ROLE OF GENETIC FACTORS IN THE PATHOGENESIS OF PRIMARY OPEN ANGLE GLAUCOMA AND POTENTIAL ROLE OF ANTIOXIDANTS TO PREVENT PRIMARY OPEN ANGLE GLAUCOMA
A THESIS SUBMITTED TO THE LIAQUAT UNIVERSITY OF MEDICAL & HEALTH SCIENCES JAMSHORO, SINDH, PAKISTAN
IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN HUMAN ANATOMY & CELL BIOLOGY
BY
DR. MUHAMMAD YAQOOB SHAHANI MBBS; D.Ophth
SUPERVISORS
DR. SAMREEN MEMON, MBBS; PhD (UK) DR. ALI MUHAMMAD WARYAH; PhD DR. ASHOK KUMAR NARSANI; FCPS
DECEMBER 2017
RESEARCH CERTIFICATE
This is to certify that Dr. Muhammad Yaqoob Shahani, the author of this thesis has performed his original research work under our guidance. We have reviewed his research work by ourselves and endorse the legitimacy of his work. We are also assuring that his work has not been submitted to any other institution for the award of
Ph.D degree other than The Liaquat University of Medical & Health Sciences,
Jamshoro. We also certify that this research document has been written under our guidance according to the format approved by The Liaquat University of Medical &
Health Sciences, Jamshoro and we recommend its appraisal for awarding Ph.D degree according to the rules and regulations of the institution.
Supervisor’s Signature: ______Prof. Dr. Samreen Memon
Supervisor’s Signature: ______Dr. Ali Muhammad Waryah Assistant Professor
Supervisor’s Signature: ______Prof. Dr. Ashok Kumar Narsani
i
Acknowledgements
I am grateful to Almighty, who gave me chance to join The LUMHS Jamshoro and got admission to pursue my PhD program.
I am thankful to my Mentor, Prof. Dr. Samreen Memon, for her kind help and assistance, during this program to fulfill the research work of my PhD degree program, very well. She has been a wonderful supervisor throughout and it would have been impossible to achieve all this without her support.
Other than Principal Guide, I am also thankful my co-supervisor Dr. Ali Muhammad
Waryah and people working in laboratory where I did my work specially Muhammad
Ali Kanrio, his overall help proved very much helpful for me.
I am also thankful to one of my respectable teachers and second co-supervisor, Prof.
Dr. Ashok Kumar Narsani for his support in enrollment and examination of individuals having POAG.
It’s very important to mention all those colleagues and friends who provided me all the support without which this all was not possible to complete. First, I am thankful to Dr.
Shakeel Ahmed Shaikh, my very good friend. I am also thankful to my sisterly colleague Dr. Umbreen Bano who remained with me during the whole period.
Secondly, I am also thankful to my dear sister Hina Shaikh, my dear friend, Yar
Muhammad Waryah, for their kind support.
I am very much thankful to my loving father, the most imperative person for me, for his guidance, prayers and support all the time.
I am also very much thankful to all my family members specially my loving wife Dr.
Shazia Yaqoob Shahani for her special care, extending her moral support and
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encouragement while pursuing my research, my cute kids, Asma Batool, Ali Hasnain and Shefa Batool.
Many thanks to all my brothers Engineer Aijaz Ali Shahani, Mubashir Ali, Ghulam
Raza and Awais Ali for their moral support.
In the end, I am very much thankful to my best friends Dr. Rafique Ahmed Jalbani, Dr.
Abdul Sattar Khan Qaimkhani and Dr. Ganesh Kumar for their special company and support. I would like to thanks my Alma Mater Liaquat University of Medical and
Health Sciences, Jamshoro, Pakistan for bearing my PhD research work expenses.
Dr. Muhammad Yaqoob Shahani December 13, 2017
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Dedication
All this research work is dedicated to my beloved mother Mrs. Hayat Khatoon, who left me during the course of my PhD program. It is because of her immense love, teachings and the way she had brought me up that I have been able to complete my studies. May
Almighty ALLAH J.J grant the highest place in Jannat-ul-Firdous to my lovely mother,
I love you and miss you, my sweet Amar.
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Publications
WARYAH, A. M., NARSANI, A. K., SHEIKH, S. A., SHAIKH, H. & SHAHANI, M. Y. 2013. The novel heterozygous Thr377Arg MYOC mutation causes severe Juvenile Open Angle Glaucoma in a large Pakistani family. Gene, 528,356-9.
Yaqoob Shahani, M., et al. (2019). "POSSIBLE PREVENTION OF REACTIVE OXYGEN SPECIES INDUCED HUMAN TRABECULAR MESHWORK CELL DAMAGE BY RESVERATROL AND ASCORBIC ACID." The Professional Medical Journal 26: 1036-1041.
"CYP1B1 mutations have low contribution to Pakistani patients with Primary Open Angle Glaucoma". Muhammad Yaqoob shahani, MBBS; Samreen Memon, Ph.D; Ali Muhammad Waryah, Ph.D; Ashok Kumar Narsani, FCPS; Hina Shaikh, BS; Shakeel Ahmed Shaikh, Ph.D; Khairuddin Shah, MMBS; Ambreen Bano, MBBS. (Submitted).
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Table of Contents
Chapter No. 01 INTRODUCTION
1.1 ANATOMY OF EYE (OPHTHALMOS) ...... 1
1.1.1 Overview…………………...... 1 1.1.2 Embryogenesis of eye ...... 2 1.1.3 Gross anatomy of eye ...... 5 1.1.4 Coats of the eyeball ...... 5 1.1.5 Trabecular meshwork ...... 12 1.1.5.1 Clinical significance: ...... 15 1.1.6 Aqueous humor (AH): ...... 15 1.1.6.1 Composition: ...... 15 1.1.6.2 Functions of aqueous humor: ...... 15 1.1.6.3 Production & drainage: ...... 16
1.2 GLAUCOMA ...... 18 1.2.1 Classification of glaucoma:...... 19 1.2.2 Primary open angle glaucoma:...... 19 1.2.3 Intraocular pressure and glaucoma: ...... 24 1.2.3.1 Ocular hypertension: ...... 24 1.2.3.2 Optic nerve head: ...... 24 1.2.3.3 Epidemiology of glaucoma: ...... 29 1.2.3.4 Clinical risk factors for glaucoma: ...... 31 1.2.3.5 Pathophysiology of glaucomatous optic cupping: ...... 33 1.2.3.6 Genetic basis of POAG: ...... 37
1.3 CATEGORIZATION OF LOCI OF GENES ASSOCIATED WITH PRIMARY OPEN ANGLE GLAUCOMA: ...... 38 1.3.1 Myocilin gene…………………………………………………………….38 1.3.2 Cytochrome C (cyp1b1) gene: ...... 41
1.4 OXIDANTS AND ANTIOXIDANTS: ...... 44 1.4.1 Free radicals & ROS: ...... 44 1.4.2 Resveratrol……………………… ...... 45 1.4.3 Ascorbate…………………………………………………………………50
Chapter No. 02 MATERIAL AND METHODS
SECTION: 01: GENETIC PHASE ...... 56 2.1 Materials used in genetic phase of this study: ...... 56 2.1.1 Field work ...... 58 2.1.2 Laboratory work at MBGD laboratory: ...... 68
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SECTION: 02: TISSUE CULTURE PHASE ...... 78 2.2 CELL CULTURE REAGENTS: ...... 78 2.2.1 Trabecular Meshwork Cell Medium (TMCM): ...... 78 2.2.2 Complete Human trabecular Meshwork Cell Medium (HTMCM): ...... 78 2.2.3 Poly L-Lysine (PLL): ...... 78 2.2.4 Dulbecco`s Phosphate Buffered Saline (DPBS): ...... 79 2.2.5 Trypsin Neutralization Solution (TNS): ...... 79 2.2.6 Trypsin – EDTA (T/E): ...... 79 2.2.7 Human Trabecular Meshwork Cells (HTMC): ...... 80 2.2.8 MTT Cell Proliferation Assay: ...... 82
2.3 H2O2 Treatment of Human Trabecular Meshwork Cells: ...... 85 2.4 Human Trabecular Meshwork Cell-Viability/ Metabolic Assay: ...... 86 2.5 HTM Cell Live Cell Assay: ...... 86 2.6 Analysis of reactive oxygen species (ROS) in HTMCs: ...... 87 2.7 Assay technique: ...... 87
Chapter No. 03 RESULTS
SECTION.01: GENETIC PHASE ...... 89 3.1 FAMILIES FOUND LINKED TO MYOCILIN GENE: ...... 91 3.2 FAMILIES FOUND LINKED TO CYP1B1 GENE:...... 101 3.3 FAMILIES FOUND UNLINKED TO MYOCILIN AND CYP1B1 GENE: ...... 108
SECTION: 02: TISSUE CULTURE PHASE ...... 131 3.4 IMPACTS OF RESVERATROL AND ASCORBIC ACID ON TM CELL METABOLISM AGAINST H2O2-INDUCED INJURY: ...... 132 3.4.1 Co-incubation of Ascorbate and Resveratrol: ...... 132 3.4.2 Pretreatment of TM cells with Ascorbate and Resveratrol ...... 134 3.5 Effects of Resveratrol and Ascorbate on Cell Viability against H2O2-Induced TM cell Injury: ...... 136 3.6 Reactive Oxygen Species (ROS) Effects of Resveratrol and Ascorbate in Co- Incubated TM Cells: ...... 137
Chapter No. 04 DISCUSSION
SECTION: 01: GENETIC PHASE ...... 140 4.1 POAG FAMILIES WITH NOVEL MUTATIONS: ...... 141 4.2 POAG FAMILY WITH REPORTED MUTATION: ...... 149
SECTION NO: 02: TISSUE CULTURE PHASE ...... 151 4.3 RESVERATROL & ASCOBRIC ACID: ...... 151
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Chapter No. 05 CONCLUSION AND FUTURE DIRECTIONS ...... 156
Chapter No. 06 REFERENCES ...... 158
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List of Tables
Table No. 1: Reaction mixtures to amplify PCR ...... 73
Table No. 2: Recipe of working Reaction mixtures for sequencing PCR ...... 76
Table No. 3: Concentration of water and Ply L-lysine for coating the culture flasks ...... 80
Table No. 4: Equipment and Materials used in this procedure...... 83
Table No. 5: Details of clinical examination of diseased persons having JOAG .. 93
Table No. 6: Details of clinical examination of diseased persons of POAG – 06 Family ...... 99
Table No. 7: Details of clinical examination of diseased persons having POAG 105
Table No. 8: Co-Incubation of HTM Cells with Ascorbate and Resveratrol against H2O2-Induced Metabolic Injury...... 133
Table No. 9: Pretreatment of TM Cells with Ascorbate and resveratrol against H2O2-Induced Metabolic Injury...... 135
Table No. 10: Impacts on cell viability of resveratrol and ascorbate against H2O2- induced TM cellular damage ...... 136
Table No. 11: Reactive Oxygen Species (ROS) Levels (IU/ml) ...... 138
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List of Figures
Figure No.1: Embryology of the human eye (Sadler and Langman, 2012)...... 3
Figure No.2: Embryology of the human eye (Sadler and Langman, 2012)...... 3
FigureNo.3: Development of human eye (Sadler and Langman, 2012)...... 4
Figure No 4: Anatomy of the Eyeball (Snell, 2012)...... 7
Figure No.5: Aqueous outflow pathway of Human eye (https://www.wikipedia.org/)...... 9
Figure No.6: Anatomy of Aqueous outflow pathway (https://www.wikipedia.org/)...... 14
Figure No 7: Aqueous outflow routes illustrated in the Cut section of the anterior chamber of eye: ...... 17
Figure No8: Distinguishing landscapes of glaucoma: ...... 21
FigureNo.9: Humphrey’s visual field presenting an advanced visual field loss (Bowling and Kanski, 2016)...... 22
Figure No.10: Width of the anterior chamber ...... 23
FigureNo.11: The structure of fibers of retina elucidating design of merging of ganglion cell fibers (described in yellow color) to the optic nerve: . 26
FigureNo.12: Pictorial demonstration of the design of merging of fibers of ganglionic cells at the optic disc, that directs the way of defects in visual fields: ...... 27
Figure No.13: C/D ratio of optic disc. A figure of left optic disc having a large C/D ratio(Bowling and Kanski, 2016)...... 28
Figure No.14: Estimations of worldwide frequency having POAG in persons with age more than forty years (Leske et al., 2007)...... 30
Figure No.15: Pictorial illustration of comparative impact applied by mechanical and vascular factors jon the progression of glaucomatous optic neuropathy at numerous intensities of Intra Ocular Pressure (Caprioli J, 1984). 35
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Figure No.16: Pictorial illustration of the probable issues influencing on the injury to optic nerve due to the Glaucoma: ...... 36
Figure No.17: Molecular structure of Trans-Resveratrol and Cis-Resveratrol (Cottart et al., 2010)...... 46
Figure No.18: Mechanism of action of Resveratrol; Adapted from (Bola et al., 2014)...... 49
Figure No.19: Molecular structure of ascorbic acid and biochemical reactions (Hacişevki, 2009)...... 52
Figure No 20: Snellin’s chart for visual acuity assessment...... 61
Figure No.21: Measurement of intraocular pressure by Goldman Aplannation tonometer ...... 62
Figure No.22: Measurement of intraocular pressure by Air Puff tonometer...... 63
Figure No.23: Performing Fundoscopy on slit lamp bio microscope with the help of +78 D lens...... 64
Figure No.24: Assessing the visual fields of the patient on Perimeter...... 65
Figure No.25: Assessing the anterior chamber angle with the help of Gonio lens. . 66
Figure No.26: Spectrophotometer used for measurement of the Optical Density. .. 71
Figure No.27: Agarose Gel Electrophoresis Results...... 72
Figure No.28: Applied Bio system thermo cycler (2720)...... 74
Figure No.29: Thermo cycling profile (Program) for sequencing reaction...... 76
Figure No. 30: Family tree of POAG-26: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members...... 92
Figure No.31: Fundus photographs of an affected individual showing increased CDR ...... 94
Figure No.32: Representative Chromatogram of MYOC sequence ...... 95
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Figure No.33: Family tree of POAG-06: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members. 98
Figure No.34: Fundus photograph of an affected individual of POAG-06 ...... 99
Figure No. 35: MYOC chromatogram of normal and diseased patient...... 100
Figure No.36: Family tree of POAG-02: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members...... 104
Figure No.37: Fundus photograph of an affected individual of POAG-02 ...... 106
Figure No.38: Chromatogram of CYP1B1 sequence: ...... 107
Figure No. 39: Family tree of POAG-01:...... 108
Figure No.40: Family tree of POAG-03:...... 109
Figure No. 41: Family tree of POAG-04:...... 110
Figure No.42: Family tree of POAG-05:...... 111
Figure No.43: Family tree of POAG-07:...... 112
Figure No.44: Family tree of POAG-08:...... 113
Figure No. 45: Family tree of POAG-09:...... 114
Figure No.46: Family tree of POAG-10:...... 115
Figure No.47: Family tree of POAG-11:...... 116
Figure No.48: Family tree of POAG-12:...... 117
Figure No.49: Family tree of POAG-13:...... 118
