDT treatment in SCCVII or EMT6 breast carcinoma mice (Cecic et al., 2001). An important part of circulating inflammation is the activation of the complement system that mediates microbial opsonization and killing and produces inflammatory peptides such as C3a and C5a (Ward et al., 2010). On the other hand, complement inhibition completely inhibited the development of PDT-induced neutrophil. The complement fragments C3a and C5a may activate neutrophilia by activating bone marrow (Kajita et 328 • Health Sciences Research Papers al., 1990). Korbelik et al. found that complement activation (increase in serum C3 level) occurs after treatment of Lewis lung carcinomas (LLC) with Photofrin®-mediated PDT. Activation of the alternative pathway of complement became apparent 1-3 days after PDT. Blocking of C3a or C5a receptors in host mice reduced the efficacy of PDT in the treatment of LLC tumors. They also showed that blocking ICAM1 with monoclonal antibodies reduced tumor treatment (Sun et al., 2002). Upregulation of ICAM1 ligands CD11b and CD11c on neutrophils was found to be associated with PDT-treated tumors. Neutrophils indicate that MHC class II molecules function as antigen-presenting cells and play a role in the development of anti-tumor immune response. Korbelik et al. showed that IL-1 and TNF-α both act as strong promoters of the early phase of PDT- induced neutrophil and play an important role in the advanced stage. Data obtained by blocking the other two cytokines, GCSF and IL-10, have shown that they contribute significantly to advanced neutrophils without significant effect in the early phase (Cecic et al., 2002). Although PDT is a local treatment, its effect is not limited to the local area, but may produce a strong acute phase response with systemic consequences (Cecic et al., 2006). The synthesis of acute phase proteins is induced by the release of pro-inflammatory cytokines. Studies in mouse models have shown that PDT causes a large increase in serum levels of acute phase reactants such as serum amyloid P components (SAP), C- reactive protein (CRP) and mannose binding lectin A (MBL-A) (Korbelik et al., 2008). ). SAP and CRP belong to pentaxin family proteins and play a role in acute immunological response (Merchant et al., 2013). It has important roles in facilitating phagocytosis and removing dying cells in PDT-treated tumors. SAP production and release is an important acute phase reactant response in mice, but in humans, CRP is a more important acute phase reactant than SAP. CRP has been shown to be dose-dependent upregulated in human lung tumor A549 cells (Merchant et al., 2013). MBL-A is another important acute phase reactant with similar functional properties to SAP (Saevarsdottir et al., 2004). The results of the study described above show that acute inflammation caused by PDT is important for the success of both systemic and local cancer therapies.

Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 329

Conclusion Some preclinical and clinical studies have shown that PDT can significantly activate both the innate and acquired immune system. Such effects on the immune system appear to be dependent on PDT treatment and the degree of PDT-induced inflammation. It has been reported that PDT treatment, which causes an increase in acute inflammation, is better in immune activation in patients with high acute inflammation. However, an increase in inflammatory mediators may, in some cases, promote tumor cell growth (Sgambato et al., 2010). Studies have shown that immune response is dependent on light intensity, photosensitive agent concentration and PDT-treated region.

330 • Health Sciences Research Papers

REFERENCES Baeuerle PA, Henkel T. Function and activation of NF-κB in the immune system. Annu. Rev. Immunol. 1994;12:141–179. Bartholomae WC, et al. T cell immunity induced by live, necrotic, and apoptotic tumor cells. J. Immunol. 2004;173:1012–1022. Bellnier DA. Potentiation of photodynamic therapy in mice with recombinant human tumor necrosis factor-α J. Photochem. Photobiol. B. 1991;8:203– 210. Bilgin MD 1999. Biomedical application of photosensitization. Doktora Tezi. Illinois Institute of Technology, Chicago, IL, USA. Bonecchi R, et al. Chemokines and chemokine receptors: an overview. Front Biosci (Landmark Ed) 2009;14:540–551. Castano, A. P., Mroz, P., & Hamblin, M. R. (2006). Photodynamic therapy and anti-tumour immunity. Nature reviews. Cancer, 6(7), 535-545. https://doi.org/10.1038/nrc1894 Hwang, H. S., Shin, H., Han, J., & Na, K. (2018). Combination of photodynamic therapy (PDT) and anti-tumor immunity in cancer therapy. Journal of Pharmaceutical Investigation, 48(2), 143-151. https://doi.org/10.1007/s40005-017-0377-x Cecic I, Parkins CS, Korbelik M. Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors. Photochem. Photobiol. 2001;74:712–720. Cecic I, Korbelik M. Mediators of peripheral blood neutrophilia induced by photodynamic therapy of solid tumors. Cancer Lett. 2002;183:43–51. Cecic I, Stott B, Korbelik M. Acute phase response-associated systemic neutrophil mobilization in mice bearing tumors treated by photodynamic therapy. Int Immunopharmacol. 2006;6:1259–1266. PMID: 16782538 DOI: 10.1016/j.intim p.2006.03.008. Dinarello CA. Historical insights into cytokines. Eur J Immunol. 2007;37(Suppl 1):S34–45. Dinarello CA. Cytokines as mediators in the pathogenesis of septic shock. Curr Top Microbiol Immunol. 1996;216:133–165. Dolmans DE, Fukumura D, Jain RK, Photodynamic therapy for cancer.Nature Reviews Cancer, 2003; 3(5): 380-387 Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 331

Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q. Phodynamic Therapy. Journal of National Cancer Institute, 1998; 90(12): 889-905. Fayter D, Corbett M, Heirs M, Fox D, Eastwood A. A systematic review of photodynamic therapy in the treatment of pre-cancerous skin conditions, Barrett’s oesophagus and cancers of the biliary tract, brain, head and neck, lung, oesophagus and skin. Health Technology Assessment 2010; 14(37):1-288. Fingar VH, Wieman TJ, Doak KW. Role of thromboxane and prostacyclin release on photodynamic therapy-induced tumor destruction. Cancer Res. 1990;50:2599–2603. Häder DP, Jori G. Photodynamic Therapy, Comprehensive Series in Photochemical and Photobiological Sciences Volume:2, Chapter 1. The Royal Society of Chemistry, Cambridge, UK. ISBN 0-85404-306-3. Haefner B. NF-κB: arresting a major culprit in cancer. Drug Discov. Today. 2002;7:653–663. Henderson BW, Donovan JM. Release of prostaglandin E2 from cells by photodynamic treatment in vitro. Cancer Res. 1989;49:6896–6900. Henderson BW, Owczarczak B, Sweeney J, Gessner T. Effects of photodynamic treatment of platelets or endothelial cells in vitro on platelet aggregation. Photochem. Photobiol. 1992;56:513–521. Gollnick SO, et al. Altered expression of interleukin 6 and interleukin 10 as a result of photodynamic therapy in vivo. Cancer Res. 1997;57:3904–3909. Gollnick SO, et al. Role of cytokines in photodynamic therapy-induced local and systemic inflammation. Br. J. Cancer. 2003;88:1772–1779. Ferrario A, et al. Cyclooxygenase-2 inhibitor treatment enhances photodynamic therapy- mediated tumor response. Cancer Res. 2002;62:3956–3961. Kajita T, Hugli TE. C5a-induced neutrophilia. A primary humoral mechanism for recruitment of neutrophils. Am. J. Pathol. 1990;137:467–477. Kennedy JC, Pottier RH, Pross DC. Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience. Journal of Photochemistry and Photobiology B: Biology 1990; 6(1-2):143-8 Kick G, et al. Photodynamic therapy induces expression of interleukin 6 by activation of AP-1 but not NF-κB DNA binding. Cancer Res. 1995;55:2373–2379. 332 • Health Sciences Research Papers

Krosl G, Korbelik M, Dougherty GJ. Induction of immune cell infiltration into murine SCCVII tumour by photofrin-based photodynamic therapy. Br. J. Cancer. 1995;71:549–555. ] Korbelik M. PDT-associated host response and its role in the therapy outcome. Lasers Surg Med. 2006;38:500–508. PMID: 16634073 DOI: 10.1002/lsm.20337. Korbelik M, Cecic I, Merchant S, Sun J. Acute phase response induction by cancer treatment with photodynamic therapy. Int J Cancer. 2008;122:1411– 1417. PMID: 18033689 DOI: 10.1002/ijc.23248. Macdonald IJ, Dougherty TJ. Basic principles of photodynamic therapy. Journal of Porphyrins and Phthalocyanines. 2001; 5(2):105-129. Magner WJ, Tomasi TB. Apoptotic and necrotic cells induced by different agents vary in their expression of MHC and costimulatory genes. Mol. Immunol. 2005;42:1033–1042. Makowski M, et al. Inhibition of cyclooxygenase-2 indirectly potentiates antitumor effects of photodynamic therapy in mice. Clin. Cancer Res. 2003;9:5417–5422. Melcher A, Gough M, Todryk S, Vile R. Apoptosis or necrosis for tumor immunotherapy: what’s in a name? J. Mol. Med. 1999;77:824–833. Nayak CS. Photodynamic therapy in dermatology. Indian J. Dermatol. Venereol. Leprol. 2005; 71: 155-160. Merchant S, Korbelik M. Upregulation of genes for C-reactive protein and related pentraxin/complement proteins in photodynamic therapy-treated human tumor cells: enrolment of PI3K/Akt and AP- 1. Immunobiology. 2013;218:869–874. PMID: 23182717 DOI: 10.1016/j.imbio.2012.10.010. O’Connor AE, Gallagher WM, Byrne AT. Porphrine and Nonporphyrine photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy. 2009;85: 1053-1074. Ormrod D, Jarvis B. Topical aminolevulinic acid HCl photodynamic therapy. American Journal of Clinical Dermatology, 2000; 1(2):133-9 Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol. 2011;29:71–109. Shattuck EC, Muehlenbein MP. Human sickness behavior: Ultimate and proximate explanations. Am J Phys Anthropol. 2015;157:1–18. Sercin OZLEM CALISKAN , Rahsan ILIKCI SAGKAN • 333

Saevarsdottir S, Vikingsdottir T, Valdimarsson H. The potential role of mannan- binding lectin in the clearance of self-components including immune complexes. PMID: 15238070 DOI: 10.1111/j.0300- 9475.2004.01437.x. Scand J Immunol. 2004;60:23–29. Scheffer SR, et al. Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo. Int. J. Cancer. 2003;103:205–211. Shaif-Muthana M, et al. Dead or alive: immunogenicity of human melanoma cells when presented by dendritic cells. Cancer Res. 2000;60:6441–6447. Sgambato A, Cittadini A. Inflammation and cancer: a multifaceted link. Eur Rev Med Pharmacol Sci. 2010;14:263–268. PMID: 20496533. Spikes, J. D. in Primary Photoprocesses in Biology and Medicine (eds Berghausen, R. V., Jori, G., Land, E. J. & Truscott, T. H.) 209–227 (Plenum Press, New York, 1985). Sun J, Cecic I, Parkins CS, Korbelik M. Neutrophils as inflammatory and immune effectors in photodynamic therapy-treated mouse SCCVII tumours. Photochem. Photobiol. Sci. 2002;1:690–695. Sun SC, Xiao G. Deregulation of NF-κB and its upstream kinases in cancer. Cancer Metastasis Rev. 2003;22:405–422. Ward PA. The harmful role of c5a on innate immunity in sepsis. J Innate Immun. 2010;2:439–445. Yenari MA, et al. Antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection. Ann. NY Acad. Sci. 2005;1053:74–83. Volanti C, et al. Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy. Oncogene. 2004;23:8649–8658. Yom SS, et al. Elevated serum cytokine levels in mesothelioma patients who have undergone pleurectomy or extrapleural pneumonectomy and adjuvant intraoperative photodynamic therapy. Photochem. Photobiol. 2003;78:75– 81.

A HOLISTIC APPROACH TO CHRONIC LEG ULCERS

Overview Of Diagnosis And Differential Diagnosis

CHAPTER 21

Sevgi AKARSU1

1 Professor Doctor, Dokuz Eylul University, Faculty of Medicine, Department of Dermatology

Sevgi AKARSU • 337

INTRODUCTION Chronic wounds resistant to healing are one of the difficulties in clinical medicine due to their complexity and prevalence. It is estimated that approximately 1-2% of the population in developed countries will suffer a chronic wound complaint at least once in their lifetime. The economic, psychosocial and health adverse effects of this condition continue to increase due to gradually aging population, high health care costs, and rising incidence of diabetes and obesity worldwide. (Augustin et al., 2014; Fino et al., 2019; Ghasemi et al.,2016; Sen, 2019). In the literature, studies on chronic wounds have focused mainly on the specific wound healing products or technologies, specific disease conditions or specific aspects of patient or wound care. However, in recent years, it has been more frequently emphasized that a simplified general approach guide based on evidence-based criteria is needed to help accurate diagnosis and proper treatment of chronic wounds. (Gupta et al., 2017; Snyder et al., 2019) The worldwide most common chronic wounds manifest in at least 60% of the patients as leg ulcers (Graham et al., 2003). Chronic leg (including the foot) ulcer is a frequent condition, causing pain, infection, social discomfort, depression and considerable costs (Bui et al., 2018; Eusen et al., 2016; Salomé et al., 2016). Prevalence rates of all leg ulcers range from 1% in the adult population to 3–5% in the elderly (Mekkes et al., 2003). Early and accurate diagnosis of the wound is essential for determining the most appropriate steps in the treatment of chronic leg ulcers. Unfortunately, correct diagnosis can be difficult due to various combinations of patient comorbidities and etiologies leading to the development of chronic ulcers. However, successful and cost-effective wound care cannot be achieved without proper diagnosis. Incorrect or delayed initial diagnosis may also harm the patient and increase the risk of serious complications such as permanent disability and amputation. (Fukaya&Margolis, 2013; Gupta et al., 2017; Richmond et al., 2013). In this chapter, it is aimed to overview the most common chronic leg ulcers in terms of diagnosis and differential diagnosis.

Factors Affecting Wound Healing Process The normal healing process of acute wounds is described in four stages: hemostasis, inflammation, proliferation and remodeling, respectively. The wounds that are not healed or that heal unusually slowly are generally 338 • Health Sciences Research Papers defined as chronic wounds as having these phases prolonged (Morton&Phillips, 2016). Yager and et al. clinically defined ulcers as chronic wounds that did not heal within 3 months and which had a slow reduction (≤1 mm/wk) in wound diameter (Yager et al.,1997). Many local and systemic factors may play a role in the formation of chronic wounds. Vascular disease (venous and arterial), neuropathy, pressure, infection, necrotic tissue on the wound bed, excessive tension of the wound, unfavourable local environment (inappropriate topical products or wound dressings, contact dermatitis, excessive dry or extremely moist wound, etc.), climatic factors, inappropriate treatment (such as pyoderma gangrenosum debridement), radiation, malignant wound and repetitive trauma are certain local factors that adversely affect wound healing (Jockenhöfer et al., 2013; Klode et al., 2011; Morton&Phillips, 2016). Associated systemic factors include advanced age, malnutrition, medications (corticosteroids, immunosuppressants, etc.), obesity, chronic medical conditions (hypoxia, vascular disease, cardiac failure, connective tissue disease, endocrine disorders, diabetes, cancer, immunosuppression, etc.) and certain habits such as alcohol consumption and smoking (Haughey&Barbul, 2017; Mc Daniel et al., 2015; Mekkes et al., 2003; Morton&Phillips, 2016) Macroscopically, wound healing depends on many factors, such as the size, depth and location of the wound, the age of the patient and the presence of a local or systemic disease. Many factors such as low oxygenation, chronic inflammation, fibroblast aging, impaired function and critical cytokine levels, growth factors and receptors, abnormal matrix metalloproteinase activity, bacterial colonization and infection contributes to the delayed healing (Rodrigues et al., 2019; Stacey et al., 2019). In culture, fibroblasts from acute wounds are mitotically competent and exhibit high mitogenic activity, while chronic wound fibroblasts have low mitogenic capacity and are aged (Shah et al., 2012). Wound fluid from acute wounds such as split-thickness skin graft donor sites induces fibroblast proliferation in culture, indicative of a growth-promoting molecular environment. In contrast, chronic wound fluid inhibits fibroblast and keratinocyte proliferation. In chronic wounds, cytokine and growth factor receptors, such as the type II TGF-beta receptor, may also be downregulated or do not function properly. Again, in chronic wounds, the levels of tissue inhibitors of metalloproteinases are decreased and matrix Sevgi AKARSU • 339 metalloproteinases are overactive. These high levels of protease destroy the tissue matrix and impair healing (McCarty&Percival, 2013; Rodrigues et al., 2019; Yager et al., 1997). Although recent research does not support a significant correlation between microbial burden and wound healing, it is known that the spectrum of wound infection varies from contamination to systemic infection, where bacteria do not proliferate or cause clinical infection. Biofilms, an important problem in chronic wounds, consist of a population of bacteria that secrete a matrix or glycocalyx and are thus difficult to protect from the host's immune response. Biofilms appear to stimulate inflammation that increases vascular permeability, wound exudate production, and fibrinous accumulation. Biofilms were detected in 60% of biopsy specimens from chronic wounds, while only 6% of acute wounds were identified. Bacteria (both planktonic and biofilms) cause inflammation, leading to the release of protease from the inflammatory cells and release of reactive oxygen species, and also release exotoxins and proteases that break down the proteins necessary for healing (Jockenhöfer et al., 2013; Jones et al., 2015; Morton&Phillips, 2016).

The Most Common Chronic Leg Ulcers Wound care authorities propose an approach based on specific criteria supported by laboratory and diagnostic tests to identify major chronic wound types. In the first evaluation, a detailed examination of the wound characteristics such as the appearance of the wound bed, the area around the wound and the wound edges, the temperature and pulses, the content and amount of the wound fluid, the wound localization and sensory perception is required. In addition, understanding the risk factors and underlying causal factors for each of the different types of chronic wounds is critical in verifying diagnoses and preparing the patient and wound for treatment (Gupta et al., 2017; Morton &Phillips, 2016; Snyder et al., 2019). Most chronic leg ulcers (90-95%) are due to venous insufficiency (45- 60%), arterial insufficiency (10-20%), diabetes mellitus (15-25%), or a combination of these (10-15%). However, rare underlying diseases may also cause or contribute to ulcer formation (Kirsner &Vivas, 2015). 340 • Health Sciences Research Papers

Venous Leg Ulcers Insufficient venous valves causing abnormal high pressure and continuous venous hypertension damage the vessels and skin over time, and minor impacts or abrasions result in deterioration of skin integrity and non-healing venous ulcers (Santler&Goerge, 2017).There are different risk factors including being male gender, previous history of deep / superficial vein thrombosis and pulmoner embolism, number of pregnancies (if female), congestive heart failure, reflux in deep veins, varicose veins, severe lipodermatosclerosis, history of previous ulcer, more than two years have passed since the previous ulcer attack, large ulcer area, malnutrition, skeletal or joint disease of lower extremities, decreased range of motion and / or ankle range of motion, advanced age, parental history of ankle ulcers, family history of venous insufficiency, smoking, diabetes, obesity, clinical venous disease that is presented with skin changes, and injection of drugs from the groin, legs and feet (Finlayson et al., 2015; Gupta et al., 2017; Kelly&Gethin, 2019; McDaniel et al., 2015; Parker et al., 2015; Pieper et al., 2009). Venous leg ulcers located typically at the midline or below the calf, on the lower leg and wrist, adjacent to or below the medial or lateral malleolus, are superficial. This shallow-looking wound is often covered with a fibrinous layer with granulation tissue. Although there are different depths inside the wound, which varies in diameter from small to large, can be individually distributed or circumferentially circular, it does not affect the deep tissues. Exudation is intense. Often accompanied by pitting edema and worsening towards the end of the day, this edema may be a precursor of ulcer. Signs of venous eczema characterized by hemosiderin pigmentation and often erythema, squam, weeping and itching may be observed around the wound. Lipodermatosclerosis is detected in the presence of long-term venous insufficiency. Leg temperature is high, consistent with chronic venous insufficiency. The pain, throbbing and weight feeling in the legs relieve with elevation and rest (Alavi et al., 2016; Gupta et al., 2017)