Figure No.50: Family tree of POAG-14:...... 119
Figure No.51: Family tree of POAG-15:...... 120
Figure No.52: Family tree of POAG-16:...... 121 xii
Figure No.53: Family tree of POAG-17:...... 122
Figure No.54: Family tree of POAG-18:...... 123
Figure No.55: Family tree of POAG-19:...... 124
Figure No. 56: Family tree of POAG-20:...... 125
Figure No.57: Family tree of POAG-21:...... 126
Figure No.58: Family tree of POAG-22:...... 127
Figure No.59: Family tree of POAG-23:...... 128
Figure No.60 Family tree of POAG-24:...... 129
Figure No.61: Family tree of POAG-25:...... 130
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List of Graphs
Graph No. 1: Co-Incubation of TM Cells with Antioxidants Protects Against Peroxide-Induced Metabolic Injury. MTT assay kit (Mitochondria function)...... 133
Graph No. 2: Pretreatment of TM Cells with Ascorbate and Resveratrol against H2O2-Induced Injury (MTT) assay kit...... 135
Graph No. 3: Co-Incubation of TM Cells with Antioxidants Also Protects Against H2O2-Induced Cell Death. (Determined by F528) ...... 136
Graph No. 4: Reactive Oxygen Species (ROS) levels (IU/ml)...... 138
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List of Abbreviations
AH : Aqueous Humor
AIDS : Autoimmune Deficiency Syndrome
ARMD : Age Related Macular Degeneration
C/D Ratio : Cup Disc Ratio
Ca+2 : Calcium
CAG : Closed Angle Glaucoma
Cl− : Chloride
CYP1B1 : Cytochrome P450 Family 1 Subfamily B Member 1
D.M : Diabetes Mellitus
DG : Developmental Glaucoma
DMSO : Dimethyl Sulfoxide
DNA : De-oxy-ribonucleic Acid
DPBS : Dulbecco’s Phosphate Buffered Saline
EDTA : Ethylene-di-amine-tetra-acetic acid
ER : Endoplasmic Reticulum
FBS : Fetal bovine serum
FCED : Fuch’s corneal endothelial dystrophy
GAG : Glycosaminoglycan
GP : Glycoproteins
H2O : Water
HCO3 : Bicarbonates
HIV-1 : Human Immunodeficiency Virus-1
HTG : High Tension Glaucoma
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HTMC : Human Trabecular Meshwork Cells
IOP : Intraocular Pressure
K+ : Potassium
K2PO4 : Monobasic Potassium Phosphate
KCl : Potassium Chloride
KDa : Kilodalton
LC : Lamina Cribrosa
LGG : Lateral Geniculate Ganglion
MBGD : Molecular Biology & Genetics department
Mg+2 : Magnesium
MSCs : Mesenchymal Stem Cells
MYOC : Myocilin
Na+ : Sodium
Na2PO4 : Sodium Phosphate
NaCl : Sodium Chloride
NHS : National Health Survey
NTG : Normal Tension Glaucoma
O.S : Oxidative Stress
OAG : Open Angle Glaucoma
OH : Ocular Hypertension
OLF : Olfactomedin
ONH : Optic Nerve Head
OPTN : Optineurin Gene
P/S : Penicillin/Streptomycin solution
PCG : Primary Congenital Glaucoma xvi
PLL : Poly-L-lysine
PO4 : Phosphate
POAG : Primary Open Angle glaucoma
RDA : Recommended Daily Allowances
RGC : Retinal Ganglion Cells
RNFL : Retinal Nerve Fiber Layer
ROS : Reactive Oxygen Species
SLE : Systemic Lupus Erythematosus
SNP : Single Nucleotide Gene Polymorphism
T/E : Trypsin – EDTA
TIGR : Trabecular Meshwork Inducible Glucocorticoid Response
TM : Trabecular Meshwork
TMCGS : Trabecular Meshwork Cell Growth Supplement
TMCM : Trabecular Meshwork Cell Medium
TNS : Trypsin Neutralization Solution
VFD : Visual Field Defects
W.H.O : World Health Organization
WDR36 : WD Repeat Domain 36
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ABSTRACT
Glaucoma, the collection of syndromes, is the 2nd leading basis of vision loss in the world. Primary open angle glaucoma (POAG), the mostly occurring form is described by optic nerve head excavation, defective fields of vision & increased or regular intraocular pressure (IOP), with no sign of closed angle on Gonioscopic examination.
Though the actual reason of POAG is unclear yet but inherited factors may perform key function in understanding actual mechanism of the disease.
This study was conducted for linkage analysis between POAG and two genes (MYOC
& CYP1B1) related with POAG in the people of Sindh province of Pakistan and elaborate the protecting properties of antioxidants (Resveratrol & Ascorbate) on cultured HTMC subjected to reactive oxygen species.
For this purpose, twenty-six families having multiple affected members diagnosed with primary open angle glaucoma (POAG) were enrolled through hospitals from different cities of Sindh, Pakistan and were numbered from POAG-1 to POAG-26. After genomic DNA isolation, exclusion studies were carried out to analyze enrolled families for linkage to MYOC and CYP1B1 genes. Out of twenty-six families, two families were found linked with the MYOCgene and one family was found linked with CYP1B1 gene.
In family POAG-26, a novel mutation for MYOC gene, c.1130 C>G resulting in p.T377R was identified where as in family POAG-06, one reported mutation for MYOC in Indian population, c.144G>T resulting in p.Q48H was found.
In another family POAG-02, a novel mutation for CYP1B1 gene, c.650 G>A resulting in p.D217N was detected.
For exploration of potential effects of antioxidants (Resveratrol & Ascorbic acid) on cultured Human Trabecular meshwork cell line, Cells were purchased from The
xviii
ScienCell Research Laboratories, Canada. Cultured HTM cells were co-incubated with varying concentrations of H2O2 in growth media for twenty-four hours. Effects of different concentrations of H2O2 on TM cell metabolism were determined by observing the cells under light microscope and detecting mitochondrial enzymes functional activity with the help of MTT assay.
It was proved that Resveratrol has more protecting properties than the Ascorbic acid, even at 4.0 mM H2O2, the TM cellular movement was 76%. Underneath co-treatment circumstances, the 1mM Resveratrol maintained TM cell metabolism. Related to H2O2- treated TM cells, resveratrol enhanced role of mitochondria up to 4.0 mM H2O2 (76%).
For evaluation of protective effects of co-incubation of Ascorbate and Resveratrol was because of metabolic alteration of Trabecular Meshwork cells, which were pre-treated by Ascorbate and Resveratrol for 24 hours followed by H2O2 treatment termed as
“pretreat-only’’.
Highly significant differences were noticed for the 1.0 mM H2O2 concentration (Chi
Square test, p=0.0001). A considerable decrease in metabolism of Trabecular
Meshwork cells was noticed estimating sixty one percent at 1.0 mM of hydrogen peroxide associated to Ascorbic acid – ninety nine percent and Resveratrol ninety nine percent (p=0.0001).
xix
Chapter No. 1:
INTRODUCTION
1.1 ANATOMY OF EYE (OPHTHALMOS)
1.1.1 OVERVIEW
The primary function of human eyeball is to deal with the light. It is a special sensory organ and being a sensory organ, the primary function of eye is vision. Eye has a sensitive nervous coat containing rods and cones which perceive light and make vision possible. Color perception and the perception of depth of vision are important physiological functions of eye. It is suggested that the human eye can discriminate about 10 million colors (Savino et al., 2012).
Non image forming light sensitive ganglions of eye receive light signals and help in the adjustment and regulation of size of pupil, biological clock and release of hormones, for example secretion of melatonin from Pineal gland (Ryan, 2013).
Cornea limits the eye ball anteriorly, and rest of it is covered by sclera. Eye ball is not like a perfect sphere. Corneal segment is smaller while the posterior sclerotic chamber is larger. Scleral chamber comprises 4/5th of eye ball; its radius measures roughly 12 mm. The junction between the cornea and sclera is a circular structure called the limbus. Iris is a colored circular sphincter muscular structure. It surrounds the pupil and looks black or green depending upon coloring pigment. The size of the pupil and amount of light entering into the eye is regulated through iris (Dagnelie, 2011, Davson,
1972,Trattler et al., 2012).
Size of pupil is adjusted by the sphincter and dilator papillae muscles. Eye interior is examined clinically by an instrument known as the “ophthalmoscope”. Light enters & falls on cornea, passes through aqueous humor, pupil, lens and vitreous humor. Shape of transparent eye lens is regulated by the contractions of ciliary muscle, which are suspended by ciliary ligaments. Light falls on the retina and stimulates rods and cones.
1
Rods and cones are photo transducers which transform light signals into electrical signals which move to the occipital lobe of head by the optic nerve (Dagnelie, 2011,
Davson, 1972, Trattler et al., 2012).
1.1.2 Embryogenesis of Eye
Human eye starts to develop at approximately 3rd week of embryonic life and continues through the 10thweek.Both mesoderm and endoderm germ layers contribute in its formation (Sadler and Langman, 2012).Developmentally, Eye develops from ectoderm and mesoderm (Figure No.1). Retina, iris, optic nerves and ciliary bodies are derived from the “neuro-epithelium”. While Lens, eyelids and cornea (epithelium) develop from the “surface ectoderm”. Sclera, cornea (connective tissue), muscles, vitreous body and blood vessels are contributed by the mesoderm germ layer(Schoenwolf and Larsen,
2009).
Eye development becomes visible as a couple of “optic vesicles”, appear on both sides of fore-brain at the completion of 4th gestational week (Figure No.2). Contact of optic vesicles and surface ectoderm brings cascade of changes essential for further development of the eye. Blood vessels enter through choroid fissure of optic vesicles
(Sadler and Langman, 2012).
Pax-6 is a principal regulator gene involved in the ocular development. Pax-6 gene expresses transcription factors and growth factors necessary for eye development. Pax-
6 gene defects result in malformations of lesser eye in mouse, defects in human ocular iris and eye-less Drosophila(Adler and Canto-Soler, 2007). Ocular growth is illustrated by several occasions of outgrowth of optic vesicles and optic grooves or sulci. Optic grooves arise from “neural folds, transmute into optic vesicles with neural tube closure”
(Eiraku et al., 2011, Sadler and Langman, 2012). Optic vesicles form the “optic cup”;
2
whose outer layer form retinal pigment epithelia, inner layer forms the retina and middle part develops into the ciliary body and iris (Eiraku et al., 2011, Fuhrmann, 2010). As the optic cup invaginates, the “lens placode” is formed by thickening of the ectoderm.
Lens placode detaches from the ectoderm eventually and develop the “lens vesicle” at the exposed side of “optic cup” (Figure No.3). More cell discrepancy and mechanical cell re-arrangement of optic cup results in full eye development (Eiraku et al., 2011,
Fuhrmann, 2010,Sadler and Langman, 2012).
Figure No.1: Embryology of the human eye (Sadler and Langman, 2012).
Figure No.2: Embryology of the human eye (Sadler and Langman, 2012).
3
FigureNo.3: Development of human eye (Sadler and Langman, 2012).
4
1.1.3 GROSS ANATOMY OF EYE
Eye ball lies within the orbital fossa. Fascial sheath separates it from the surrounding fat and bone. It is tethered by ligaments and muscles and comprises of 3 coats from insides to outside; (Figure No. 4).
Fibrous coat- the sclera
Vascular (pigmented) coat – choroid, ciliary body and iris (uveal tract)
Nervous coat- retina (Fuhrmann, 2010, Snell, 2012,Standring, 2016).
1.1.4 Coats of the Eyeball
Fibrous Coat
Fibrous coat comprises of glassy transparent anterior part called the cornea and opaque posterior coat called the sclera.
The Cornea
Cornea is glassy transparent and avascular coat which occupies anterior portion of
eye ball. It is answerable for the “light refraction”. Anteriorly, this one is free and
posteriorly, cornea is in exposure with the aqueous humor that provides essential
nutrients to it. There is no lymphatic drainage of cornea. Tear film is present on its
anterior surface (Snell, 2012, Standring, 2016).
It is highly sensitive structure being innervated by long ciliary nerves, branches of
ophthalmic division (V1) of the trigeminal nerve (V).
5
Functions of the Cornea
Tear film nurtures the anterior corneal epithelial cell layer
Refractive medium of the eye
Both anterior and posterior surfaces of cornea add to light refraction.
The Sclera
Sclera is a white opaque coat due to presence of collagen fibers and of blood vessels.
Posteriorly, it is fused with the duralsheath of optic nerve, which pierces it at back
pole. Lamina cribrosa, the scleral area is pierced by the optic nerve fibers. Ciliary
nerves, ciliary arteries and concomitant veins, “the venae vorticosae” also pierce
the sclera coat.
Limbus, also called corneo-scleral junction, is a circular area of junction between
the cornea and sclera (Snell, 2012, Standring, 2016).
6
Figure No 4: Anatomy of the Eyeball (Snell, 2012).
7
The Pigmented Vascular Coat
The pigmented vascular coat comprises of 3 anatomical structures;
1. The choroid,
2. The ciliary body, and
3. The iris.
Collectively these three structures are called as the uveal tract. The choroid is highly vascular coat. It shows inner vascular layer and an outer pigmented layer. Ciliary body comprises of ciliary processes, ciliary ring and the ciliary muscles. Ciliary body is continuous anteriorly with iris and posteriorly with the choroid. Ciliary processes are corrugated folds or ridge like structures (Figure No. 5). On the posterior surface they are attached with suspensory ligament of the lens. Ciliary ring is defined as posterior part of the ciliary body. Ciliary striate are present on its posterior surface. Ciliary striate look as shallow grooves. Ciliary muscle is comprised of 2 sorts of muscle fibers; one the meridianal fibers and the other the circular fibers. Ciliary muscle is special type of single unit smooth muscle fibers. Meridian fibers extend from sclero-corneal junction to the ciliary processes. Meridian fibers are more in number and lie external to the fewer circular fibers.