Arterial Insufficiency Ulcers It is associated with atherosclerotic disease of middle- and large- diameter arteries. Hypertension and decreased arterial blood flow lead to tissue hypoxia and damage. Thromboangitis obliterans, vasculitis, pyoderma gangrenosum, thalassemia and sickle cell disease are comorbid Sevgi AKARSU • 341 conditions that contribute to the formation of arterial ulcers. Associated risk factors include peripheral vascular disease, coronary artery disease, poor glycemic controlled diabetes, obesity, smoking, hypertension, dyslipidemia, family history, advanced age and sedentary life (Hess, 23(9), 2010; Gupta et al, 2017) Arterial ulcers usually occur between the toes or on the toe tips, the outer part of the ankle or on pressure points such as the lateral edges of the foot. These quite deep, punched-out round and well-circumscribed ulcers may affect the underlying tendon, muscle or bone structures. The typically non-hemorrhagic wound bed may be yellow, brown, gray or black. Exudation is minimal and limited edema is present. The skin and nails of the affected extremity are atrophic in appearance. The skin is pale, shiny, stretched and thin, and hair growth is minimal or absent. The lower extremity turns red when it is sagged and it becomes pale with elevation (dependent rubor). Foot / leg pain, especially during exercise, at rest or during the night, is very severe and relieves with sagging. The extremity pulses, which are felt cold by palpation, are unnoticeably absent or absent (Fukaya&Margolis, 2013; Gupta et al., 2017; Kirsner &Vivas, 2015).

Diabetic Foot Ulcers These ulcers associated with neuropathy and/or peripheral arterial disease on lower extremities in diabetic patients are located in the lower ankle. Diabetic foot ulcers, which affect 4-10% of diabetic patients annually, are the leading cause of hospitalization and amputation in these patients. 50-60% of patients also have signs of infection at the time of admission to the hospital. These wounds are most commonly seen on the plantar surface of the foot, which is subject to repeated trauma. Callus develops in areas of abnormal pressure due to deformities such as neuropathy, Charcot's foot, or limited joint movement (Morton&Phillips, 2016). Excessive exudation may occur due to concomitant infection, heart failure, kidney disease and lymphatic disease (Gupta et al., 2017). Autonomic deficiencies in diabetes lead to decrease in sweating and changes in blood flow regulation (Morton&Phillips, 2016). As a result, distal extremity oxygenation is impaired, resulting in a warm, dry and pink skin prone to fissurations (Morey et al., 2019). Consequently, distal extremity oxygenation is impaired, resulting in a warm, dry and pink skin prone to fissuration and splitting (Doupis&Veves, 2008). 342 • Health Sciences Research Papers

Diabetic foot ulcers are generally classified as neuropathic (54%), ischemic (10%) or neuroischemic (34%). It is important to distinguish between ischemia and neuropathy at the time of the diagnosis, because the complications are different and therefore require different therapeutic strategies. Although a diabetic foot ulcer can develop as a result of neuropathy and unintentional recurrent trauma, the presence of underlying arterial insufficiency may prevent wound healing (Gupta et al., 2017). Neuropathic diabetic foot ulcer; occurs in relation to hyperglycemia, peripheral sensory neuropathy and recurrent mechanical walking forces. Risk factors such as deformity, high plantar pressures, peripheral arterial disease, advanced age, obesity, hypertension and and previous history of amputation facilitate wound formation (Gupta et al., 2017; Oyibo et al., 2002; Parisi et al., 2016; Pendsey, 2010). They are typically seen on the weight-bearing areas of the foot, such as the metatarsal caps and heels, and on the back of the clawed toes. It usually has a thick hypertrophic callus and is accompanied by dry skin, brittle nails, hammer toes, fissures, bullae, Charcot joint and digital necrosis. The wound is surrounded by callus, the wound edges are undermined or macerated, and the wound bed is pink and granulated. The lower extremity is warm and sweating decreased. Sensorial and deep tendon reflexes are absent. Pulses are fast and bounding (Gupta et al.,2017; Oyibo et al.,2002). Ischemic diabetic foot ulcer; occurs in relation to hyperglycemia, peripheral vascular disease-induced ischemia, coronary artery disease, and cerebrovascular disease. Risk factors such as prolonged disease duration, poor glycemic control, peripheral vascular disease, the presence of retinopathy and smoking facilitate wound formation (Gupta et al.,2017; Pendsey, 2010). Ischemic ulcers, typically seen at the toes, nail edges, sides of the foots and between the toes, are often accompanied by digital necrosis and particularly dry gangrene on the toes. There is a small amount of pale granulation tissue or tightly adherent yellowish crusting in the wound bed. Hair loss on the dorsum of the foot, pallor with elevation and redness with sagging occur. The foot is painful at rest, accompanied by intermittent limping and burning in the arch or distal part of the foot. The lower extremity is cold and there is no palpable pulse. (Gupta et al., 2017). Neuro-ischemic diabetic foot ulcer; occurs in relation to hyperglycemia, occlusive vascular disease (the main factor), peripheral Sevgi AKARSU • 343 sensory neuropathy, trauma and inappropriate shoes. Risk factors such as prolonged disease duration, poor glycemic control, presence of neuro- ischemic foot, presence of retinopathy, smoking and other signs of atherosclerotic vascular disease and lack of vibration sensation are factors that facilitate wound formation (Gupta et al., 2017; Parisi et al., 2016) It is located on the feet and at the edges of the fingers or behind the heel, especially the medial surface of the first metatarsophalangeal joint and the lateral surface of the fifth metatarsophalangeal joint. Callus formation is minimal, prone to necrosis, and especially dry gangrene is observed in the toe. Minimal granulation is present on the wound bed and the surrounding skin is hairless, thin and shiny. Variable degrees of sensory loss is also felt. As with ischemic diabetic foot ulcer, the foot is cold and the pulse cannot be taken (Gupta et al., 2017; Oyibo et al., 2002).

Pressure Ulcers / Injuries These ulcers are generally characterized by localized damage to the skin and/or underlying tissue on a bone protrusion caused by intense and/or prolonged pressure or shear and/or friction with pressure. Unchangeable pressure can lead to ischemia, cell death and tissue necrosis. The tolerance of soft tissue to pressure and shear may also be affected by microclimate, nutrition, perfusion, comorbidities and condition of soft tissue (Edsberget al., 2016). Immobility, sensory loss, vasopressor use, stroke, advanced age, low body mass index (<20), multiple traumas, musculoskeletal disorders / fractures, gastrointestinal bleeding, unstable and / or chronic medical conditions (such as diabetes, renal cancer, chronic obstructive pumonary disease, congestive heart failure, dementia), a history of pressure ulcer / injury, preterm infants, recently being anesthetized surgically with more than 3 hours, excessive moisture and increase in temperature may play a role in the formation of pressure ulcers / injuries (Tachibana et al., 2016). In recent years, the National Pressure Ulcer Advisory Panel has proposed the use of the term pressure injury instead of the previously used pressure ulcer to describe both pressure ulcers in both intact and ulcerated skin. Pressure injury has been redefined as localized damage to the skin and underlying soft tissue, usually on a bone protrusion or in association with a medical or other instrument. Accordingly, pressure ulcers / injuries are classified as follows (Edsberget al., 2016; Tachibana et al., 2016):

344 • Health Sciences Research Papers

 Stage 1: Non-blanchable erythema of intact skin  Stage 2: Partial-thickness skin loss with exposed dermis  Stage 3: Full-thickness skin loss  Stage 4: Full-thickness skin and tissue loss  Unstaged: Indeterminate full-thickness skin and tissue loss  Deep tissue pressure injury: persistent, dark red, maroon, or purplish discoloration  Medical device-related pressure injury  Mucosal membrane pressure injury These wounds, which are usually seen on the skin on malleoli, trochanters, heels or sacrum, show moderate and persistent pain that increases in severity by wound diameter. The base of the wound bed may be undermined or tunneled, covered with crust and/or eschar, and often have rounded wound edges. They affect the underlying deep tissues, and in stage 4 injuries, even the fascia, muscle, tendon, ligament, cartilage and bones can be exposed. Exudation may be observed in relation to the wound size. The skin around the wound may be indurated, erythematous, masere or healthy. Edema may be a precursor to a stage 1 pressure ulcer/injury. Localized temperature or coldness is felt in the affected area. (Gupta et al.,2017; Tachibana et al., 2016).

Less Common Atypical Chronic Leg Ulcers If the ulcer does not fall into any of the abovementioned ulcer types, if it is in an atypical localization, its clinical appearance or symptoms are beyond expectations, and if it does not respond to conventional treatment or has not been treated properly after three months, an atypical etiological factor may be considered to play a role (Hess, 23(10), 2010; Jockenhöfer et al., 2016; Kirsner&Vivas, 2015; Körber et al., 2011; Mekkes et al., 2003; Misciali et al, 2013; Shanmugam, 2017; Sławiński et al., 2019). Many different conditions in the differential diagnosis are listed below. *Infection-related Bacterial: Erysipelas bullosa, necrotizing fasciitis (Streptococcus haemolyticus), botryomycosis (commonly Staphylococcus aureus), gas gangrene (Clostridium species), ecthyma gangrenosum (Pseudomonas aeruginosa), septic embolism, bacterial endocarditis, anthrax (Bacillus anthracis), diphtheria (Corynebacterium diphteriae), meningococcemia (Neisseria meningitides), bartonellosis (Bartonella bacilliformis), glanders Sevgi AKARSU • 345

(Burkholderia mallei), malakoplakia (commonly E. coli), tularemia (Francisella tularensis), yaws (Treponema pallidum pertenue), lues maligna (lues III, gummata) Mycobacterial: Leprosy (Mycobacterium leprae), buruli ulcer (M ulcerans), tuberculosis (M tuberculosis causing ulcerating cutaneous tuberculosis, lupus vulgaris, and papulonecrotic tuberculid) Viral: herpes simplex, varicella zoster, cytomegalovirus Fungal: bullous tinea pedis, granuloma trichophyticum, eumycotic mycetoma, chromoblastomycosis, coccidiomycosis, sporotrichosis, histoplasmosis, blastomycosis Protozoal: Leishmaniasis, amoebiasis (Entamoeba histolytica), acanthamoeba Others: Tropical ulcer (Bacteroides, Borrelia vincenti and other bacteria), osteomyelitis (several microorganisms), complications by secondary wound infections, toeweb infection *Medication-induced Steroid ulcus (intralesional injection), methotrexate, chemotherapeutics, immunosuppressives Hydroxyurea, granulocyte-colony stimulating factor, halogens, ergotamin Paravasal injection of cytostatic and other drugs Bacillus Calmette-Guerin vaccination *Malignancy-related Squamous cell carcinoma (Marjolin ulcer), basal cell carcinoma, melanoma (including acral and amelanotic types), pseudoepitheliomatous hyperplasia Merkel cell carcinoma, malignant fibrous histiocytoma, lymphoma, leukaemia, cutaneous T-cell and B-cell lymphoma, Hodgkin disease Kaposi and pseudo-Kaposi sarcoma Sarcoma,lymphosarcoma,rhabdomyosarcoma, haemangiosarcoma, lymphangiosarcoma 346 • Health Sciences Research Papers

Internal malignancy metastasis *Medical conditions Diabetes mellitus Hypertension (ulcus hypertensivum, Martorell ulcer) Neuropathic conditions Diabetes, leprosy, alcohol neuropathy, tabes dorsalis, syringomyelia, spina bifida, paraplegia, paresis, multiple sclerosis, poliomyelitis Blood disorders Polycythemia vera, sickle cell anemia, other forms of anemia, thalassemia, hereditary spherocytosis, glucose-6-phosphate dehydrogenase deficiency, essential thrombocythemia, thrombocytopenia (including thrombotic thrombocytopenic purpura), granulocytopenia, leukemia, monoclonal dysproteinemia (Waldenstrom’s macroglobulinemia, myeloma), polyclonal dysproteinemia (cryofibrinogenemia, purpura, hyperglobulinemia, cold agglutinins) Microcirculatory disorders Blood transfusion reactions, Hypertension, Raynaud phenomenon, scleroderma, Increased blood viscosity (increased fibrinogen level, paraneoplastic, paraproteinaemia, leukaemia) Autoimmune conditions Scleroderma, rheumatoid arthritis, cutaneous lupus erythematosus, inflammatory bowel disease (including metastatic Crohn’s disease) Metabolic diseases Diabetes mellitus, necrobiosis lipoidica, porphyria cutanea tarda, gout, calciphylaxis, calcinosis cutis, homocysteinuria, prolidase deficiency, hyperoxaluria Nutritional disorders Caloric, protein, vitamin, mineral deficiencies Sevgi AKARSU • 347

*Physical or chemical injury Pressure (decubitus), pressure by shoes, orthopaedic appliances, compression bandages Trauma (including burns, bites, and postsurgical injury), freezing, electricity Roentgen damage, intra-articular injection of Yttrium-90 Chemical (corrosive agents), sclerotherapy Factitial (including dermatitis artefacta, automutilation, malingering, and Munchausen by proxy) *Primary ulcerating skin conditions Ulcerative pyoderma gangrenosum Panniculitis (including erythema induratum, polyarteritis nodosa) Bullous diseases (including bullous pemphigoid, pemphigus, bullous lichen planus, porphyria cutanea tarda) Necrobiosis lipoidica, sarcoidosis, Stevens Johnson syndrome, toxic epidermal necrolysis *Vascular disorders Venous insufficiency and dependency Venous valve insufficiency in the deep (usually post-thrombotic) or superficial venous system Venae communicantes insufficiency Congenital hypoplasia ⁄ aplasia of venous valves Weakness of the venous wall (collagen disorders) Arteriovenous anastomosis, angiodysplasia Compression or obstruction of veins (tumours, enlarged lymph nodes, pelvic vein thrombosis) Ulcerating thrombophlebitis, ruptured varices Dependency syndrome (immobility, arthrosis, rheumatoid arthritis, paresis, paralysis, orthopaedic malformations) 348 • Health Sciences Research Papers

Arterial occlusion Peripheral arterial disease (arteriosclerosis) Arterial thrombosis ⁄ macrothromboembolism and microthromboembolism (fibrin, platelets) Fat embolism (hypercholesterolemia, hyperlipidemia) Detachment of cholesterol containing plaques from aorta, aneurysm or atrium (atrial fibrillation) Thromboangiitis obliterans (Buerger disease) Arteriovenous anastomosis (congenital ⁄ traumatic) Trauma, rupture, infection, vascular procedures Fibromuscular dysplasia Vasculitis Small-vessel: leukocytoclastic vasculitis, microscopic polyangiitis, granulomatosis with polyangiitis (formerlyWegener granulomatosis), Churge-Strauss, Henoche-Schönlein purpura, cryoagglutination (cryoglobulins, cryofibrinogen), and Behçet disease Medium-sized vessel: polyarteritis nodosa Large vessel: giant cell arteritis (polymyalgia rheumatica, Takayasu arteritis) Vasculopathy Hypercoagulopathic disorders: Factor V Leiden, prothrombin G20210A mutation, methyltetrahydrofolate reductase mutation, cystathione-beta-synthase mutation (homocysteinemia), dysfibrinogenemia, antithrombin III deficiency, protein C or S deficiency, plasminogen deficiency, elevated factors VIII, IX, or X, tissue factor pathway inhibitor deficiency, lupus anticoagulant, anticardiolipin (antiphospholipid syndrome), Disseminated intravascular coagulation and purpura fulminans Sneddon syndrome Cholesterol emboli Sevgi AKARSU • 349

Calciphylaxis Warfarin-induced necrosis (and heparin necrosis) Livedoid vasculopathy Degos disease (malignant atrophic papulosis)

Useful Tests for Diagnosis and Differential Diagnosis For all patients with leg ulcers, firstly assess whether arterial flow is sufficient. If the dorsalis pedis and posterior tibialis pulses are not palpable or very weak, more objective tests such as Doppler and/or ankle-brachial index (ABI) should be performed. The cut-off ABI value at rest was determined as ≤ 0.9 for diagnosis of peripheral vascular disease. Since ABI values may be incorrectly elevated due to calcification and glycosylation in veins in elderly or diabetic patients, additional tests such as transcutaneous oxygen measurements (<40 mmHg considered abnormal) or toe-brachial index (<6 considered abnormal) may be required. If initial tests are abnormal, more complex and specific tests such as Doppler arterial waveforms and pulse volume recordings can be performed. Sensory perception determined by 10-g monofilament test, Achilles tendon reflex and 128 Hz tuning fork test used for sensory neuropathy evaluation in diabetic foot ulcers may be abnormal. Other causes of peripheral neuropathy such as alcoholism, vitamine deficiencies (B1, B6, B12, thiamine, niacin) connective-tissue diseases, uraemia, liver disease, medications, leprosy, hepatitis C, HIV, trauma to nerves and paraneoplastic syndromes, should also be investigated in patients with sensation loss (Kirsner&Vivas, 2015; Snyder et al., 2019). Venous and arterial doppler studies should be performed to assess the presence of venous reflux, vein thrombosis and superficial venous disease in patients with suspected venous leg ulcers and to ensure the adequacy of arterial circulation to initiate appropriate compression therapy (Alavi et al., 2016). Levels of factor VIII and IX, factor V Leiden, homocysteine, fibrinogen, protein C, protein S, antithrombin III, prothrombin and antiphospholipid antibodies should be investigated for thrombophilia in young patients (aged <40 years) diagnosed with deep vein thrombosis, thrombophlebitis, livedo racemosa or livedoid vasculopathy (Kirsner&Vivas,2015). 350 • Health Sciences Research Papers

The wound healing process requires an adequate intake of calories and proteins to increase anabolism, nitrogen and collagen synthesis, and healing. To assess nutritional status, CBC, albumin, prealbumin, vitamin C and zinc levels should be evaluated. Recent consensus views suggest that laboratory markers such as albumin, prealbumin, and total protein are not reliable on their own, but can be used to complete comprehensive nutritionally focused physical examination results (Gupta et al., 2017; Haughey&Barbul, 2017). In order to narrow the differential diagnostic spectrum in patients with uncertain diagnosis, punch biopsy should be taken from the tissue containing wound surface and wound edge for histopathology and culture. Although histopathology can help in the diagnosis of ulcers associated with vasculitic, vasculopathic, infectious and malignant etiology, it is non- specific in the pyoderma gangrenosum. According to biopsy results, further laboratory tests may be required to determine the underlying and related diseases (Gupta et al., 2017, Hess, 23(10), 2010).