Ciliary muscle gets its nerve supply by parasympathetic nerve fibers. Ciliary ganglion receives pre-synaptic nerve fibers of oculomotor nerve and supplies post ganglion nerve fibers as short ciliary nerves to the ciliary muscles.
Ciliary muscle contraction provides a mechanism for increasing the refractive power of eye lens by making it more convex through contraction of suspensory ligament (Snell,
2012, Standring, 2016,Willoughby et al., 2010).
8
Figure No.5: Aqueous outflow pathway of Human eye (https://www.wikipedia.org/).
9
The Iris and Pupil
Iris is a pigmented diaphragm which is thin and contractile. It shows a middle opening known as the Pupil. Anatomically, the iris lies between the cornea on one side and the lens on the other side. It is bathed by the aqueous humor(Banks and Held, 2012,
Willoughby et al., 2010).
Peripheral margin of iris is attached anteriorly to the ciliary body. The space between cornea and lens is divided by the iris into anterior and posterior chambers.
Iris comprises of involuntary single unit muscles fibers. One layer is circular called sphincter papillae and other layer is radial called dilator papillae. Sphincter and dilator pupillae muscle fibers regulate the light entry into eye balls and supplied by the sympathetic and parasympathetic nervous system. Parasympathetic nerve fibers of oculomotor nerve innervate the sphincter pupillae by short ciliary nerves originating from the ciliary ganglion. Radial fibers, the dilator pupillae, are innervated by the sympathetic nerve of long ciliary nerves. Sympathetic nerve fibers originate in the superior cervical ganglion.
Sphincter pupillae makes the pupil constricted in bright light and during the accommodation reflex. Dilator pupillae dilates the pupil in dim light. Pupil gets dilated also due to fight or flight reaction of sympathetic nerve stimulation as occurs during fear.
The Nervous Coat
Nervous coat of eye is called the retina. Retina shows an inner nerve fiber layer, and outer pigmented layer. External part is associated with the choroid, while internal part is related to the vitreous body. Posterior ¾ of retina contains receptors. Anterior edge of retina is serrated called the “oraserrata”. Nerves and receptors terminate at the
10
oraserrata(Banks and Held, 2012, Snell, 2012, Standring, 2016,Willoughby et al.,
2010).
Anterior part of retina is non-receptive; it shows few pigmented cells within deep columnar epithelium. Anterior part of retina covers the ciliary processes and posterior part of iris(Banks and Held, 2012, Snell, 2012, Standring, 2016,Willoughby et al.,
2010).
The Vitreous Body
Vitreous body fills the vitreous cavity, a space behind the lens and inner eye coat.
Vitreous body is jelly like transparent structure.
A narrow channel that runs through the vitreous body from back of lens to the optic disc is known as the “hyaloid canal”. Hyaloid canal is a remnant of hyaloid artery of fetal life, which regresses even before birth.
Vitreous body supports the eye lens and neural part of retina and contributes to the refractive power of eye. It holds the nerve fiber layer of retina against its pigmented layer (Banks and Held, 2012, Snell, 2012, Standring, 2016,Willoughby et al., 2010).
The Lens
Ocular lens is a bilaterally curved translucent structure placed inside the capsule. It is located in front of vitreous body and behind the iris. Eye lens comprises of;
Elastic transparent capsule,
Cuboidal epithelium, &
Lens fibers
Lens capsule is an elastic structure which encircles it completely. Anterior lens surface shows the Cuboidal epithelium. While lens fibers are lined by cuboidal epithelium on
11
lens equator. Bulk of lens comes from the lens fibers(Banks and Held, 2012, Snell,
2012, Standring, 2016,Willoughby et al., 2010).
The flexible lens capsule is under strain, bringing on the lens continually to attempt to expect a globular instead of a circle shape. The equator of the lens is attached to the ciliary processes by the “suspensory ligament”. Pulling effect of the emanating strands of the suspensory ligament tends to keep the flexible lens smoothed so that the eye may be focused for the distant objects(Banks and Held, 2012, Snell, 2012, Standring,
2016,Willoughby et al., 2010).
1.1.5 TRABECULAR MESHWORK
Trabecular Meshwork (TM) is a special tissues network situated in base of cornea close to the ciliary body and drains aqueous humor from the frontal ocular cavity. Trabecular meshwork tissue is spongy and is lined by trabeculocytes. TM is the major route of aqueous drainage which then drains into the Schlemm's canal and eventually into venous system.
Trabecular meshwork is a network of fibers at the irido-corneal angle between the frontal ocular cavity and scleral venous sinus. Pores are present among the fibers, through which flows the aqueous humor. It has 2 parts; the area connected with sclera called “corneo- scleral area” and the area close towards iris called the uveal part
(Figure No 6).
Synonyms: Following synonyms are used for the trabecular meshwork (Dua et al.,
2014, Keller and Acott, 2013).
Reticulum trabecularesclerae,
Gerlachvalvula,
Hueck ligament,
Ligamentumanularebulbi, 12
Pectinate ligaments of iridocorneal angle,
Pillar of iris,
Trabecular network,
Trabecular reticulum
Trabecular meshwork is divisible into three parts with highly different ultra-structure:
Inner uveal meshwork: lies close to the angle of anterior chamber. It shows thin
cord-like trabeculae which are orientated in a radial fashion. It encloses the
trabecular spaces which are larger than the corneo-scleral meshwork.
Corneo scleral meshwork: Corneo scleral meshwork shows elastin fibers oriented
as thin, flat and perforated sheets. Fibers are arranged in a laminar pattern which is
considered the ciliary muscle tendon.
Juxta-canalicular tissue (Cribriform meshwork): This part is present close to the
Schlemm's canal. Tissue consists of connective tissue rich
in glycosaminoglycan (GAG) and glycoproteins (GP). Juxta-canalicular meshwork
is concealed over by a layer of endothelial cells.
Uveo-scleral route is an alternative path of aqueous drainage. Almost, 5-10% of
aqueous flows through this pathway. Anti-glaucoma drugs such as prostaglandins
increase the Uveo-scleral pathway flow of aqueous humor (Dua et al., 2014, Keller
and Acott, 2013).
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Figure No.6: Anatomy of Aqueous outflow pathway (https://www.wikipedia.org/).
14
1.1.5.1 CLINICAL SIGNIFICANCE:
Trabecular meshwork blockage results in a clinical condition called the “glaucoma”, which is characterized by an increased intraocular pressure (Dua et al., 2014, Keller and
Acott, 2013).
1.1.6 AQUEOUS HUMOR (AH):
AH is a translucent, fluid secreted by ultra-filtration of blood plasma by ciliary processes. Compared to plasma it has low protein content. Ciliary epithelium of ciliary processes secretes the aqueous humor. It circulates in anterior and posterior chambers of eye ball (Goel et al., 2010, Johnson et al., 2017).
1.1.6.1 COMPOSITION:
It is composed of 98% Water (H2O), Glucose, Proteins, Crystalline lens proteins,
Amino acids, Electrolytes, Ascorbic acid, Glutathione, Immunoglobulin, Bicarbonates,
Chloride, Sodium, Potassium, Calcium, Magnesium (Goel et al., 2010, Johnson et al.,
2017, Saraswathy et al., 2016). This is also composed of Phosphate, Pyruvate, Lactic acid, Plasminogen, Growth factors inhibitors, Oxygen, Urea and Creatinine(Goel et al.,
2010, Johnson et al., 2017, Saraswathy et al., 2016).
1.1.6.2 FUNCTIONS OF AQUEOUS HUMOR:
It functions to Inflates eye ball, acts as refractive media, responsible for creating intraocular pressure, delivers nutrition to the avascular structures such as lens, cornea, trabecular meshwork, anterior vitreous. Ascorbic acid acts as anti-oxidant, increases Immunity- Immunoglobulin’s(Goel et al., 2010, Johnson et al.,
2017,Saraswathy et al., 2016).
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1.1.6.3 PRODUCTION & DRAINAGE:
Aqueous humor is an ultra-filtrate of blood plasma. It is secreted through the ciliary processes in hollow cavity in the back of eye by the ciliary body (pars plicata). This fluid passes by way of pupil within anterior chamber and eventually anterior chamber angle. From here, it circulates via trabecular network to the canal of Schlemmn. After
Schlemm`s canal, it drains by 2 pathways (Figure No 7).
Directly into aqueous vein towards episcleral vein, or
Secondarily, via collecting routes towards episcleral vein through intrascleral
plexus and
Ultimately into orbital veins(Cheng et al., 2009, Do, 2014).
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Figure No 7: Aqueous outflow routes illustrated in the Cut section of the anterior chamber of eye:
a) Via the TM. b) Via the Uveo-scleral pathway. c) Via the iris (Kanski et al., 2011).
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1.2 GLAUCOMA
Glaucoma is the chronic, heterogeneous set of syndromes categorized by elevated IOP that results in deterioration of optic disc and flaws in the field of vision, if remains untreated (Kwon et al., 2009).It is 2ndprincipal cause for impaired vision worldwide(Kulkarni, 2012).It is categorized broadly in three main categories: Angle
Open glaucoma (OAG), Angle Closure Glaucoma (CAG) and Developmental
Glaucoma (DG). These types are further subdivided into primary and secondary glaucoma. Among all types, angle open glaucoma is the usually occurring form (Kwon et al., 2009).The etiology of glaucoma is because of the collapse of ganglionic cells of retina, which results in a typical damage in field of eyesight and cupping in optic nerve head (ONH) (Wiggs et al., 2012). Glaucoma is closely associated with elevated IOP.
Various experiments have explored the function of raised IOP in the mechanism of action of OAG (Ekstrom, 2012). If the intraocular pressure is in-between 10 to 21 mm
Hg, then it will be considered as with in normal limits. This is a recommended value but it is unnecessary that if this value exceeds these limits, the disease is suspected.
Although, value above 21 mmHg is suspicious of the disease (Mansouri et al., 2012).
There is variation in IOP in different times of the day. Although there are no any safer levels of the IOP, glaucomatous damage can occur in IOP level of 12 mm Hg, but on the other hand, that damage can be absent even on 30 mm Hg (Lee et al., 2012b).
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1.2.1 CLASSIFICATION OF GLAUCOMA:
Glaucoma may be categorized into three main types:
Open Angle Glaucoma(OAG)
Angle Closure Glaucoma (ACG)
Developmental Glaucoma.
Open angle glaucoma must be differentiated from Angle closure glaucoma for treating the patients. Furthermore, glaucoma is further sub-classified into primary or secondary glaucoma. There is no association of systemic disorders with primary glaucoma like
POAG which are accountable to raised endurance to aqueous humor drainage. But there is an association of eye or general diseases with secondary glaucoma which causes the raised endurance to aqueous humor drainage way.
POAG is further sub-categorized in:
High Tension Glaucoma (HTG)
Normal Tension Glaucoma (NTG)
Ocular hypertension (OH)
The very important risky issue for POAG is ocular hypertension (American Academy of, 2012).
1.2.2 Primary Open Angle Glaucoma:
High Tension Glaucoma (HTG):
In high tension glaucoma, the essential risky issue for the development and advancement of the disease is IOP. The grade of injury to the ganglionic cells of retina with their axons is dependent upon the levels of intraocular pressure (Figure No 8). The characteristic features of HTG are:
On Gonioscopic examination:
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Open angle of anterior chamber
IOP >21 mm Hg.
Cupping of the optic disc (Figure No 8).
Presence of defect in field of vision.
Age of onset > 40 years (Jan et al., 2012).
Normal Tension Glaucoma (NTG):
NTG is the very significant sub type of POAG. In this type, there is progressive cupping of the optic nerve head which leads to defects in field of vision. However the IOP may be within its normal ranges (10-21 mm Hg); (Figure No 9). The causative factor may be other than IOP like it may occur due to vascular changes (Hirooka et al., 2012).
The characteristic features of NTG are:
On Gonioscopic examination,
Open angle of anterior chamber (Figure No 10).
Presence of optic disc cup
Presence of defective field of vision
IOP within its standard values (10-21 mm Hg).
Some studies suggest that there is a vascular factor which plays significant function in mechanism of action of normal tension glaucoma. The decrease blood supply of eye can cause the loss of ganglionic cells of retina (Hwang et al., 2012).
There are many other factors that can perform key function in pathogenesis of NTG however the exact cause is still not clear. It has been reported that in population of
United States there is a prevalence of up to two percent above 40 years of age and between five and seven percent of the population have self-reported cases of POAG
(Ogunyemi, 2011).
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Fig 1.1
a) b)
c) d)
Figure No8: Distinguishing landscapes of glaucoma: a) Normal view of Cut segment of the optic nerve. b) Analogous anterior aspect of the optic nerve head. c) Cut segment of the optic nerve head of an individual having Glaucoma. d) The analogous anterior aspect of the optic nerve head presenting total cupping (Bowling and Kanski, 2016).
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FigureNo.9: Humphrey’s visual field presenting an advanced visual field loss (Bowling and Kanski, 2016).
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Open Angle Closed Angle
b) c)
Figure No.10: Measurement of chamber of anterior ocular segment:
(a) According to Shaffer’s grading, grade 4 is widest (open) angle whereas grade 0 is the narrowest (closed) one.
(b) Cut section of anterior ocular area which shows open angle as found in Primary open angle glaucoma.
(c) Cut section of anterior ocular part which shows a closed angle as found in Primary closed angle glaucoma (Bowling and Kanski, 2016).
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1.2.3 INTRAOCULAR PRESSURE AND GLAUCOMA:
Elevated intraocular pressure is the leading predisposing factor in the pathogenesis of glaucoma, and a 2nd leading source of loss of sight in the world. The regulation of intraocular pressure depends upon the excretion and drainage of aqueous humor. (Tam et al., 2013).
Following are the factors which can affect the intraocular pressure:
The amount of excretion of aqueous humor through ciliary epithelium.
Drainage of the aqueous humor via irido-corneal angle.
The intravenous pressure presents in the episcleral veins (Ho et al., 2014);
Predominantly, raised IOP is directly proportional with the resistance in routes of
the aqueous outflow (Moosa et al., 2014).
1.2.3.1 OCULAR HYPERTENSION:
In this type of glaucoma, the IOP is raised above normal ranges of 21 mm Hg, with no any features for Glaucoma like optic disc cupping & defective visual fields. An important predisposing factor in advancement of POAG is Ocular hypertension which remains directly related with increased levels of IOP(De Moraes et al., 2012, Demirel et al., 2012,Russell et al., 2012).
1.2.3.2 OPTIC NERVE HEAD:
Ganglionic cells of retina axons contain the nerve fiber layer (NFL) of retina; the inner most retinal layer. The optic nerve is made up of axons converged at the optic nerve head. There are 1.2 million axons in single Optic nerve (Figure No. 11).The fibers of ganglionic cells leave the eye to pass across the sieve like lamina cribrosa (LC) and here it is covered with a myelin sheath and go to synapse in the lateral geniculate ganglion (LGG) (Bowling and Kanski, 2016).
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Distribution patterns of these axonal fibers help in the understanding of defective field of vision resulting in the Optic disc cupping due to Glaucoma. Their arrangement of conjunction indicates patterns of defective field of vision (Figure No. 9).
The Macular fibers travelling straight to the head of the optic nerve, forming the papilla-macular bundle, and nasal fibers moreover come in a straight route towards the head of the optic nerve. Temporal fibers run in an arcuate way around the papilla- macular bundle to access at 2nd cranial nerve. It has got a concavity in its center, the optic cup which does not have any type of neural tissue present in it. On the other hand, some emerging axonal fibers are present in the neuro-retinal rim, which can be well- defined with ratio between external margin of the disc and external edge of cup (Figure
No. 12). The normal rim has a pink color and uniform width. The degree of the glaucomatous optic cupping may be expressed with cup-disc (C/D) ratio (Schmidl et al., 2012, Bowling and Kanski, 2016).
The diameter of the cup can be measured both in horizontal and vertical apices.
Standard vertical cup-disc ratio is 0.3 or less and the ratio more than this will be in pathological conditions (Ekstrom, 2012); (Figure No. 13).
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FigureNo.11: The structure of fibers of retina elucidating design of merging of ganglion cell fibers (described in yellow color) to the optic nerve: The macular fibers goes straightaway to the head of optic nerve creating the papillomacular bundle, and the nasal fibers of retina are also run in the straight way to ON. Fibers present temporally towards macular area pursue arcuate pathway round the papillomacular bundle for joining the optic nerve(Bowling and Kanski, 2016).
26
FigureNo.12: Pictorial demonstration of the design of merging of fibers of ganglionic cells at the optic disc, that directs the way of defects in visual fields: In this figure: a) Injury to the infra-temporal nerve fibers of retina b) Leads to injury in superior arcuate field t like presented in a Humphery’s field of vision (Bowling and Kanski, 2016).
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Figure No.13: C/D proportion of optic disc. Figure of eye (OS) having a large C/D ratio(Bowling and Kanski, 2016).
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1.2.3.3 EPIDEMIOLOGY OF GLAUCOMA:
Glaucoma is an important public health issue nowadays, which is also the 2nd principal reason of loss of sight worldwide (Ekstrom, 2012). As mean age of population is increasing globally, patients of glaucoma will increase accordingly. It has been anticipated that there were 60.5 million individuals having POAG by 2010 and that figure of cases will reach 79.6 million in 2020, which results in the consensual sightlessness to 8.4 million and 11.2 million persons in subsequent years correspondingly (Ray and Mookherjee, 2009).Most recently, world health organization
(WHO) declared that if 10 crore persons are suspected to have glaucoma, over 2 crore persons are suffering as of glaucoma and over 50 lac persons are sightless due to glaucoma (Figure No 14).
Approximately 70% patients having glaucoma are present in the emerging nations. It has been expected that 2/3rd of those sightless persons are due to the POAG and the remaining are due to the primary angle closure glaucoma. According to the united states national eye institute, about 2.2 million people are affected from glaucoma in united states (I, 2009). In national health survey (NHS) held in 2003, it was estimated that in Pakistan, the prevalence of blindness is 2.7% (1,140,000 blind adults) and surprisingly it was found that in Pakistan, the 4th leading cause of blindness is glaucoma
(Iqbal et al., 2011).
29
Figure No.14:Estimations of worldwide frequency having POAG in persons with age more than forty years (Leske et al., 2007).
30
Primary open angle glaucoma is very dangerous amongst all types of glaucoma because it is asymptomatic, and the visual loss can occur before the diagnosis of the disease so the early diagnosis of the disease is very much important (Kenneth Bentum Otabil,
2013).
1.2.3.4 CLINICAL RISK FACTORS FOR GLAUCOMA:
Medical supervision is necessary for the people who are at high risk. With the help of this, there will be early detection of the glaucoma and can be treated properly within time. Clinically, the predisposing factors can be classified intraocular factors, general factors and systemic predisposing factors (Takai et al., 2012).
OCULAR RISK FACTORS:
Intraocular Pressure (IOP):
Proper function of intraocular pressure in association with other predisposing factors and their importance in the etiology of Glaucoma is still unclear and is a hot issue to discuss yet. Though, intraocular pressure is the major predisposing factor positively worked on it so far. By the mid of the twentieth century, the Glaucoma was classified and managed on the basis of intraocular pressure to avoid the consequences of
Glaucoma (Takai et al., 2012).
Features of the head of optic nerve:
Shape of the 2nd cranial nerve is important in detecting risk of having glaucoma. The more the enlarged shape of disc the more are chances of glaucoma. Optic disc hemorrhages also have close relation to the consequences of progression of defective field of vision. Optic nerve head is the primary site which may be on high risk in glaucoma (Mansouri et al., 2012).
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Central corneal thickness:
Previous studies have revealed a close association between central thickness of cornea and the POAG. This is a major predisposing factor in developing the POAG but the exact mechanism of the disease still needs more to elaborate (Dastiridou et al., 2013,
Ishikawa et al., 2011).
Myopia:
Most of the studies yet have found a close association of myopia with POAG. But some research experiments have become unsuccessful to prove linkage between myopia and
Primary open angle glaucoma, as the features seen in myopia like C/D Ratio and visual field defects resembles with POAG (Kimura et al., 2012).
Other Predisposing Factors:
Age
Aging plays an important role in development of Primary Open Angle Glaucoma. The anatomy of the lamina cribrosa changes with the process of aging and disease
(Coudrillier et al., 2012, Kang et al., 2008,Sigal et al., 2012).
Race
POAG is very common in black population than in white population. Most of the studies have suggested that frequency of POAG is greater in black population as compared to white population (Budenz et al., 2013, Lee et al., 2012a).
Family history:(Speckauskas et al., 2012, Abegao Pinto et al., 2012).
Family history is a major predisposing factor for POAG. Individuals who are 1st degree relatives of the people having glaucoma have 3-9 folds more risk than other people.
Exact cause of the disease is not still clear but it is thought that genetic factor might perform a vital function in the mechanism of action for the POAG (Osman et al., 2012).
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General risk factors:
Diabetes mellitus (D.M):
Some studies proved that there is more prevalence of raised IOP and POAG in the people having diabetes mellitus, but this is still controversial due to lack of more authentic findings.
Blood pressure and migraine:
Different studies have found that there is presence of peripheral vasospasm, systemic hypotension and migraine in the patients having POAG (Gemenetzi et al., 2012).
1.2.3.5 PATHOPHYSIOLOGY OF GLAUCOMATOUS OPTIC CUPPING:
Apoptosis
The exact mechanism of the disease is not understood, the research carried out till now suggests that there are some environmental factors which in association with genetic factors influence the disease, that is why this is also known as the multifactorial disease.
Exact cause of POAG is not understood but the final fate of the disease is apoptosis of ganglionic cells which leads to the cupping of the optic disc. Apoptosis is thought to be the possible mechanism of cellular death of axons in patients with POAG (Oshida et al., 2010). Any functional impairment of trabecular meshwork (TM) cells can affect the normal function of TM which may elevate the IOP and can cause glaucoma.
Advancing damage in Trabecular Meshwork cells is observed in individuals affected with POAG which is because of the exposure of free radicals for very long time.
Reactive oxygen species (ROS) have the capability to change the normal role of TM cells (Izzotti et al., 2010). In the progression of POAG, the oxidative stress (OS) plays a key function. Initially it damages TM cells, leading to alteration in nitric oxide and endothelium homeostasis which results in the ganglion cell death. It has been proved
33
that there is no any involvement of environmental factors of oxidative stress like cigarette smoking or radiation in the pathogenesis of glaucoma (Nucci et al., 2013).
Mechanical and vascular theories of POAG:
Although more experimental studies are needed to know the accurate mechanism of
POAG, but there are two theories for the explanation of the optic disc cupping due to
Glaucoma. Muller was a scientist who presented Mechanical theory in 1858, which states that ganglion cell death occurs in the result of mechanical force produced due to the raised IOP above physiological levels, and Von Jaeger presented a Vascular Theory in 1858 which states that the optic nerve damage occurs because of compromised vascularity of the optic nerve which causes its ischemia (Figure No 15).
Although the precise mechanism responsible for glaucomatous optic neuropathy is still not clear, it has been suggested that some other factors also contribute which include excitatory damage induced by excessive glutamate secretion, lack of growth factors, toxic damage induced by increased nitric oxide production, free radical mediated damage and immune-mediated toxic injury (Nucci et al., 2013); (Figure No. 16).
34
Figure No.15: Pictorial illustration of comparative impact produced through mechanical and vascular factors for advanced optic neuropathy due to glaucoma on numerous intensities of Intra Ocular Pressure (Caprioli J, 1984).
35
Figure No.16:Pictorial illustration of the probable issues influencing on the injury to optic nerve due to the Glaucoma:
Elevated intraocular pressure usually causes retinal ganglionic cell death due to trophic insufficiency. Some other factors have also been implicated which include local ischemia, immunological disorders, over-excitation of glutaminergic system and variation in number of glial cells or astrocytes (Weinreb and Khaw, 2004).
36
1.2.3.6 GENETIC BASIS OF POAG:
Most of the scientists stayed interested in the hereditary nature of glaucoma. In 1842,
BENEDICT highlighted the familial nature of glaucoma, when he observed POAG in two siblings. In 1869, VON GRAFE discovered the significance of heritage in the etiology of glaucoma and he consigned to the families who were suffering from glaucoma in their 3rd to 5th generation (Fingert, 2011b).Several studies proved a major part of genetic factors in the advancement of several ocular disorders comprising Fuch’s corneal endothelial dystrophy (FCED), exfoliation syndrome, age related macular degeneration (ARMD) and POAG. Mechanism of action of genetically inherited ocular disorders can be understood by elaborating the pathogenic genes (Li et al., 2009).Some genes are revealed however that are associated with POAG. Myocilin is the first gene to be discovered which is associated with greater than 10% of JOAG cases and 4% of the POAG cases. There are 504 amino acid glycoproteins coding for the Human
Myocilin gene, which is present within the cells or extracellularly in approximately all tissues of eye but especially in the trabecular meshwork (Menaa et al., 2011). Initially
Myocilin was called as a trabecular meshwork inducible glucocorticoid response
(TIGR) gene. Myocilin is present into aqueous humor (AH) therefore the quantities of
MYOC proteins are elevated into glaucomatous AH and it works as a marker for glaucomatous patients (Cheng et al., 2012). Till date, nine loci have been identified for
POAG only. Many genes like WDR36 and CYP1B1 have been found linked to the
POAG but Myocilin gene was noticed as responsible for 3-4% POAG cases. The mechanism of this disease is not clear yet and still more work is required to discover these mechanisms of progression of the illness (Fourgeux et al., 2009, Gasten et al.,
2012).
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1.3 CATEGORIZATION OF LOCI OF GENES ASSOCIATED WITH PRIMARY OPEN ANGLE GLAUCOMA:
Three groups of glaucoma loci are formed; GLC1 indicates to Primary Open Angle
Glaucoma, GLC2 indicates to primary angle closure glaucoma and GLC3 indicates to congenital glaucoma. For every novel locus, a capital letter of alphabet is attached after
GLC. For instance, 1st Locus for POAG is GLC1A. There is an autosomal dominant inheritance pattern is present all 9 loci of POAG (Gomes et al., 2012).
1.3.1 MYOCILIN GENE
Discovery
Myocilin gene (MYOC) was found by Stone et al (1997). They denoted term Myocilin
Gene as “GLAC1A”, 1 which was mapped on chromosome 1. GLC indicates
Glaucoma, 1 denotes the POAG and A denotes 1st discovered association for the glaucoma (Alward, 2000, Lohano, 2016,Stone et al., 1997).
Trabecular meshwork cells of human origin were cultured and treated with high dose of drug dexamethasone in-vitro. Gene expression was compared with controls trabecular meshwork cells in this experiment. A new protein was noticed in the steroid treated trabeculocytes. This protein was denoted as “Trabecular Meshwork Inducible
Glucocorticoids Receptor Protein (TIGR)” (Polansky et al., 1997).
Myocilin gene (MYOC) was found by Stone et al (1997). They denoted term Myocilin
Gene as “GLAC1A”, 1 which was mapped on chromosome 1. GLC indicates
Glaucoma, 1 denotes the POAG and A denotes 1st discovered association for the glaucoma (Alward, 2000, Lohano, 2016,Stone et al., 1997).
Later on, with continuous research, the gene responsible for increased TIGR protein was isolated and denoted as Myocilin gene. The Myocilin closely resembles to the retinal cytoskeleton proteins closely related to its homology to non-muscle Myocilin
38
found in the photoreceptors. This gene was denoted officially by the “Human Genome
Database Nomenclature Committee” (1998). Now it is known as Myocilin and TIGR was omitted completely (Kubota et al., 1997).
Gene Structure and Protein
Myocilin gene is located at chromosome 1 (1q24.3 – 1q25.2). It shows 3 exons and 2 introns. Its length is approximately 17 kb and 782 base pairs.
Exon 1.
It encodes non muscle “myosin” like domains which holds signal peptide for
Myocilin secretion, and other leucine zipper motif which is necessary for self-
aggregation of Myocilin (Wang et al., 2015).
Exon 2
It encodes for a central “linking domain” which is spliced by calpain II (Wang et
al., 2015).
Exon 3
It encodes for “olfactomedin (OLF)-like globular domain” which is attached at the
C terminal (Wang et al., 2015).
Humanoid Myocilin Gene transcription produces acidic glycoprotein, which
contains 504 amino acids. Its isoelectric pH is 5.24 having a molecular weight of
55.3-kilodalton (kDa) (Caballero and Borras, 2001).