Approach to the Patient with Chronic Leg Ulcers In the patient and wound evaluation, certain stages should be followed with a general holistic method and systematic multidisciplinary approach regardless of the wound type. First, the etiology of the wound should be accurately defined by a thorough evaluation of both the patient and the wound. A multidisciplinary treatment team should be established to address patient-related factors and underlying causes of the wound. Once the wound has been correctly diagnosed, a full agreement should be reached with the patient in terms of compliance with the treatment and the goals of treatment should be determined. The wound should be cleaned and debrided properly, infection and / or persistent inflammation (if any) should be managed and moisture balance should be maintained. After the patient and the caregivers are trained about the treatment plan, they should be treated with appropriate treatments and dressings chosen according to the wound characteristics. Patient compliance and adherence to the care plan should be questioned regularly throughout the wound healing process. Once the wound has healed, a proactive follow-up plan should be established to help prevent wound recurrence. (Gupta et al., 2017; Kirsner &Vivas, 2015; Morton&Phillips, 2016; Richmond et al., 2013) Following the diagnosis and consideration of the underlying factors, the goals of treatment should be determined before starting treatment. Sevgi AKARSU • 351

Achieving this can be difficult, especially in cases where the diagnosis is not fully definitive. Two or more causes may coexist in 10-15% of chronic wounds. Diseases such as venous insufficiency, peripheral arterial disease and diabetes mellitus, usually associated with leg ulcers, are associated with significant patient morbidity and mortality. Although optimal nutrition is an important component in all stages of wound healing, obese patients should be encouraged to record and reduce calorie intake, eat nutritiously, and exercise to lose weight (Gupta et al., 2017; Haughey&Barbul, 2017). Evaluating wounds based on healing tendencies facilitates the development of more realistic treatment objectives and treatment plans. Clinicians need to determine whether the underlying cause is treatable, whether blood flow is adequate, the presence of concomitant conditions (ability to adapt to treatment and/or adequacy of resources), or the use of medications that prevent healing. In addition, elderly, demented and / or nursing care patients may not respond to or tolerate curative treatment for too long. In any case, wound care decisions should be made according to the benefit and well-being of the patient. In some cases, this means switching to a palliative treatment plan where symptoms are alleviated and soft care is the primary objective. In case of delay in wound healing, obesity, nicotine use and the associated risks should be considered when determining cost-effective treatment strategies (Gupta et al., 2017; Morton & Phillips, 2016) In summary, determination of chronic wound etiology can often be very difficult even with careful systematic evaluation due to underlying multiple causes, borderline diagnostic markers and mixed etiologies. The most difficult to diagnose and treat are usually those that do not fall into the typical category of chronic ulcers (venous, arterial, diabetic and / or pressure ulcers) due to various comorbid conditions. Therefore, a detailed history of both patient and wound origin should be obtained and evaluated comprehensively before determining further investigations and treatment strategies in such patients.

352 • Health Sciences Research Papers

REFERENCES: 1. Alavi, A., Sibbald, R.G., Phillips, T.J., Miller, O.F., Margolis, D.J., Marston, W., et al. (2016). What's new: Management of venous leg ulcers: Approach to venous leg ulcers. J Am Acad Dermatol, 74(4), 627-40. 2. Augustin, M., Brocatti, L.K., Rustenbach, S.J., Schäfer, I., Herberger, K. (2014). Cost-of-illness of leg ulcers in the community. Int Wound J, 11(3), 283-92. 3. Bui, U.T., Finlayson, K., Edwards, H. (2018). Risk factors for infection in patients with chronic leg ulcers: A survival analysis. Int J Clin Pract, 72(12), e13263. 4. Doupis, J., Veves, A. (2008). Classification, diagnosis, and treatment of diabetic foot ulcers. Wounds, 20(5), 117-26. 5. Edsberg, L.E., Black, J.M., Goldberg, M., McNichol, L., Moore, L., Sieggreen, M. (2016). Revised National Pressure Ulcer Advisory Panel Pressure Injury Staging System: Revised Pressure Injury Staging System. J Wound Ostomy Continence Nurs, 43(6), 585-597. 6. Eusen, M., Brenaut, E., Schoenlaub, P., Saliou, P., Misery, L. (2016). Neuropathic pain in patients with chronic leg ulcers. J Eur Acad Dermatol Venereol, 30(9), 1603-5. 7. Finlayson, K., Wu, M.L., Edwards, H.E. (2015). Identifying risk factors and protective factors for venous leg ulcer recurrence using a theoretical approach: A longitudinal study. Int J Nurs Stud, 52(6), 1042-51. 8. Fino, P., DiTaranto, G., Pierro, A., Kacjulite, J., Codolini, L., Onesti, M.G., et al. (2019). Depression risk among patients with chronic wounds. Eur Rev Med Pharmacol Sci, 23(10), 4310-4312. 9. Fukaya, E., Margolis, D.J. (2013). Approach to diagnosing lower extremity ulcers. Dermatol Ther, 26(3), 181-6. 10. Ghasemi, F., Anooshirvani, N., Sibbald, R.G., Alavi, A. (2016). The point prevalence of malignancy in a wound clinic. Int J Low Extrem Wounds, 15(1), 58-62. 11. Gupta, S., Andersen, C., Black, J., de Leon, J., Fife, C., Lantisli, J.C., et al. (2017). Management of chronic wounds: Diagnosis, preparation, treatment, and follow-up. Wounds, 29(9), S19-S36. 12. Graham, I.D., Harrison, M.B., Nelson, E.A., Lorimer, K., Fisher, A. (2003). Prevalence of lower-limb ulceration: a systematic review of prevalence studies. Adv Skin Wound Care, 16(6), 305-316. Sevgi AKARSU • 353

13. Haughey, L.,Barbul, A. (2017). Nutrition and lower extremity ulcers: Causality and/or treatment. Int J Low Extrem Wounds, 16(4), 238-243. 14. Hess, C.T. (2010). Arterial ulcer checklist. Adv Skin Wound Care, 23(9), 432. 15. Hess, C.T. (2010). Checklist for differential diagnosis of lower-extremity ulcers. Adv Skin Wound Care, 23(10), 480. 16. Jockenhöfer, F., Gollnick, H., Herberger, K., Isbary, G., Renner, R., Stücker, M., et al. (2013). Bacteriological pathogen spectrum of chronic leg ulcers: Results of a multicenter trial in dermatologic wound care centers differentiated by regions. J Dtsch Dermatol Ges, 11(11), 1057-63. 17. Jockenhöfer, F., Gollnick, H., Herberger, K., Isbary, G., Renner, R., Stücker, M., et al. (2016). Aetiology, comorbidities and cofactors of chronic leg ulcers: retrospective evaluation of 1000 patients from 10 specialised dermatological wound care centers in Germany. Int Wound J, 13(5), 821- 8. 18. Jones, E.M., Cochrane, C.A., Percival, S.L. (2015). The effect of pH on the extracellular matrix and biofilms. Adv Wound Care (New Rochelle), 4(7), 431-439. 19. Kelly, M., Gethin, G. (2019). Prevalence of chronic illness and risk factors for chronic illness among patients with venous leg ulceration: A cross- sectional study. Int J Low Extrem Wounds, 18(3), 301-308. 20. Kirsner, R.S.,Vivas, A.C. (2015). Lower-extremity ulcers: diagnosis and management. Br J Dermatol, 173(2), 379-90. 21. Klode, J., Stoffels, I., Körber, A., Weindorf, M., Dissemond, J. (2011). Relationship between the seasonal onset of chronic venous leg ulcers and climatic factors. J Eur Acad Dermatol Venereol, 25(12), 1415-9. 22. Körber, A., Klode, J., Al-Benna, S., Wax, C., Schadendorf, D., Steinstraesser, L., et al. (2011). Etiology of chronic leg ulcers in 31,619 patients in Germany analyzed by an expert survey. J Dtsch Dermatol Ges, 9(2), 116-21. 23. McCarty, S.M.,Percival, S.L. (2013). Proteases and delayed wound healing. Adv Wound Care (New Rochelle), 2(8), 438-447. 24. McDaniel, J.C., Kemmner, K.G., Rusnak, S. (2015). Nutritional profile of older adults with chronic venous leg ulcers: a pilot study. Geriatr Nurs, 36(5), 381-6. 25. Mekkes, J.R., Loots, M.A., Van Der Wal, A.C., Bos, J.D. (2003). Causes, investigation and treatment of leg ulceration. Br J Dermatol, 148(3), 388- 401. 354 • Health Sciences Research Papers

26. Misciali, C., Dika, E., Fanti, P.A., Vaccari, S., Baraldi, C., Sgubbi, P., et al. (2013). Frequency of malignant neoplasms in 257 chronic leg ulcers. Dermatol Surg, 39(6), 849-54. 27. Morey, M., O'Gaora, P., Pandit, A., Hélary, C. (2019). Hyperglycemia acts in synergy with hypoxia to maintain the pro-inflammatory phenotype of macrophages. PLoS One, 14(8), e0220577. 28. Morton, L.M., Phillips, T.J. (2016). Wound healing and treating wounds: Differential diagnosis and evaluation of chronic wounds. J Am Acad Dermatol, 74(4), 589-605. 29. Oyibo, S.O., Jude, E.B., Voyatzoglou, D., Boulton, A.J.M. (2002). Clinical characteristics of patients with diabetic foot problems: changing patterns of foot ulcer presentation. Practical Diabetes Int, 19 (1), 10–12. 30. Parker, C.N., Finlayson, K.J., Shuter, P., Edwards, H.E. (2015). Risk factors for delayed healing in venous leg ulcers: a review of the literature. Int J Clin Pract, 69(9), 1029-30. 31. Parisi, M.C., MouraNeto, A., Menezes, F.H., Gomes, M.B., Teixeira, R.M., de Oliveira, J.E., et al. (2016). Baseline characteristics and risk factors for ulcer, amputation and severe neuropathy in diabetic foot at risk: the BRAZUPA study. Diabetol Metab Syndr,8, 25. 32. Pendsey, S.P. (2010). Understanding diabetic foot. Int J Diabetes Dev Ctries,30(2), 75–79. 33. Pieper, B., Templin, T.N., Kirsner, R.S., Birk, T.J. (2009). Impact of injection drug use on distribution and severity of chronic venous disorders. Wound Repair Regen,17(4), 85-91. 34. Richmond, N.A., Maderal, A.D., Vivas, A.C. (2013). Evidence-based management of common chronic lower extremity ulcers. Dermatol Ther, 26(3), 187-96. 35. Rodrigues, M., Kosaric. N., Bonham, C.A., Gurtner, G.C. (2019). Wound healing: A cellular perspective. Physiol Rev, 99(1), 665-706. 36. Salomé GM, de Almeida SA, de JesusPereira MT, Massahud MR Jr, de OliveiraMoreira CN, de Brito MJ, et al. (2016). The impact of venous leg ulcers on body image and self-esteem. Adv Skin Wound Care, 29(7), 316-21. 37. Santler, B., Goerge, T. (2017). Chronic venous insufficiency - a review of pathophysiology, diagnosis, and treatment. J Dtsch Dermatol Ges, 15(5), 538-556. Sevgi AKARSU • 355