39
Expression Site
Myocilin mRNA can be found in both extra and intra-ocular tissues. It is expressed in the vitreous body, aqueous humor, lamina cribrosa, choroid, 2nd cranial nerve head, ciliary body, iris, retina, sclera and mostly in the trabecular meshwork. Extra expression sites include the trabeculocytes and sclera (Ohlmann et al., 2003, Swiderski et al.,
2000,Torrado et al., 2002).
Extra-ocular sties of expression include the kidney, gastric tissues, colon, trachea, thyroid tissue, skeletal muscle, nerves, Schwann cells, brain parts, sciatic nerve and heart etc. (Goldwich et al., 2005, Joe et al., 2014).
Physiological Function
The exact physiological role of Myocilin gene is obscured and unidentified. It may be linked to some structural proteins in the cytoplasm which remains to be identified. It is speculated to function as a “molecular chaperone”.
Myocilin protein may bind to cell membrane or extracellular proteins of trabeculocytes which create the resistance to the flow of aqueous humor. Myelination of the peripheral nerve fibers is accomplished by the Myocilin gene expressed protein (Kwon et al.,
2013a, Kwon et al., 2014).
Myocilin gene causes expression of bone marrow-derived mesenchymal stem cells
(MSCs) to differentiate into “osteoblasts” has been evidenced in-vivo experiments. In- vivo evidence suggests it stimulates the Osteogenesis (Kwon et al., 2013b, Salowe et al., 2015).
Myocilin gene is proposed to increase skeletal muscle mass in transgenic mice and is suggested responsible for skeletal muscle hypertrophy (Kwon et al., 2013b).
Myocilin gene expresses Myocilin protein in Endoplasmic reticulum which induces strain and apoptosis of cells (Anholt, 2014). 40
At angle of eye ball, Myocilin contributes to the survival of trabeculocytes. This causes an increase in resistance to flow of aqueous humor leading to increased risk of glaucoma by causing alteration in the structure of drainage passage way (Tomarev and Nakaya,
2009).
Mutation and Mechanism of Disease
Greater than 273 transmutations of MYOC gene are reported thus implicated in 93 genetic disorders. Many transmutations are located in 3rd exons that encodes to the
“olfactomedin domain” (Borras, 2014, Kumar et al., 2016).
Glaucoma linked Myocilin gene mutated OLF domain has been isolated from the
POAG (Wiggs, 2012). Peculiar Myocilin gene mutations include Pro370Leu (P370L) and Gln368Stop (Q368X).Genetically, the glaucoma is heterogeneous (Fan and Wiggs,
2010, Fingert, 2011b). More than 20 gene loci have been reported for POAG (Huang et al., 2014, Quigley, 2011).
1.3.2 CYTOCHROME P4501B1 (CYP1B1) GENE:
CYP1B1 gene embraces more than 70 mutations isolated from the patients of primary congenital glaucoma (PCG). CYP1B1 belongs to the cytochrome P450 (CYP450) which is a super-family of heme proteins (Hasler, 1999). The approximate weight of
CYP1B1 is about 50 kDa. Most of the Cytochrome P450 proteins are bounded by a membrane. They are also attached with organelles’ membranes such as endoplasmic reticulum, mitochondria, etc.(Stoilov et al., 2001). CYP1B1 exhibits high catalysis of several polycyclic aromatic hydrocarbons, which powerfully induces Pulmonary and
Mammary malignancies (Shimada et al., 1996).
There is involvement of CYP1B1 in the endogenous steroid genesis like 17β-estradiol.
High expression of CYP1B1 has been noted in the lungs, uterus, prostate gland, breast, etc. A single nucleotide gene polymorphism (SNP) in CYP1B1 (R48G, A119S, and 41
V432L) shows seriously transformed kinetics of estradiol hydroxylation. The SNPs are closely related to the carcinogenesis (Aklillu et al., 2002, Shimada et al., 2001,Tanaka et al., 2002).
CYP1B1 expression in eye Tissues:
CYP1B1 protein is present in numerous eye tissues of Humans including cornea, ciliary body, iris, and retina. The expression of CYP1B1 gene and proteins has never been found in the TM. However, increased manifestation of CYP1B1 gene is noted in the eyes of fetus matched with the eyes of adults. Thus the role of CYP1B1 has been speculated in the eye growth and evolution of eye tissues (Doshi et al., 2006).
CYP1B1 mutation spectrum:
For PCG, More than 70 distinct mutations are reported for CYP1B1 gene. The prevalence of CYP1B1 gene mutations differs worldwide;
20% in people of Japan (Mashima et al., 2001).
33.3% in people of Indonesia(Sitorus et al., 2003).
44% with in people of Indian (Stoilov et al., 2002).
50% within the people of Brazil (Chakrabarti et al., 2010).
100% within the people of Saudi Arabia (Bejjani et al., 2000); and Slovakian
Gypsies (Plasilova et al., 1999).
Majority of mutations are missense mutations. Missense mutations interfere with folding of proteins, heme synthesis, and interaction with the redox partner (Stoilov et al., 2001).
Prevalent Mutations in CYP1B1:
G61E mutations of CYP1B1 are common among Saudi Arabians (69.3%) and Iranian
(29%). Other mutations include the R390H and R368H which are discovered which
42
possess high rate of recurrence (20.1% and 10.6%, respectively) in Iran population.
E387K mutation of CYP1B1 was found among Slovakia Gypsies (Plasilova et al.,
1999). 4340delG mutation in CYP1B1 has been found in the Brazil. 4340delG mutations manifests phenotypically with very high IOP ranging from 25 to 55 mmHg during first month of life (Kaur et al., 2011).
In Indians, the R368H mutation of CYP1B1 was found predominantly. However, this gene mutation was not related to acuteness of Glaucoma(Reddy et al., 2004).
CYP1B1 Mutations in pathogenesis of Primary Congenital Glaucoma:
Precise function of CYP1B1 gene with normal eye development and PCG is not known.
Various experimental studies had been reported to explain the underlying mechanism.
Hollander et al reported that the CYP1B1 mutations might interfere with the angle development leading to its dysgenesis which was observed histologically. This was diagnosed from six cases of PCG with increased IOP (Hollander et al., 2006). They concluded that the mutations of CYP1B1 gene may be explored by histological findings(Achary et al., 2006).
A Study conducted by Jansson et al (Bidinost et al., 2006); that the constancy and enzymatic activity of CYP1B1 may be affected by two missense mutations (G61E and
R469W). They reported that these mutations decrease CYP1B1 enzyme metabolic activity by 50-70% at cellular level (Jansson et al., 2001).
(Bagiyeva et al., 2007) studied two mutations (R117W and G329V); proteins who reported decrease in enzymatic activity of transmuted proteins. They reported that it happened due to the delayed CYP1B1 transport via endoplasmic retinaculum in cases of transmuted CYP1B1 which further lowers enzyme activity. Another previous study concluded that this may have a function in the mechanism of action for PCG
(Chakrabarti et al., 2006). 43
1.4 OXIDANTS AND ANTIOXIDANTS:
1.4.1 FREE RADICALS & ROS:
Free radicals & Reactive Oxygen Species are highly instable type of molecules which have unpaired electrons in their shells. Due to their instability, they are highly reactive
& enhance oxidative reactions with other molecules for instance; lipids, DNA & proteins to become stabilized. ROS & free radicals are important for human body because they regulate our immune system. Immune cells like macrophages & neutrophils produce ROS in inflammatory conditions during antigen-antibody reactions to eliminate the antigens.
Nature has provided a vital network of antioxidants to human body which neutralizes the highly reactive species & helps in regulation of defense system of body. When the imbalance between oxidants & antioxidants concentration occurs, the impairment of cellular functions in human body take place. It has been proved from different studies that presence of ROS & free radicals in our body is not harmful but is very necessary factor for body’s protective mechanism. Only they become harmful when there is excessive production of free radicals take place. So, the oxidants & antioxidants both are essential to maintain the process of homeostasis (Oter et al., 2012).
According to the “mitochondrial theory of aging”, mostly the free radical & ROS production take place in mitochondria of any cell which are responsible for damage to mitochondrial DNA which ultimately leads to the process of aging. Due to these mutations, disturbance occurs in normal functions of cells (Tourtas et al., 2012).
TM is very important human ocular tissue which drains 90% of aqueous humor through anterior chamber of human eye ball. TM also produces outflow resistance which eventually known as intraocular pressure (IOP). Increase in IOP leads to Glaucoma
44
which occurs due to the improper function of the TM cells. Different studies have highlighted the changes in the morphology of Trabecular Meshwork tissue of patients having primary open angle glaucoma (POAG) which leads to a hypothesis. According to that hypothesis, the changes in morphology of TM cells occurred because of production of too much quantity of oxidative stress & Reactive Oxygen Species in TM tissue. Also the highly impaired function of TM cells has been reported in patients of
POAG which causes accumulation of ROS in the cells.
Other glaucoma studies have reported the increased level of oxidative stress & decreased antioxidant potential in POAG patients. Increased production of Reactive
Oxygen Species & oxidative stress is suspected in pathogenesis of POAG (Tanito et al.,
2012).
1.4.2 RESVERATROL
Resveratrol (3, 5, 41-trihydroxy-trans-stilbene) is a stilbenoid of plant origin, which is a natural phenol and generated by plants in reaction to an infection by pathogens such as fungi, etc. (Abu-Amero et al., 2016, Guerrero et al., 2009).
Biochemically, the Resveratrol has a stilbenoid structure comprising of 2 aromatic rings linked through a methylene bridge (Figure No 17).Resveratrol exists in 2 natural forms namely; the Cis-Resveratrol and trans-Resveratrol. Grapes and grape juices are rich in trans- resveratrol-3-O-glucoside. Cis-resveratrol is present in wines (Langcake and
Pryce, 1977).
45
Figure No.17: Molecular structure of Trans-Resveratrol and Cis-Resveratrol (Cottart et al., 2010).
46
Research on the Resveratrol commenced with the “French Paradox” of describing the red wine as beneficial for the cardiovascular health. This was because of high concentrations of Resveratrol which prevented against the cardiovascular disease.
French people consumed high saturated fat but cardiac disease was still low (Lippi et al., 2010). Subsequently, research was concentrated on the anti-oxidant activity of
Resveratrol. It is suggested to exert effective anti-oxidative and anti-inflammatory activity. Cardio-protective (Wu and Hsieh, 2011), neuroprotective (Richard et al.,
2011), chemotherapeutic (Gusman et al., 2001), and anti-aging effects (de la Lastra and
Villegas, 2005); have been reported.
Discovery
Takaoka (1940) was the first who discovered the Resveratrol from “hellebore roots”
(Veratrum grandiflorum O. Loes). Following this, Monomer (1960) isolated it from the Japanese knotweed (Polygonum cuspidatum). Previously it was used as a traditional remedy oriental medicine. It had been used for the fungal skin diseases, athlete’s foot, gonorrhea, dermatitis, etc. It was possible only in 1990 that the active compound
Resveratrol was detected from wine. Later on, Jang et al discovered its mechanism of anti-oxidative effects (Abu-Amero et al., 2016).
Food Sources
Natural polyphenols are powerful anti-oxidants which are found mostly in plants. Skin of grapes, cranberries, blueberries, raspberries, and mulberries is rich source of
Resveratrol. Cocoa, peanuts, grape juice and red wine are other particularly rich sources of Resveratrol. Red wine contains many polyphenols other than Resveratrol.
Nowadays, synthetic analogues of Resveratrol and its isomers are available (Abu-
Amero et al., 2016, Aggarwal et al., 2004).
47
Resveratrol conjugates differ in their radical groups such as hydroxyl, methoxyl, halogenate, glycosylate and esters. Stilbeic double bonds, stereoisomers and oligomers are most potent anti-oxidants (Abu-Amero et al., 2016, Aggarwal et al., 2004).
Mode of Action
(Jang et al., 1997); performed experiments on the Resveratrol for detecting its mechanisms of action in the yeast model. They concluded that the Resveratrol activates sirtuin deacetylases in the yeast (Figure No 18).
48
Figure No.18:Mechanism of action of Resveratrol; Adapted from (Bola et al., 2014).
49
Resveratrol and Eye Diseases
Different experiments have been performed and reported the beneficial effects of
Resveratrol in the ophthalmological disorders. Major protection against eye disease is attributed to its;
Anti-oxidative activity,
Angiogenic activity,
Anti-cancerous activity,
Anti-inflammatory activity, &
Vasodilator effects
Many data has been printed concerning potential defensive function of Resveratrol in many ocular disorders like glaucoma, diabetic retinopathy, macular degeneration, and cataract, etc. (Abu-Amero et al., 2016).
1.4.3 ASCORBATE
Ascorbate, usually renowned as Vitamin C, have vital role in humans and other species as well.
Collagen production is a famous instance of Ascorbate facilitated reaction (Hacişevki,
2009).
Scurvy was described by compromised wound restoration and loss of re-union of bone fractures. The deficiencies were accredited to ascorbate insufficiency in scurvy. This is a potent agent which under goes oxidization & produce dehydro-ascorbic acid & mono- ascorbic acid. Such oxidized products are concerned with coupling reactions during oxidative phosphorylation & the electro-chemical actions across plasma membrane
(Packer and Fuchs, 1997).
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Ascorbate is associated with redox coupling reactions of biological significance. For instance, ascorbate is consumed in production of neuro-transmitters and hormones like epinephrine (Arrigoni and De Tullio, 2002). It interacts with several proteins of cellular membrane; recommend its part in neuromodualtion (Aguirre and May, 2008, Arrigoni and De Tullio, 2002).
Molecular structure
L-ascorbic acid is di-basic acid having a enediol category formedin 5 membered heterocyclic lactone ring. Instinctual and biochemical characteristics remain some way associated with its molecular configuration (Aguirre and May, 2008, Arrigoni and De
Tullio, 2002); (Figure No 19).
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Figure No.19: Molecular representation of ascorbic acid and its oxidation (Hacişevki, 2009).
52
Biosynthesis
Certain animals and some bacteria can synthesize ascorbate via glucuronic acid pathway, the main detoxification pathway. Various animals can convert D– glucose to
L– ascorbic acid, but others do not have certain enzymes and unable to biosynthesize ascorbate. Humans and other animals are unable to biosynthesize ascorbate naturally due to an enzymatic deficit identified as the “L-gulono-γ-lactone oxidase”, which has a role in its final phase (Nishikimi et al., 1994).
Metabolism
Metabolism of Ascorbic acid is mostly performed into the liver cells and to certain levels performed in parenchyma of kidneys. Primary path of ascorbic acid results ininjury of 2 electrons (Arrigoni and De Tullio, 2002).