38. Sen, C.K. (2019). Human wounds and its burden: An updated compendium of estimates. Adv Wound Care (New Rochelle), 8(2), 39-48. 39. Shah, J.M.,Omar, E., Pai, D.R., Sood, S. (2012). Cellular events and biomarkers of wound healing. Indian J PlastSurg, 45(2), 220-8. 40. Shanmugam,V.K., Angra, D., Rahimi, H., McNish, S. (2017). Vasculitic and autoimmune wounds. J Vasc Surg Venous Lymphat Disord, 5(2), 280- 292. 41. Sławiński, P., Radkowski, M., Lewandowicz, A., Targowski, T. (2019). Mixed-etiology leg ulcers in a patient on long-term glucocorticoid therapy. Reumatologia, 57(3), 173-177. 42. Snyder, R.J., Jensen, J., Applewhite, A.J., Couch, K., Joseph, W.S., LantisIi, J.C., et al. (2019). A standardized approach to evaluating lower extremity chronic wounds using a checklist. Wounds, 31(5 Suppl), S29-S44. 43. Stacey, M.C., Phillips, S.A., Farrokhyar, F., Swaine, J.M. (2019). Evaluation of wound fluid biomarkers to determine healing in adults with venous leg ulcers: A prospective study. Wound Repair Regen, 27(5), 509-518. 44. Tachibana, T., Imafuku, S., Irisawa, R., Ohtsuka, M., Kadono, T., Fujiwara,H., et al. Wound/Burn Guidelines Committee. (2016). The wound/burn guidelines - 2: Guidelines for the diagnosis and treatment for pressure ulcers. J Dermatol, 43(5), 469-506. 45. Yager, D.R., Chen, S.M., Ward, S.I., Olutoye, O.O., Diegelmann, R.F., Kelman Cohen, I. (1997). Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Repair Regen, 5(1), 23-32.

WAR SURGERY AND NURSING CARE

CHAPTER 22

Sonay GÖKTAŞ1, Elif GEZGİNCİ2, Ayşenur ATA

1 Assoc. Prof. 2 Asst. Prof.

Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 359

THE ROLE OF NURSES IN THE HISTORICAL PROCESS OF WAR The historical development of nursing care dates back to ancient times. There is evidence that interventions such as tooth extraction, trepanation and amputation were performed during the Neolithic period (Ökdem, Abbasoğlu & Doğan, 2000). In an Indian book written in 3500 BC, it is stated that nurses are responsible for the care of patients. In ancient Greece, women were responsible for the care of patients. Hygieia, the daughter of Aesculapius, a well-known physician and a god in mythology, was identified as the person responsible for the care of the patients in the temple (Ökdem et al., 2000; Torun, 2014). In Islam, women took part in patient care and gave importance to nutrition, hygiene, avoiding infectious people, taking care of oral and dental health and preventing infection. In addition, it is stated that blood draw was among the health protection measures. The first Field Hospital in the history of Islamic medicine was founded by the nurse Rufeyde binti Sa’d al-Ansari. Rufeyde laid the foundations of the triage system applied in modern medicine by dividing and grading the tents she established (Ökdem et al., 2000; Torun, 2014). Hebrew in 2000 BC improved themselves in the field of medicine. Treatment methods included many applications such as the prohibition on the consumption of pork, medication with herbs, male circumcision and gluing leeches on body. In order to prevent infection, practices such as burning the belongings of people with infectious diseases have also been seen. In medieval Europe, during the Christian era, men chosen as a helper in church were called Deacon and women Deaconess. They provided assistance and care to the poor and ill. The Deaconesses did not receive any training and assisted people only by the skills adopted from their womanhood and motherhood (Ökdem et al., 2000; Torun, 2014). With the crusades of Westerners, the hospitals of the Islamic world were recognized, and hospitals were built in accordance with this model. The malnutrition of the soldiers and poor hygiene conditions during the Crusades led to a rapid increase in infections and infectious diseases, resulting in the need for hospitals and caregivers. To meet this requirement, many groups of priests, musketeers and volunteers have emerged. The noble ladies among the volunteers were also responsible for the care of the wounded soldiers during the war. Order of Saint Lazarus and the Teutonic 360 • Health Sciences Research Papers

Knights, who were responsible for the maintenance of wounded soldiers during the war, were among these organized groups. The Beguines organization was established in Belgium in 1184 as a result of the grouping of civilian women and provided care for orphans and wounded people. Women also took part in the “Florence Sisters” organization, founded in Florence in 1226, and achieved successful results in the care of patients (Torun, 2014.) With the adoption of Christianity by Russia in 988, monasteries emerged, where the care of the patients was planned. In Russia, the “Merciful Sisters” and the “Holy Trinity Society” were founded by Grand Duchess Alexandra Nikolaevna in 1844. The Merciful Sisters were first trained in patient care and then started their duty. In this way, the first formal nursing education was established in Russia (Sorakine, 2005). Florence Nightingale came to Üsküdar during the Crimean War and settled in Selimiye Barracks, and in the first week following her arrival, she taught the nurses what they were going to do and started the planned activities. During this period, the first group of volunteers went to the Crimean Peninsula, cared for many wounded on the battlefield, worked to cope with illnesses and helped many soldiers to regain their health (Sorakine, 2005; Hatipoğlu, 2011). Many wounded in the war, rather than the battle, died mostly due to reasons such as cholera, high fever, poor environmental conditions and nutritional disorders. Florence Nightingale defined the necessity of determining the effects of these factors on the health of individuals and reduced the mortality rate as a result of environmental practices. Within that period, she collected data on the disease and death rates of the soldiers and the factors affecting these rates and presented the results with tables and pie charts which were an advanced data presentation type for those days (Burns & Grove, 2005). In the following years, women served in many wars such as Gallipoli Campaign, Balkan, Korean, Iraq, Afghanistan and First and Second World Wars. It can be said that especially the Balkan War played an important role in Turkish women's nursing. In these wars, women laid the foundation of nursing by performing many applications such as wound care and debridement, environmental sanitation, triage, and nasogastric intubation. Many nurses took part in the First and Second World War and 7500 nurses participated in the Vietnam War and treated the wounded soldiers. Nurses working in hospitals during the Iraq War also took the necessary Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 361 precautions to prevent infection and reduced the infection rates (Reineck, Finstuen, Connelly & Murdock 2001; Leininger, Rasmussen, Smith, Donalt & Coppola 2006; Bebiş & Serpil 2013). The importance of nursing in the history of our country has emerged with wars. The need for nursing services came to light again in 1911 with the casualties lost in the Tripoli War due to the lack of health interventions, and it was decided to take the necessary measures. For this purpose, in 1911, with the support of Dr. Besim Ömer Pasha, six-month nursing courses were started by the Hilal-i Ahmer Society (Red Crescent Society). Turkish women like Safiye Hüseyin Elbi, Kerime Salahar and Münire İsmail who completed these courses worked as volunteer nurses in Gallipoli Campaign and Balkan War. When the Hilâl-i Ahmer Society called for women from Istanbul to look after wounded soldiers during the Balkan Wars, Safiye Hüseyin and her sister Nesime were among the first applicants. Elbi and her sister Nesime were first assigned to the establishment of hospitals by collecting beds and quilts as donations, and then, due to their excellent knowledge of English, they were assigned to Asar-ı Atika Hospital where English physicians worked. Safiye Hüseyin, who provided outstanding services in this hospital where seriously injured people were treated, thus took her first step into nursing. Without distinction, she cared for all injured Turkish and foreign soldiers (Yılmaz Gören & Yalım, 2016; Leblebicioğlu, 2017) During the Balkan War and World War I, she led the honorary nursing of Istanbul women. When the Gallipoli Campaign started, since many ferry boats were converted into hospital ships, Safiye Hüseyin Elbi was assigned as the chief caregiver by Besim Ömer Pasha at the Resit Pasha Ferry, which was prepared for the treatment of injured soldiers in Çanakkale (Bilgin, 2008).

TRIAGE AND THE ROLE OF NURSES IN WAR Triage, derived from the French verb “trier”, is used to sort, select and classify (Tarhan & Akın, 2016; Şimşek, 2018). It was used to meet and classify the medical needs of soldiers during the Napoleonic Wars to help them return to the war environment, and to identify the soldiers to be transported in the United States army to remove the wounded from the battlefield (Tarhan et al., 2016). In Turkey, however, triage is defined as “the rapid selection and coding of the patients requiring primary treatment and transportation in cases of large number of sick and wounded both at 362 • Health Sciences Research Papers the scene and at all health institutions they are sent to” (Karcıoğlu & Topaçoğlu, 2017). Triage during the war is based on the principle that limited resources are best utilized for the largest number of people. The priority is given to wounded soldiers who can move and are in a less life-threatening condition, not to the wounded who have a greater risk of death. The wounded soldiers taken from the battlefield must be triaged properly and need to be sent quickly to the operating room and emergency response areas with helicopters and ambulances (Bridges, 2003; Bebiş et al., 2013). In the study conducted in 2017, Firouzkouhi et al. expressed the difficulties they had and stated that they could not use triage effectively during the Iran-Iraq War because of the challenging environment, inadequate health care team and limited opportunities. The nurses played a major role in triage and applied triage to the wounded in emergency tents, camps, field hospitals and finally in transferred hospitals. Because of the insufficient nurses in the war zones, civilian nurses were asked for help. One of the attenders tells: ‘The triage during the war was very different from what was told in the books. We used to apply triage based on the survival of the patients. Patients with slight injuries and who are able to walk were transported with vehicles after the initial treatment. The first stage of our triage was to save the lives of those seriously injured. We chose patients who needed surgery. For example, patients with wounds on the neck and abdomen were given priority. Clinical service was applied to the wounded people with internal bleeding. Vascular cases were also accepted. Patients with fractures were first treated, then prepared and finally transferred. The availability of the vehicles to transport the patients (ambulances, buses with or without seats, helicopters…) was checked and triaged.’ Physicians and nurses have problems due to certain situations while applying triage during the war. The continuation of the treatment of the wounded during the battle, emergency resuscitation and triage at the same time causes some disruptions. These problems lead to increased mortality, disruption of interventions and discontinuation of treatment. Nurses, on the other hand, themselves, had to suture the chest tube, take part in resuscitation and intubate due to the lack of physicians (Frouzkouhi, Baroujeni, Kako & Abdulhamimohammed, 2017). Nurses’ interventions after triage include practices such as; Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 363

 Obtaining intravenous access and drawing blood,  Administering tetanus prophylaxis, antibiotics, analgesics and prescribed drugs,  Dressing and splinting the wounded,  Indwelling urinary catheter (Sinan, 2010). The model used during triage is START (Simple Triage and Rapid Treatment) and jump START model is used in paediatric groups. In this model, wounded in green colour are defined as slightly injured and treated as an outpatient. Wounded in yellow colour represent a group of patients with significant wounds that do not need immediate treatment. They are not in immediate danger of death but should be kept under observation. The wounded in the red area are the emergency patient group that requires immediate intervention. The wounded who require basic and advanced life support, and intubation and mechanical ventilation are in the red group. The wounded / patient group in black colour include the dead patients not requiring any intervention. The main purpose of this triage system is to identify the patients with red colour and to intervene as soon as possible. Colour coding the patients during the classification of the injured accelerates the process about which colour group they will be included. Table 1 represents the START model (Karcıoğlu et al., 2017). Table 1: START model

364 • Health Sciences Research Papers

MECHANISMS OF INJURY IN WAR Physicians and nurses come across with many different injuries during the war. These include traffic accidents caused by military vehicles, mutilated body parts, blunt trauma resulting from explosion-related building collapses, penetrating trauma due to weapons used in combat, blast injuries due to mines and explosives, burns and injuries caused by unconventional weapons (Derici, 2010; Aslan & Olgun 2014).

EMERGENCY ORGANIZATION IN WAR The organization of emergency starts from the moment the wounded are approached during conflict. Appropriate treatment and care are provided for the wounded who were transported during the war by using the available facilities. The first organization is the establishment of proper triage. The triage practitioner must organize the interventions in a very cool and fast manner. First, questions like “Do you hear me? Are you okay?” are asked to the wounded in order to determine the state of consciousness and the Glasgow Coma Scale is used to assess the level of consciousness (Aslan et al., 2014; Tarhan et al., 2016). After appropriate triage is performed, the circulation, airway and respiration of the wounded are primarily evaluated. Parameters to be considered while evaluating the circulation need to be in the form of pulse, blood pressure, capillary refill, skin colour and body temperature. If the injured person does not have a pulse during the short examination, if there is cyanosis and loss of consciousness, basic life support should be started immediately (Karcıoğlu et al., 2017). In case of bleeding, it should be evaluated whether the injured person is in shock. Early signs include tachycardia, hyperventilation, cold and pale skin; late symptoms include hypotension, filiform pulse, shallow breathing, impaired consciousness, and pupillary dilatation. It is critical that nurses evaluate patients for these symptoms. Vital signs should be closely monitored, pupil reflexes and consciousness level should be checked (Vincent & Backer, 2013). If there is a head tilt / chin lift or neck trauma in the management of the airway, jaw thrust is performed. Oropharyngeal airway is used to make an effective airway. If there is deterioration or loss of consciousness, intubation is planned. After cardiopulmonary arrest, airway obstruction is the second most common cause of death related to trauma. In the evaluation of the airway which is the entrance gate of the oxygen Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 365 transmission chain, which results in organ failure and death if absent; obstruction causes such as foreign bodies, tongue, teeth, oedema, blood and other secretions are observed. Excessive secretion control and drainage monitoring through aspiration should be performed carefully in obstructions. After providing airway patency of the injured, respiration rate, depth and rhythm should be evaluated (Fatma & Olgun, 2014).

WAR SURGERY AND NURSING War injuries are different from traumatic injuries. Due to the risk of infection and insufficiency in health services, large tissue damage caused by war injuries are treated under more difficult conditions than injuries caused by trauma. Limited facilities during the war impede treatment. There should be an efficient surgeon and nurse in limited treatments. Surgeon’s understanding the problem of the wounded, having a foresight and being able to find early solutions in a situation of limited resources provides effective treatment. It includes multi-surgical approaches rather than multi- disciplinary approaches (Derici, 2010). The nurse also has an important role in providing treatment, care and environmental conditions during the war. It is important that they perform appropriate triage for the wounded, coordinate their care and treatment and make full use of their facilities. Nurses need to have sufficient experience and ability to produce solutions to the crisis in an extraordinary situation (Bebiş et al., 2013). Another situation seen in the war is to take precautions against chemical, biological, radiological and nuclear attacks. Those exposed to these attacks should be sorted out using the appropriate triage method and be isolated and sent to a separate unit (Karcıoğlu et al., 2017) Performing surgical interventions to the wounded in the war as soon as possible reduces the mortality. Many injury mechanisms are seen during the war. Mutilation of body parts caused by explosion, penetrating trauma caused by firearm injuries, and injuries caused by chemical attacks can be seen (Karcıoğlu et al., 2017). The surgeon must be an expert in this field and be skilled and efficient. The continuation of conflicts during the war provides treatment for the wounded, but can also cause disruptions (Bebiş et al., 2013; Karcıoğlu et al., 2017; Frouzkouhi et al., 2017). In case of any attack on hospitals, security units should be reached, and these officers should wait at the exit points. Nurses should have the 366 • Health Sciences Research Papers necessary clinical skills when dealing with the wounded and be able to provide a high quality of care in line with the current opportunities. If an experienced nurse dies or is unable to perform the duty, another nurse must come into the position and maintain the treatment (Duncan et al., 2005; Bebiş et al., 2013). The inadequate number of health members and the limited availability of health care facilities reduce the success rate of the treatment. The lack of clean and sterile surgical instruments used, and the contamination of the disintegrated tissues increase the risk of infection and cause delayed wound healing and infectious diseases (Katoch & Rajagopaları, 2011; Frouzkouhi at al., 2017). Nurses should pay attention to the environmental sanitation in order to prevent these negative events and use the best possible means to apply wound care under aseptic conditions. Nurses should have sufficient knowledge and equipment regarding the injury mechanisms of the wounded. They have important roles such as administering basic life support at the time of the event, providing treatment and nursing care in accordance with the standards (Qureshi 2002; Nies & Mceven 2011; Bebiş et al., 2013).

HEAD TRAUMA AND NURSING CARE Kinematics of head trauma is the injury called whiplash which is caused by the acceleration and deceleration. Traumatic brain injury is defined as “blunt or penetrating head trauma that disrupts normal brain function.” (Oyasanya et al., 2017). Traumatic brain injury as a result of blunt and penetrating head trauma is primarily caused by impact injury, and secondary by the hemorrhage and oedema causing increased intracranial pressure (Tilmen, Akçil & Tunalı, 2015). Hypoxia and hypotension, which cause serious problems as a result of head trauma, should be urgently intervened. Oxygenation should be performed to prevent hypoxia and adequate hydration should be provided for hypotension. Crystalloid, colloid and / or blood products are used to replace lost intravascular volume. The state of consciousness needs to be checked, urgent surgical intervention should be performed in case of epidural and subdural hematoma (İzci & Tehli, 2017). Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 367

THORACIC TRAUMA AND NURSING CARE Thoracic traumas are classified as primary and secondary thoracic traumas. Primary thoracic traumas are listed as sternal fractures, oesophageal injuries, rib fractures, scapular fracture, and pulmonary laceration. Secondary thoracic traumas, however, include pneumothorax, hemothorax, sail sign and tension pneumothorax (Öncel, Sunam & Bayır, 2013). Pneumothorax occurs as a result of air collection to the pleural cavity. It is seen as open or closed pneumothorax (Öncel et al., 2013). The patient with pneumothorax has severe respiratory distress and is cyanotic. The open part should be covered with a sterile drape. Inhaled air enters the damaged lung during inspiration, causing paradoxical collapse. A slight expansion occurs during the expiration; this leads to the formation of cavities that damage the lung (Liman & Taştepe, 2006). Tube thoracostomy is performed by closing the wound on three sides and away from the chest wall. The patient's respiratory rate and depth are monitored, surgical debridement is performed, and the wound is closed (Dennis, Bellister & Guillamondegui, 2017). Hemothorax is caused by blood collection in the pleural cavity as a result of blunt and penetrating traumas. During the examination, the lung sounds are decreased or absent, and there is dull percussion sound. Chest X-ray is used for diagnosis (Dennis et al., 2017). In patients with hemothorax, at least two vascular access should be established, and fluid replacement should be performed. In case of moderate and severe hemothorax, tube thoracostomy is performed from the anterior or midaxillary line to the 5th intercostal space. Blood transfusion planning should be made according to the amount of blood and fluid lost. Thoracotomy should be considered if the loss is 20mg / kg (Öncel et al., 2013). Sail sign is a chest trauma in a free movement in the chest wall which is caused by the breakage of two or more adjacent ribs at two points and is characterized by the formation of paradoxical breathing. Appropriate ventilation and adequate hydration should be provided first. The chest wall does not expand and rise during inspiration, but it is filled with air while expiration (Öncel et al., 2013; Aslan et al., 2014; Dennis et al., 2017). The diagnosis of tension pneumothorax is made according to the patient's clinical findings. Pleural cavity is filled with air pressure because 368 • Health Sciences Research Papers the air inhaled through inspiration cannot be exhaled with expiration. This accumulation of air pushes the mediastinal organs towards the hemithorax, causing compression of the lungs and large vessels. In this case, it is urgently necessary to perform tube thoracostomy to the 5th intercostal space from mid or anterior axillary lines (Öncel et al., 2013).