When electrons have been lost, it eventually synthesizes, "Dehydro-ascorbic acid," that can ultimately be metabolized into physiologically unreactive "2, 3-diketogulonic acid." All the above metabolites usually are excreted by urine(Ashwell et al., 1961,
Huang et al., 2000).
Requirement of Ascorbate in Diet
The suggested dosages range from 30 – 10,000 mg daily in humans. Usually 10 mg/day is adequate for prevention of scurvy. Daily requirement is sixty milligram daily for non- smoker people. Low levels of Ascorbate have been observed in smokers. Therefore in smokers, the RDA has been recommended up to 100 mg (Levine et al., 2001).
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Ascorbic acid – Physiology
Since very long, Ascorbic acid was determined as a reduction agent in cellular metabolism.
Ascorbate is protective against oxidative stress. It neutralizes reactive oxygen species
2- - such as O , H2O2, OH , etc. It also exercises bactericidal and anti-viral activity due to its pro-oxidant activity, as various reports have shown (Arrigoni and De Tullio, 2002).
Ascorbate in human disease:
Many disorders are related to low ascorbate levels in blood plasma like diabetes mellitus, cataract, cancer, AIDS, systemic lupus erythematosus etc. (Evans et al., 2000,
Figueroa-Mendez and Rivas-Arancibia, 2015, Jaruga et al., 2002, Ravindran et al.,
2011, Tu et al., 2015, Valero et al., 2002).
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Chapter No. 2:
MATERIAL AND METHODS:
Setting:
Molecular Biology & Genetics department (MBGD) Laboratory at Medical Research
Centre, Liaquat University of Medical & Health Sciences, Jamshoro, Sindh, Pakistan.
Study Design:
Descriptive (cross sectional) and experimental study.
Genetic Part of study:
26 families of POAG with two or more alive and affected members in their families were enrolled.
Tissue culture part of study:
Human Trabecular Meshwork Cells (HTMCs) were utilized in this phase of research work, obtained by, “The ScienCell Research Laboratories”.
Study Protocol
Present study protocol was categorized into;
Genetic Phase
Tissue culture Phase
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SECTION: 01: GENETIC PHASE
2.1 MATERIALS USED IN GENETIC PHASE OF THIS STUDY:
Plastic and Glassware:
1. Sterilized Gloves
2. Adjuster Pipettes
3. Falcon Tubes
4. PCR Tubes
5. Screw Tubes
6. Eppendorf Tubes
7. Beakers
8. Pipette Tips
Chemicals and Reagents:
1. EDTA
2. Tris EDTA
3. TNE Buffer
4. 10% SDS
5. Proteinase K
6. Nacl 6M
7. Low Preservation TE Buffer
8. dNTPs
9. Taq Polymerase
10. MgCl2
11. Distilled Autoclaved H2O
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12. Absolute Ethanol
13. 70% Ethanol
14. Iso-propanolol
15. Agrose Powder
16. Ethidium Bromide
17. Primers
18. Flourescent labeled Dyes
19. Formamide
20. Liz size standard (Genescan 500)
Electronic Research Instruments:
1. Centrifuge Allegra X-22R centrifuge machine (Beckman Coulter)
2. Shaking Water Bath (Wise Bath)
3. Oven (Binder)
4. Flaking Ice chiller (Thermo)
5. Refrigerator -80oC (Revco)
6. Incubator (Binder)
7. DU-800 UV-visible spectrophotometer (Beckman Coulter)
8. Thermocycler (ABI 2720)
9. Thermal cycler Px2
10. Genetic Analyzer (ABI 3130)
11. Refrigerator Centrifuge Z 300 (Labnet)
12. Electronic Balance AY220 (Shimadzu)
13. Microwave oven MW3000 (HLL LandgrafLaborsysteme)
14. Gel Documentation System (BIORAD)
15. Magnetic Stirrer 1000 (JENWAY) 57
16. pH Meter 3510 (JENWAY)
17. Heat Block (Labnet)
18. MinRun Electrophoresis System GE-100 (Hangzhou Bioer.
Genetic phase of this study consisted of two very essential parts:
Field work
Laboratory work at Molecular Biology and Genetics Department, LUMHS.
2.1.1 Field work
Recognition and registration of patients having two or more members suffering
from Primary open angle glaucoma (POAG).
Collection of Blood samples of patients and healthy individuals.
Clinical investigations of patients of enrolled families.
Identification and Enrolment of Families:
Families having two or more individuals affected with Primary open angle glaucoma
(POAG) were recruited from different eye hospitals of Sindh, Pakistan. After taking history, detailed information including their contact numbers and detailed postal addresses were taken. Diagnosed cases of POAG were selected for inclusion in the study protocol. Illness story, complete family tree illustration and form of heritage were verified by personal visit to their homes. Individuals of the family were questioned to draw pedigree and to verify the genetic nature of disorder. Other relatives affected with
POAG were also included in this study after taking consent. The pedigrees were drawn
® on the white paper at the time of visit and were again re-drawn using Cyrillic program
® ® (Cyrillic for Windows .210) and Macromedia FreeHand software.
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Consent form:
Selected participants and family members were informed about the purpose of study, losses and gains and once they were ensured that the there was no harm of any kind; they were asked to sign the consent form. Volunteers who signed the consent form were included in the study protocol.
Confidentiality of Patient`s Personal Data:
Patient’s information and family data was kept confidential. Only concerned persons were allowed to check if necessary. Record of enrolled families was maintained properly including;
Manually drawn pedigrees with their cell numbers and complete home addresses
Informed consent form signed by patients or guardians,
Clinical examination findings
Data of every family was kept in the lock and key within their separate files labeled with family numbers.
Patient and Family history:
Patient and family history was noted in detail. Each family was interviewed separately.
History of trauma, medical disorders or surgical procedures was also enquired. Eye problems were interviewed in detail including inflammatory eye disease, or any other ophthalmological problem. Surgical eye procedures such as cataract, Trabeculectomy, retinal detachment or any other procedure in family members was asked for and noted in Proforma. History of detailed medical disorders and eye disease was taken to ensure any research bias. Each family member was interviewed for any eye problem. In those with history of glaucoma; the age of onset, duration of problem, symptoms and
59
treatment were asked in particular. Use of anti-glaucoma drugs and surgical procedures were noted in the Proforma.
Clinical Assessment/Examination:
Following completing patient and family history of eye problems, the affected subjects were requested for eye examination.
Visual acuity (VA)
Visual acuity was checked by using Snellen`s chart (Figure No 20). Light perception was checked by a torch. Counting fingers and hand movements were also the procedures for checking VA, depending upon the level of visual function.
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Figure No 20:Snellin’s chart for visual acuity assessment.
61
Tonometry
Intraocular pressure (IOP) of patients and normal family members was measured using
Applanation tonometry (Figure No. 21) or Pneumotonometry (Air Puff
Tonometry)(Figure No. 22). IOP of affected individuals was beyond the normal limits i.e., ≥ 22 mm Hg in most of the cases and was within normal limits in all normal individuals i.e., 10-21 mm Hg.
Figure No.21: Measurement of intraocular pressure by Goldman Aplannation tonometer
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Figure No.22: Measurement of intraocular pressure by Air Puff tonometer.
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Examination of Optic nerve head
Fundoscopy was performed under “slit lamp bio microscope” by using +78 D lens
(Figure No. 23). Glaucomatous optic discs, cupping and cup-disc ratio was measured during Fundoscopy. In difficult cases, OCT was performed for examination of RNFL for confirmation of the disease.
Figure No.23: Performing Fundoscopy on slit lamp bio microscope with the help of +78 D lens.
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Examination for Visual field defects
Visual field defects were assessed by using Goldman Perimetry and Humphrey field
Analyzer (Figure No. 24). This test was used in Glaucoma patients for evaluation of the limits of visual fields and to determine the progression of disease.
Figure No.24: Assessing the visual fields of the patient on Perimeter.
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Gonioscopy
Examination of angle of anterior chamber by Gonioscope is known as Gonioscopy.
Each patient was examined by a slit lamp bio microscope using Goldman 3 D
Gonioscopy lenses (Figure No. 25). Gonioscopy was performed at Department of
Ophthalmology of LUMHS tertiary care hospital which is fully equipped with modern techniques and instruments. Findings were confirmed by a Consultant ophthalmologist.
Figure No.25: Assessing the anterior chamber angle with the help of Gonio lens.
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Blood sampling
All affected and normal members in each family were requested for blood sampling.
Cubital fossa was examined for a prominent vein, once localized; the part was washed with a alcohol swab. Approximately 10 ml whole venous blood was saved in 50 ml sterile falcon tubes. 400 µl of 0.5 Molar EDTA was already present in each Sterilin falcon tube. Blood samples from normal individuals from every family were also taken as controls. Blood samples were stored in -80°C freezer at MBGD, LUMHS Jamshoro for subsequent analysis.
Ethical approval
The present study strictly followed the rules of “Declaration of Helsinki”. The researcher had to file an application to ethical review committee of institute for the approval of the, The research project was endorsed by members of ERC and Board of
Advanced studies of LUMHS, Jamshoro, Sindh, Pakistan.
Supervision
Supervision of the study was carried out by the supervisor and co-supervisors. Where ever felt necessary, the concerned supervisor was requested to solve the problem.
Pre structured Performa
A pre-structured Proforma was designed and was used for data entry of every patient and normal members.
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2.1.2 Laboratory work at MBGD laboratory:
The laboratory work was performed at MBGD Lab at Medical Research Centre,
LUMHS, Jamshoro. The Laboratory is fully equipped with modern equipments, genetic studies. Genetics study included the following steps;
DNA extraction
Measurement of DNA concentration
Estimation of DNA by Agarose gel electrophoresis
Primer designing and optimization for POAG genes.
Amplification of DNA by PCR in ABI 2720 (Applied Bio systems).
Sequencing of the gene for Analysis of mutations.
Extraction of DNA from blood samples
Blood samples were stored at t -200C before DNA extraction. DNA extraction was
carried out according to the standard non-organic method (Grimberg et al., 1989).
Steps performed for DNA extraction from Blood:
Before starting DNA extraction, frozen blood samples were thawed.
30-35 ml of Tris EDTA buffer (TrisHCl 10mM, EDTA 2mM) was added into 10ml blood and was blended through upturning many times. Whole process of centrifugations was carried out at 25oC). Centrifugation of Blood samples was performed at 3000 rpm up to 20 minutes and 15-20 ml supernatant was abandoned,
Spot was developed in base of the falcon tube after every centrifugation was dissolved by tapping it gently. After discarding the supernatant and dissolving the pellet, Tris
EDTA (DNA washing buffer) buffer was added for next centrifugation.
Same steps were reiterated till the pellet converted into slight pink (Usually we do washing of blood up to 4 times and the pellet should be light pink by that time).
68
when Pellet became light pink, it was re-suspended into 6ml TNE Buffer (Tris HCL
10mM, EDTA 2mM, NaCl 400mM; TNE buffer is usually taken in a quantity of
0.6ml/ml of original blood volume), 10% SDS and proteinase K. The blood models were kept in incubator for whole night at 37°C in shaker. Placing the samples in incubator for 24 hours, broad dissolution of the pellet was assessed. In case the pellet was not dissolved properly, proteinase-K was added as per requirement until dissolved.
Again the samples were placed within incubator on 37oC for 2-3 hrs or still the complete absorption of pellet. Tubes were kept with in ice & 1ml soaked NaCl (6M) was added. Tubes were shaken briskly and placed within ice again for 10-15 min. The samples were centrifuged again for 15mins at 3000 rpm to remove the salts and proteins settled down in the shape of pellet. The supernatant was decanted into conical bottom
15 ml appropriately marked falcon tube. Again the labeled conduits were centrifuged at 3000 rpm up to fifteen minutes. Then upper fluid within tubes was poured out in a
50ml labeled falcon tube whereas the initial falcon tubes containing pellet of salts and proteins were discarded. Now the uniform volume of iso-propanol was inserted and tubes were inverted softly till DNA was noticeable. Labeled samples were placed at normal temperature up to 10 minutes. Again falcon-tubes were kept in centrifuge on
3000 rpm up to 10 minutes. Then upper fluid within tube was removed cautiously and
DNA pellet was splashed by 10ml 70% ethanol. The labeled samples were then centrifuged on 3000 rpm up to ten minutes and seventy percent ethanol was decanted cautiously. Air drying of DNA pellet on thirty seven degree Celsius within incubator or on normal atmosphere was done and 1.5ml low T.E. (Tris HCl 10mM, EDTA
0.2mM) was inserted.
Labeled samples were kept within thirty seven degree Celsius shaker bath up to 24 hours to disseminate the DNA, Bands of Para film round covers of samples were 69
wrapped. Samples were placed in a shaker bath at 70°C (Heat Shock) up to 1 hour for inactivation of nucleases. The samples were air-conditioned at normal atmosphere and spanned shortly. DNA was aliquoted in labeled screw tubes and stored at -20oC within
Cryoboxes, which were given numbering & labeling or at -70oC for long term storage.
Measurement of DNA concentration (Quantification of DNA):
Quantification of DNA was carried out via two methods;
1. On single cell beam spectrophotometer at the wavelength of 260 nm.
2. Second by 1.2% Agarose gel in gel electrophoresis.
Measurement of the Optical Density (Spectrophotometry):
Concentrations of DNA were attained by calculating the optical-density (O.D) on two hundred and sixty nanometers consuming 1/100 dilution in quartz cuvette. Proportion of calculations measured on two hundred, sixty nanometers and two hundred, eighty nanometers wavelengths showed the integrity of nucleic acid. Quality of De-oxyribo
Nucleic acid is basically dependent on the point of absorbance (A) on two hundred and sixty nanometers is double than that at two hundred, eighty nanometers when uncontaminated DNA was present in the final outcome. In case of any contamination like protein, there was bit extra absorbance which decreased absorbance ratio among two hundred, sixty and two hundred, eighty nanometers (Figure No. 26).
70
Figure No.26: Spectrophotometer used for measurement of the Optical Density.
71
Agarose gel electrophoresis estimation:
A 1.2% (w/v) agarose gel was made by cooking 0.3 gram agarose powder
(HyAgaroseTMACT Gene USA) in 25 ml of Tris borate EDTA for two minutes in microwave oven. 2µl Florescent Ethidium Bromide dye was added for DNA visualization, as it intercalates between molecules of DNA and makes it visible under
Ultra violet light. The liquid gel was then poured into gel tray which contained different sized combs. T gel was left on room-temperature up to thirty mins to solidify. Then combs were taken away and gel was placed in electrophoresis tank GE-100
(HANGZHOU BIOER Technology co. LTD) in such a way that it was submerged with
TAE buffer. Now 2µl 1x DNA loading dye (Bromophenol Blue) was taken. It was added and mixed with 3µl diluted DNA and loaded into the corresponding wells. Gel electrophoresis was performed on hundred millivolt at eighty to hundred milli-ampere up to 3-4 minutes until the diluted DNA product came out of the wells for a sufficient distance. Then the gel was examined in UV illuminator for its purity. The pure DNA was appeared as a single sharp and bright band under UV light (Figure No. 27).
Figure No.27: Agarose Gel Electrophoresis Results.
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Primer Designing and Selection:
Various Bioinformatics tools like UCSC (University of California Santa Cruz)
Genome browser, ENSEMBL genome browser and Primer3 (V.4.0) were used to design and select the Primers for sequencing of MYOC and CYP1B1 gene.
PCR Amplification
The desired segments of DNA will be amplified by PCR at different optimized conditions for each pair of sequencing primers (PF and PR), using Applied Bio system thermo cycler 2720 (Figure No. 28). Agarose gel electrophoresis was performed to assess the quality of amplification. The reaction mixture volume of 25 µl for amplification was prepared by using following recipe in Table No.1.
Table No. 1: Reaction mixtures to amplify PCR Reaction mixtures for amplification of PCR.
Ingredient Final Stock Working Concentration Concentration Template DNA 50ng 25 ng/µl 4 µl Primer Forward 0.40µM 8.0 µM 1 µl Primer Reverse 0.40µM 8.0 µM 1 µl dNTPs 200µM 1.25mM 2.5 µl PCR Buffers 1X 10X 2.5 µl Taq Polymerase 1-2 Units 2 Units/ µl 0.4 µl
dH2O q.s to 25 µl
73
PCR reactions were accomplished by 4µl of 25ng of template DNA in twenty-five microliter reaction volume. PCR programs used for MYOC and CYP1B1 gene fragment amplification included touch-down 64°C-54°C, multicemb 54°C, 58°C and 59°C.
Illustration of these programs is shown in Figure No. 29.
Figure No.28: Applied Bio system thermo cycler (2720).
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Assessment of PCR Amplification using Agarose Gel Electrophoresis:
1.5% agarose gel was used to assess the amplification of DNA with sequencing primers.
Preparation of 1.5% Agarose Gel:
A quantity of 1.5 g (0.38 g of Agarose powder /25ml of buffer to make 1.5% Agarose gel) of Agarose powder was taken by weighing into electric balance (SHIMADZU corporation, Japan) and was put into a 250ml glass beaker. 25 milliliter from 1x TBE buffer was added and Gel was cooked (3-4min) till it was ready to boil in Microwave oven, mixing it once or twice to prepare a pure solution.
Then it was cooled down to 55°C. After that, 2µl Ethidium Bromide (5mg/ml) was inserted and intermingled (F.C of Ethidium bromide = 0.5g /ml). Gel suspension was dispensed in Gel Caster, which was placed in gel caster up to 15-20 minutes in room temperature until it was completely solidified.
Three microliter from PCR artifact was evaluated on 1.2% agarose gel for verification of required amplification & to ensure integrity of PCR product aforegoing for sequencing PCR. The PCR product was then treated to confiscate un-incorporated nucleotides and oligo-nucleotides by an ethanol precipitation method. Reaction mixture was formed by whole PCR product, 80µl 99% distilled ethanol and 20µl injection water.
Sequencing PCR
Following recipe was used to form the working reaction for performing the sequencing
PCR (Table No.4).
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Table No. 2:Recipe Recipe of ofworking working Reaction Reaction mixtures mixtures for for sequencing sequencing PCR. PCR
Ingredients Required Volume Diluted DNA sample 3µl
Big Dye Terminator 1µl
Primer (3.2 µM) 3 µl
5x sequencing buffer 1 µl
dH2O q.s to 10 µl Total Reaction Volume 25 ul
Figure No. 29: Thermo cycling profile (Program) to sequence a reaction.
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Preparation of product for sequencing on ABI Prism 3130 Genetic analyzer:
To precipitate the product, 20 μl 95 percent ethanol was used, 1 μl EDTA( 125 mm), 1
μl 3 M pH sodium acetate: 5.6 was poured within the 10ul sequencing reaction plate and placed on room temperature for 30 minutes.
Plate was placed within centrifuge for 20 minutes on 3500 rpm. After centrifugation, supernatant was decanted via overturning sequencing plate over paper towel. Each well was added 100ul of 70 percent ethanol, and the plate was re-centrifuged on 3500 rpm up to thirteen minutes. At the end, ethanol was decanted as described over, pellets were air dried and digested within 10ul deionized Hi- Di Formamide (ABI).The samples were denatured on ninety five degree Celsius up to five minutes & quickly cooled through keeping them within ice prior to processing into the ABI PRISM 3130 Genetic
Analyzer for sequencing.
The sequencing results were then analyzed using Chromas software for mutational analysis for each result and checked for segregation within each family.
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SECTION: 02: TISSUE CULTURE PHASE
2.2 CELL CULTURE REAGENTS:
2.2.1 Trabecular Meshwork Cell Medium (TMCM):
Trabecular Meshwork Cell Medium (TMCM) was obtained from ScienCell Research
Laboratories, Canada. TMCM was a complete growth medium for the optimal culture of normal trabecular meshwork cells (TMC) in vitro when used along with Trabecular
Meshwork Cell Growth Supplement (TMCGS), and fetal bovine serum (FBS). This culture medium was physically liquid and sterile. It contained amino acids (essential and non-essential both), trace minerals, organic and inorganic compounds, growth factor and hormones, and FBS in low concentration of 2% only. This culture medium was HEPES and HCO3-buffered having pH of 7.4 in an incubator incubated with atmospheric air.
2.2.2 Complete Human trabecular Meshwork Cell Medium (HTMCM):
For culture of TMCs, HTMCM was prepared by adding 10 ml of FBS, 5 ml of TMCGS and 5 ml penicillin/streptomycin solution (ScienCell Research Laboratories, Canada) to 500 ml of TMCM. When ready, HTMCM was kept on four degree Celsius & was used within 15 days of preparation.
2.2.3 Poly L-Lysine (PLL):
Poly-L-Lysine (PLL) is a synthetic, positively charged amino acid chain. In biological culture media it increases cell bonding by changing surface charges of culture substrate.
PLL is usually consumed like coating agent to enhance cell adhesion, to improve survival of primary cells and to support the neurite growth. PLL contains polymers of
70 -150kDa. It was supplied as sterile-filtered liquid in the concentration of 1 mg/ml.
Product was stored at 2 – 8oC and was used within 6 months from the date of purchase.
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2.2.4 Dulbecco`s Phosphate Buffered Saline (DPBS):
The DPBS was obtained from The ScienCell Research Laboratories. It is an isotonic, colorless, non-toxic buffer with pH 4 ± 0.2, Osmolarity 90 ± 20 mOsm and endotoxin concentration <0.5 EU/ml.This sterile buffer was used for rinsing of cells without causing cell damage. DPBS was sterilized through a filter of 0.2μm pore size. It is devoid of Ca++, Mg++and phenol red. It contained Potassium Chloride (KCl) 0.2 g/L,
Monobasic Potassium Phosphate (K2PO4) 0.2 g/L, Sodium Chloride (NaCl) 8.0 g/L,
Sodium Phosphate (Na2PO4)1.15 g/L. It was stored at room temperature and used for
15 days after opening the bottle.
2.2.5 Trypsin Neutralization Solution (TNS):
Trypsin Neutralization Solution (TNS) was obtained from The ScienCell Laboratories.
It is phosphate and HEPES-buffered saline solution with 7.4pH. 10% FBS is present as trypsin inhibitor and cell protecting. It is free from Ca2+ and Mg2+. It was stored at room temperature. It was used to neutralize the effects of Trypsin/EDTA solution. It was stored at -200C. It was thawed before use and once thawing was performed, the solution was kept at 4°C and used for 1 month.
2.2.6 Trypsin – EDTA (T/E):
Trypsin – EDTA (T/E) was purchased from The ScienCell Research Laboratories.
0.25% T/E is a sterile, phosphate and HEPES-buffered saline solution with 7.4pH at room temperature. It contains 0.25% trypsin and 1mM EDTA. Trypsin is a serine protease enzyme. EDTA is a divalent ion chelator as for the divalent ions such as
Ca++ and Mg++. Trypsin was used to enhance the trypsinization reaction.
0.25% Trypsin/EDTA solution was used to detach adherent cells from a culture surface.
T/E was stored at -200C after receiving. Once thawing was performed, this solution remained longstanding up to thirty days on 4°Celsius. 79
Coating Procedure of Culture Flasks:
2 Optimal recommended concentration was 2μg/cm for normal cell attachment. Table
No.5 was used as guideline for proper requirement per flask. Appropriate volume of
H2O and PLL solution was taken with the help of pipette into each flask. The flask was swirled to encourage proper covering. Then the flask was incubated at 37° C for 1 hour.
The PLL solution was removed. Flask was rinsed twice with sterile H2O before addition of medium and cells.
TableConcentration No. 3: Concentration of water and of Poly water L-lysine and Plyfor coatingL-lysine the for culture coating flasks. the culture flasks
Water PLL (µL)
T-25 5 50 (1 mg/ml)
T-75 10 150 (1 mg/ml)
T-175 13 350 (1 mg/ml)
2.2.7 Human Trabecular Meshwork Cells (HTMC):
HTMC were obtained from The ScienCell Research Laboratories. HTMC cells were isolated form the “Juxta-canalicular” and “corneo-scleral” regions. Cell count was approximately >5 x 105cells/1ml volume in each vial. Vial was thawed at 37oC in water bath and cells were returned to the culture very quickly.
Procedure for performing Cell Culture:
Chilled ampoule was kept within thirty seven degree Celsius water bath. Vial was hold
& rotated smoothly till complete thawing of contents. Vial was removed from the water bath immediately after thawing. It was wiped down with 70% ethanol and was transferred to the sterile field. Cap was removed carefully without touching the internal threads. The contents of vial were resuspended and dispensed gently into the PLL 80
coated culture flask. Cap was replaced; vessel was rocked to distribute the cells evenly.
Gas exchange was allowed if necessary by loosening the cap of the flask. Culture flask was returned back to the incubator. Culture was kept for minimum16 hours. It was refreshed next day for removing of DMSO and any of the unattached cells. This Procedure was repeated every other day thereafter. The supplemented culture medium was refreshed on the next morning from the cryopreserved cells after establishing a culture.
Thereafter, the culture-medium was replaced after each 3rd day till culture confluency reached up to 70%. Once culture confluency reached up to 70%, the medium was changed on alternate day until the 90% confluency was achieved.
Subculture was performed when the culture reaches ≥90% confluency. PLL coated vessel (2 μg/cm2) was prepared one day before the subculture was initiated. Complete medium was warmed in 250C water bath. The Trypsin/EDTA solution, DPBS, and TNS were also warmed at 250C in water bath. Cells were rinsed with DPBS solution. 8 ml of DPBS was added, followed by Trypsin/EDTA solution (2 ml) into the flask. The flask was rocked gently to expose all cells completely to the Trypsin/EDTA solution.
Flask was incubated at 370C in incubator for the period of 1-2 minutes. A microscope was used for monitoring the cell morphology changes. Fifty milli-liter cone shaped centrifuging tube was put within five milli-liter of FBS during incubation,
Trypsin/EDTA solution was transferred to the 50 ml conical centrifuge tube. Flask was incubated for 1-2 minutes at 370C. The flask was taped gently from sides to dislodge any cell on the surface and this procedure was performed at the end of incubation.
Microscope was used to ensure that all the cells were detached. 5 ml of TNS solution was added to the flask. Detached cells were transferred into the 50 ml conical centrifuge tube. Flask was rinsed again with 5 ml TNS to collect any of residual cells trapped
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behind. Flask was examined under microscope for checking the successful cell harvesting. Number of cells was looked left behind (<5% was admissible).
50 ml of conical centrifuge tube was placed within centrifuge on one thousand rpm up to five mins, and then cells were re-suspended in the cell-culture media. Cultured cells were counted and placed on a freshly PLL coated vessel and the cell density was counted. Then, the cells were plated to the center of 96 well plates in triplicates, once
90% confluence was observed, and kept for 2 hours for the attachment of cells. 100μl complete Culture medium was poured into every well and plates were placed in incubator for 24 hours for growth of the cells. After 24 hours, cells were examined under inverted light microscope for growth of cells. 100μl of culture medium only in controls and H2O2 with different concentrations (0.0 mM H2O2, 0.5 mM H2O2, 1.0 mM
H2O2, 2.0 mM H2O2, 4.0 mM H2O2) were added. After 24 hours, cells were examined for any changes. Cultures were observed for morphological changes under light microscope and were left for 2 more days. On day 5, the cell viability was assessed by
MTT cell proliferation assay.
2.2.8 MTT Cell Proliferation Assay:
MTT Assay is the most frequently used assay worldwide for assessing the viability of cells. This was first described by Tim Mosmann in 1983.Cell proliferation is examined by the reduction of tetrazolium salts and is now a widely accepted reliable method.
Metabolically active proliferating cells reduced the yellow tetrazolium MTT (3-4, 5- dimethylthiazolyl-2-2, 5-diphenyl-tetrazolium bromide) by dehydrogenase enzymes which generate NADH and NADPH. Resultant purple formazan (intracellular) is made soluble and quantified by a Spectrophotometry. The MTT Cell Proliferation Assay was used to calculate the “cell proliferation rate”. If the proliferating cells are undergoing apoptosis or necrosis, the cell viability is reduced and this is estimated by MTT assay. 82
The MTT reagent showed low absorbance values in the absence of cells. A linear relationship is established for the cell number and signals produced, this allows most accurate quantification of proliferating cell numbers.
The MTT Reagent was kept at 4°C and was ready to use in the dark for up to 18 months, provided no contamination happens. Care was taken not to contaminate the MTT reagent with cell culture media while pipetting the solution. During this experiment,
10µL volume of MTT reagent was removed and remaining was tightly closed and returned back to 4°C temperature in the dark. Detergent reagent was stored at 4°C.
Bottle was warmed for 5 minutes at 37°C. Then it was mixed by inverting gently to avoid frothing. Detergent reagent remains stable for 18 months at the room temperature.