SPINAL CORD INJURIES AND NURSING CARE Injury leads to disruption of motor and sensory neurons in the spinal cord. This results in partial or complete paralysis, loss of function and deterioration in quality of life. In a complete cervical injury, autonomic reflex disappears as a result of parasympathetic nerve activation of smooth muscles in blood vessel walls, vasodilatation occurs in vessels and bradycardia is seen. Hypotension is a sign of neurological shock. In acute phase, sinus bradycardia, arterial hypotension, vasodilatation and venous stasis are observed and in chronic phase cardiovascular deterioration, autonomic dysreflexia, increased troponin and cardiac rhythm disturbances are seen (Hagen, Rekond, Gronning & Faerestrand, 2012). The degree of respiratory complication depends on the spinal cord injury and motor impairment according to ASA classification. Due to the muscle paralysis, adequate cough and secretion cannot occur and may cause impaired respiratory function (Tollefsen & Fondenes, 2012). Oedema and bleeding result in pressure on the microvascular circulation. Surgical spinal decompression reduces this pressure and reduces the risk of secondary hypoxia and ischemia. Fractures that do not respond to reduction, such as unstable vertebral fractures, are among the indications for surgery (Ahuja, Martin & Fehlings, 2016). ABDOMINAL TRAUMA AND NURSING CARE

Injuries occur as a result of direct or movement related stress and laceration. Perforation in hollow organs causes infection as a result of bleeding or contamination (Sarsılmaz & Kocataş, 2016). As a result of abdominal trauma, organs such as liver, spleen and kidney can be damaged and cause various complications. Blunt or penetrating trauma mechanisms are seen in liver injury. Because of its increased blood flow, in case of a trauma, the vascular system in the liver can cause bleeding and shock (Taghavi & Askari, 2018). Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 369

It is important that patients are closely monitored for signs of bleeding and shock after injury. Splenic trauma is the most commonly injured organ due to blunt trauma. As the spleen is a vascularized organ that is very sensitive to trauma due to its spongy structure, blood loss can be seen as a result of vessel injury. Since trauma may result in blood loss, the first evaluation of the wounded should be performed according to circulatory, airway and respiratory parameters. The only treatment of the injury is splenectomy (Sarsılmaz et al., 2016; Waseem & Bjerke, 2018). Renal trauma causes injury to the parenchyma or vessels. This results in bleeding and perforation. Tenderness, pain and ecchymosis in the abdomen and back indicate kidney damage. Another symptom is haematuria. If haematuria is present, urine output should be monitored and recorded. Blood gas and biochemistry tests should be checked as there will occur fluid-electrolyte imbalance due to the kidney damage (Singh & Sokraj, 2018). CONCLUSION Wars are conflicts that threaten human life, order of a country and negatively affect health systems. As hospitals, which are one of the most affected places in the war, cannot provide adequate treatment and quality care, an increase in morbidity and mortality rates can be seen. Intervention at the scene during the war can save the lives of many wounded. Adequate knowledge, skills and equipment of the health care team will enable the rapid intervention of the injured and prevent the complexity to be experienced. Nurses, which have an important place among health professionals in extraordinary situations such as war, need to make the most of their facilities and provide optimal care by applying triage in the most effective and fast way.

370 • Health Sciences Research Papers

REFERENCES Ahuja, CS. Martin, AR. Fehlings, M. (2016). Recent Advances In Managing a Spinal Cord Injury Secondary to Trauma. F1000Research, (F1000 Faculty Rev) 5:1017,1-12. Allender, J.A., Rector, C., Warner, K.D. (2011), Community Health Nursing: Promoting and Protecting the Public’s Health. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins. Baltacı, T. (2010). Hemşirelik Mesleğinin Dünü Bugünü. Sağlık ve Yaşam 19 Mayıs Üniversitesi Dergisi, (32) 8. Bebiş, H. Özdemir, S. (2013). Savaş, Terör ve Hemşirelik, Florence Nightingale Hemşirelik Dergisi, (1),57-68. Bilgin, İ. (2008). Çanakkale’nin Kadın Kahramanı: Safiye Hüseyin. İstanbul: Timaş Yayınları. Bridges, E. (2003). Military and Disaster Nursing. Critical Care Nursing Clinics of North America, 15(2), 13-14. Burns, N., Grove, SK. (2005), The Evolution of Research in Nursing, Burns N, Grove SK. In The Pracrice of Nursing Research Conduct,Critique, and Utilization 5th edition. USA: Elsevier, Chatoupis, K., Popodopoulou, G., Kaskarelis, I. (2013). New Technology in The Management of Liver Trauma. Annals of Gastroenterology, 26, 41-44. Dennis, B.M., Bellister, S.A., Guillamondegui, O.D. (2017). Thoracic Trauma. Surgical Clinics of North America, 97, 1047-1064. Derici H. (2010). Savaş Yaralanmalarının Cerrahi Tedavisi, Eryılmaz M. (Çev. Ed.) Savaş Cerrahisi, Cilt 1, Tuna Matbaacılık: Ankara. Dilmen, Ç., Akçıl, E., Tunalı, Y. (2015). Travmatik Beyin Hasarında Yoğun Bakım Tedavisi. Turkish Journal of Anaesthesiology and Reanimation, 43,1-6. Duncan, R., Pansoy, C., Towe, L., Nosalik, B., Sundem, B., Snyder,L,. Garcıa, D., Kıdwell, G. (2005). Grace Under Fire. Nursing, 35(5), 62-64. Firouzkouhi, M., Baroujeni, A., Kako, M., Abdulhamimohammed, A. (2017). Experiences of Civilian Nurses İn Triage During the Iran-Iraq War. Chinese Journal of Traumatology, 20(5), 288-292. Hagen, E., Rekond, T., Gronning, M., Faerestrand, S. (2012). Cardiovascular Complications of Spinal Cord Injury. Tidsskr Nor Laegeforen, 132(9),1115-1120. Sonay GÖKTAŞ, Elif GEZGİNCİ, Ayşenur ATA • 371

Hatipoğlu, S. (2011). Selimiye’den Geleceğe Notlar. Hacettepe Üniversitesi Sağlık Bilimleri Fakültesi Hemşirelik Dergisi, 103-111. İzci, Y., Tehli, Ö. (2017). Terörde ve Savaşta Kraniyal ve Spinal Kord Yaralanmaları. Okmeydanı Tıp Dergisi, 33, 21-29. Karcıoğlu, Y., Topaçoğlu, H. (2017). Savaş ve Terör Afetlerinde Acil Servis Triyajı. Okmeydanı Tıp Dergisi, 33, 1-8. Kenar, I. (2004). Kitle İmha Silahlarına Karşı Savunmanın Boyutu. TSK Koruyucu Hekimlik Bülteni, 3(10), 243-259. Leblebicioğlu, H. (2017). 18. Yüzyıldan Günümüze Türkiye ve Amerika’da Askeri Hemşireliğin Tarihsel Gelişimi. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, 6(4),268-274. Myburgh, J., Mythen, M. (2013). Resuscitation Fluids. The New England Journal of Medicine, 369, 1243-1251. Nies, M. A., Mceven, M. (2011). Natural and man-made disaster. Community/Public health nursing: Promoting the health of populations. 5th ed., Elsevier, St. Louis, 561-583. Oyesanya, T.O., Brown, R.L., Turkstra, L.S. (2017). Caring for Patients with Traumatic Brain Injury: A Survey of Nurses’ Perceptions. Journal of Clinical Nursing, 26 (11), 1562-1574 Ökdem, Ş., Abbasoğlu, A., Doğan N. (2000). Hemşirelik Tarihi, Eğitimi ve Gelişimi. Ankara Üniversitesi Dikimevi Sağlık Hizmetleri Meslek Yüksekokulu Yıllığı, (1)1, 5-11. Öncel, M., Sunam, G., Bayır, A. (2013). Toraks Travmalarına Acil Yaklaşım. Arşiv Kaynak Tarama Dergisi, 22(1), 110-129. Öztekin, D., Uğraş, G. (2009). Kafa Travması Geçiren Hastada Hemşirelik Girişimleri. Fırat Sağlık Hizmetleri Dergisi, 4(11), 146-157. Sarsılmaz, A., Karakoç, E. (2016). Abdominal travma. Türk Radyoloji Derneği Seminerleri,4, 299-312. Sinan, H. (2010). Kitle Yaralanmalarında Hastane Triyajı, Eryılmaz M. (Çev. Ed.) Savaş Cerrahisi, Cilt 1, Tuna Matbaacılık: Ankara. Singh, S., Sookraj, K. (2019). Kidney Trauma, Stat Pearls Publishing. Accessed: https://www.ncbi.nlm.nih.gov/ books/ NBK532896/ Sorokina, T.S. (2005). Nursing in The Russian-Turkish Wars (1853–1856,1877– 1878). 38. Uluslararası Tıp Tarihi Kongresi Bildiri Kitabı. Ankara: Türk Tarih Kurumu Yayınları. 372 • Health Sciences Research Papers

Taghavi, S. Askari, R. (2019), Liver Trauma, Stat Pearls Publishing Accessed: https://www.ncbi.nlm.nih.gov/ books/NBK513236/ Tarhan, M. Akın, S. (2016). Triyaj Uygulamalarında Hemşirenin Rolleri. Celal Bayar Üniversitesi Sağlık Bilimleri Enstitüsü Dergisi, 3(2), 170-174. Tollefsen, E., Fondenes, O. (2012). Respiratory complications associated with spinal cord injury. Tidsskr Nor Laegeforen, 132(9):1111-4 Torun, S. (2014). Kırım Savaşı’nda Hasta Bakımı ve Hemşirelik. İstanbul: Akademisyen Kitabevi. Vincet, J., Backer, D. (2013). Circulatory Shock. The New England Journal of Medicine, 3, 1726-1734. Waseem, M. Bjerke, S. (2019). Splenic Injury, Stat Pearls Publishing. Accessed: https://www.ncbi.nlm.nih.gov/ books/NBK441993/ Word Health Organization Health Topics (2006). Accessed: http://who.int/health_topics/en Yılmaz Gören, Ş., Yalım N.Y. (2016). Hemşirelik Tarihinde Bir Öncü “Safiye Hüseyin Elbi”. Lokman Hekim Dergisi, 6(2), 38-45.