Table No. 4: Equipment and Materials used in this procedure.
Micro titer plate reader with 650- and 570- Micro titer plate (flat-bottomed)
nm filters
Inverted Microscope Sterile tube (5 ml)
Multi- channel pipette Serological pipettes
370C incubator Sterile pipette tips
Laminar flow hood
Determining Optimal Cell Counts:
Cells were harvested to produce cell suspension by centrifugation. Adherent cells were released from surface by trypsinization and or scrapping. Cells were resuspended at 1x
106 per ml in 96 well plates. Three control wells of medium were included along to
0 provide blanks for the absorbance readings. Cells were incubated in 37 C at 5% CO2
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incubator and 100% humidity for 12 hours. 100 µL of MTT reagent was added to each well including blanks and controls after aspiration of culture medium. Plate was returned to cell culture incubator for 2 hours. Cells were viewed periodically under an inverted microscope for the intracellular “Punctate Purple Precipitate”. Once intracellular “Punctate Purple Precipitate”was visible clearly, 100 µL of Detergent reagent was added to all wells. It was swirled gently. Plates were left in the darkness for 2 hours at room temperature. Plate cover was removed, and absorbance of each well was measured including of blanks at 570 nm in a micro titer plate reader. Average values from triplicate readings were determined. Average value was subtracted from blank. Absorbance was plotted against cell number/ml. Then the absorbance values of test samples were divided by values of control samples and the results obtained were multiplied by 100 to get percentage of cell viability or proliferation.
Calculation was performed:
Absorbance values lower than the control cells indicated a decrease in the rate of cell proliferation. Contrarily, the higher absorbance values were interpreted as an increase in rate of cell proliferation.
(abs sample – abs blank) % viable cells = x 100 (abs control – abs blank)
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2.3 H2O2 Treatment of Human Trabecular Meshwork Cells:
Human TM cells were assessed mostly at 2-3 days of post plating. The
Resveratrol and Ascorbate were diluted in proper concentrations in the fresh culture media. The HTMC were kept in CO2 incubator in 100 mM antioxidant
0 solutions at 37 c temperature and 5% concentration of CO2 with relative humidity of 100% for 24 hours. Cells were incubated as co-incubated and pre- treated cell groups.
TM Cell Groups: a. Control group – TM cells incubated with PBS. b. Resveratrol group – TM cells were incubated with Resveratrol. c. Ascorbic acid group – TM cells were incubated with ascorbic acid.
The antioxidant solutions were suctioned & substituted by new antioxidant
solutions. The H2O2 was used in the range of 0.0, 0.5, 1.0, 2.0 and 4.0 mM
concentrations and put into the 5% CO2 incubator at temperature of 370C.
This was allowed for 24 hours. After that the HTMC cells were assayed for
the:
TM cell – metabolic viability assay
TM cell – Live cell assay
TM cell - Reactive Oxygen Species (ROS).
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2.4 Human Trabecular Meshwork Cell-Viability/ Metabolic Assay:
After the exposing to hydrogen per oxide, the mitochondrial function of HTMC was estimated via the MTT assay. Excessive H2O2 was removed by washing HTM cells twice which might intervene to organic reduction reactions of MTT assay. HTMC were placed within incubator in 100μl of 0.5 mg/ml MTT digested within culture medium up to one hour on 370C. MTT medium was suctioned & 100μl dimethyl sulfoxide was added to make the purple formazan reaction product soluble. The absorbance was interpreted om five hundred and forty nanometer for each sample in synergy 4 Multi-
Mode Micro plate Reader (MMMR) using the Gen-5 plate Reader Control. Data analysis was performed on BioTek, Winooski, VT software. At the MTT absorbance of 100%, HTMC cells were normalized and results were presented as Mean ± SD (n
=6 experiments).
2.5 HTM Cell Live Cell Assay:
TM cell live-cell assay was checked by uptake and retention of the fluorescent calcium- binding dye “Calcein-AM”. Once suctioning from medium, the HTMC were bathed two times within PBS solution for removing excessive amount of hydrogen peroxide which may interfere with biological reduction reactions. HTMCs were incubated in
100µl of 2 mM concentration of “Calcein-AM” which was prepared in PBS for twenty minutes at the temperature of 25oC. Calcein-AM fluorescence (F528) quantification was estimated by band-pass filters (excitation=485 ± 20nm, emission=528 ± 20nm) in synergy 4 MMMR using the Gen-5 plate Reader control. Data analysis was performed on BioTek, Winooski VT software. The F528 Fluorescence was normalized for 100%
HTMC cells and were presented as Mean ± SD (n =6 experiments).
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2.6 Analysis of Reactive Oxygen Species (ROS) In HTMCs:
The Glory-ROS Elisa kit was used for analysis of ROS in HTMCs. This kit was purchased by Glory Science Co limited.
2.7 ASSAY TECHNIQUE: Step 1:
Standard: Whole chemicals were brought on the normal temperature.
Diluting the standard:
50μl standard attenuation was pipetted out into every tube,100μl standard (900IU/ml) was pipetted into the 1st tube, Took 100μl from 1st tube into 2nd. Fifty microliter from the 2nd tube was pipetted to 3rd tube. Following dilution series was obtained finally as displayed below;
Tube 0 1 2 3 4 5 IU/ml 900 600 400 200 100 50
Step 2: Empty wells were fixed individually. 40μl of sample dilution was added to the sample well. Followed by adding 10μl testing sample to each sample producing a final dilution of 5-fold. Sample was pipette to the wells. Touching the wells was avoided strictly and admixed smoothly.
Step 3- Incubation: The samples were concealed at 37OC for 30 minutes. They were concealed by the help of sticky ribbon given.
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Step 4- Configuration liquid: The washing solution was diluted 20 to 30- times through distilled H2O,
Step 5- Washing: The sticky strips were removed and fluid was decanted. Washing buffer was pipetted into every well for thirty seconds & repeated for five times,
Step 6- Add enzyme: HRP-Conjugate reagent was pipette into 50μl to every well, other than blank well.
Step 7- Incubation: Step 3 was repeated.
Step 8- Washing: Step 5 was repeated.
Step 9- Color: 50ul Chromogen Solution A and 50 ul Chromogen Solution B were pippeted into every well. Samples were avoided form light for fifteen minutes and kept at 37OC temperature,
Step 10- Stop the reaction: 50μl of Stop Solution was pippeted into every well.
Stopping of reaction changed blue to yellow color.
Step 11- Assay: Absorbance at 450nm after Pipetting was noticed. The blank well was taken as zero.
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Chapter No: 03
RESULTS
SECTION.01: GENETIC PHASE
This study was approved by Board of Advance Studies & Research (BASR), LUMHS,
Jamshoro, Sindh, Pakistan. Families with POAG having two or more affective members were identified from various eye hospitals from Sindh, Pakistan. A total of 26families were enrolled for this study. These families were personally visited detailed information was taken from their normal as well as affected individuals to verify the genetic nature of the disease (POAG). Detailed questions were asked from the family members regarding medical as well as surgical history of the eye. History of any other systemic disease was also asked.
Questions related to POAG like age of onset of the disease, symptoms and progression of the disease were also asked from the affected individuals as well as from their parents to classify the disease.
All the affected individuals of each enrolled family underwent detailed ophthalmological examination like assessment of visual acuity (VA), measurement of intraocular pressure (IOP), cup disc ratio (C/D ratio) and assessment of angle of anterior chamber (Gonioscopy) to verify the phenotype of the disease.
After getting informed consent, from all the involved individuals, the 10 ml blood was drawn from all affective individuals & other healthy members as well in different generations.
.Initially rough pedigrees were drawn on the white paper at the time of visit, but
® pedigrees were re-drawn with the help of Cyrillic program (Cyrillic for Windows .210)
® ® and Macromedia FreeHand software.
Genomic DNA was isolated from collected blood by using nonorganic method. Optical density (OD) was taken to measure the concentration of DNA. Primers were intended
89
with help of Ensemble Genome Browser bioinformatics tool. Then optimization of primers was performed. After optimization, the sequence of nucleotides was analyzed by using Chromas bioinformatics tool.
Genomic DNA was segregated from collected blood. Linkage analysis was accomplished on particular members of each pedigree by typing at least four STR markers for 14 reported POAG (GLC1A-N) and three PCG (GLC3A-C) loci (Table
No.2).
All families with POAG were directly sequenced for any association with Myocilin or
CYP1B1 gene. Of 26 POAG diagnosed families, 3 were found linked to various genes.
One family (POAG-02) was found associated with CYP1B1 gene on GLC3A at chromosome 2. The mutation (p.D217N) found in the CYP1B1 gene is novel and has not been reported earlier. One family (JOAG-26) segregated with novel MYOC mutation (p.T377R) where as one family (POAG-06) was found linked to a reported
MYOC variant but has not been previously reported from Pakistan in any glaucoma affected individual. This study is first of its kind with a cohort of 26 families having two or more than affected individuals from Sindh province of Pakistan.
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3.1 FAMILIES FOUND LINKED TO MYOCILIN GENE:
POAG-26
A novel mutation (p.T377R) was found to be segregated in POAG-26. This family was registered from Liaquat University Tertiary Care Eye Hospital. Family tree was sketched
& complete information was received. All seventeen individuals with eight patients in three consecutive offspring were involved in this research. Pedigree analysis revealed that legacy was autosomal dominant type. All affected individuals were undergone complete checkup and severe open-angle glaucoma characteristic features were detected. The average illness onset was twenty four years (ranging between eight to forty years) placing the disorder in Juvenile-onset of open angle glaucoma. Complete story disclosed that half of the patients were NPL in both eyes due to development of illness, earlier to the identification and any treatment.
Patient V: 4 was found glaucomatous up to 10 years of period whose IOP of 25/25 mm
Hg (OD/OS) before taking medications. Fundus examination showed cup disc ratio of
0.8/1.0 & VA 6/36 (OD) and PL-ve (OS). Glaucoma surgery was performed in right eye & IOP was measured up to 8 mmHg. Surgery was performed in two eyes of both affected individuals (IV: 4, V: 3) and IOP measured normal, C/D ratio was increased and low VA was recorded.
Two patients (III: 2, Age seventy years and III: 5, Age sixty five years) had onset of decreased visual acuity during late thirty years of age which resulted in loss of vision in early forty years of age. Clinical ophthalmological assessment exposed both eye opaque corneas in both patients, due to which other findings like IOP, gonioscopy and fundoscopic examination could not be carried out. Patients (IV: 3 and IV: 6) were completely NPL in their early twenty years of age. There were no corneal opacities in
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these patients, optic disc cupping observed in both eyes of the patient IV: 6 with C/D ratio of 1.0/1.0. Patient IV: 3 had corneal opacity bilaterally. (Table No.7).
Sequence analysis of MYOC gene revealed a heterozygous transversion from C to G at nucleotide number 1130 in exon 3 of the gene leading to replacement of threonine byarginine at position 377 of the protein. This is a novel variant and has not been reported earlier. This variant segregated in the family with the disease phenotype and it was also screened in 120 ethnically matched normal control individuals. Bioinformatics tools like Polyphen-2 and SIFT described the variant probably damaging.
Figure No. 30: Family tree of POAG-26: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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Table No. 5: Details of clinical examination of diseased persons having JOAG Clinical data of patients with JOAG.
Patient ID Gender/age Onset age BCVA IOP mm Hg C/D ratio Gonioscopy Surgery OD/OS OD/OS OD/OS III: 2 F/70 32 y NPL/NPL NR NR OA NIL III: 5 F/65 30 y NPL/NPL 15, NR NR OA NIL IV: 3 M/40 18 y NPL/NPL 15, NR NR OA NIL IV: 4 M/50 40 y 3/60, 3/60 10, 11 0.9, 0.9 OA B/L Trabe IV: 6 M/30 18 y NPL/NPL NR 1.0,1.0 OA NIL IV: 7 M/35 25 y PL + ve1/60 12, 8 NR 0.9 OA L/T Trabe V: 3 M/22 18 y 6/12, 6/9 22,10 0.7, 0.4 OA B/L Trabe V: 4 M/10 8 y 6/36, NPL 8, 25 0.8, 1.0 OA R/ Trabe
NR: Not Recordable. BCVA: Best Corrected Visual Acuity. C/D ratio: Cup/Disc. PL: Perception of Light. NPL: No Perception of Light. Trabe: Trabeculectomy. OD: right eye, OS: left eye. OA: Open Angle.
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A B
Figure No.31: Fundus photographs of an affected individual showing increased CDR
A- Left eye optic disc showing 0.7 C/D ratio and peri-papillary atrophy.
B- Right optic disc showing 0.9 C/D ratio, and nasalization of vessels and peri-
papillary atrophy.
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Figure No.32: MYOC sequence Chromatogram
Representative Chromatogram of MYOC sequence
A- A heterozygous C to G Substitution at codon 377. B- Normal sequence from an unaffected member
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POAG.06
A reported mutation (p.Q48H) was found in MYOC gene in family POAG-06. This family was enrolled from Hyderabad region of Sindh province. There were thirteen patients in three consecutive generations. Ten patients were already died whereas three were alive. Once complete story of illness was noted down, the age of onset of disease was found (all patients developed POAG after 40 years). On sequencing, a reported transmutation (p.Q48H) for Myocilin gene was discovered to segregate in the family.
The mutation was first reported in 2002 (Mukhopadhyay et al., 2002a). Direct Sanger sequencing revealed c.144G>T transversion which led to replacement of glutamine with histidine at codon number 48. Although it is a reported metamorphosis but is being registered first time from Sindhi speaking Pathan family of Pakistan. Pedigree analysis showed the discrimination of illness as an autosomal dominant configuration in a large consanguineous family, having 13 affected individuals in 3 consecutive generations (Fig.33). Of thirteen patients; only 3 were found alive whereas 10 were died. All three affected individuals were examined and diagnosed as a case of POAG.
The mean age for commencement of illness was forty six years (forty one to fifty years;
Table no.9). It was found from family story of illness that one patient had become blind due to late diagnosis. At the age of 41 years, the patient IV:2 was found diseased with primary open angle glaucoma. At that time his intraocular pressure was 28/32 mmHg
(OD/OS).
When the fundoscopic examination was performed, cup disc ratio was found as 0.8/0.7 and after correction the VA was assessed as 6/36 (OD) and (OS). When the angle of anterior chamber was assessed with the help of Gonioscopic lens, the angle was found open. The filtration surgery was performed in both eyes and after that the intraocular pressure was noticed as 12 mmHg (OD) and 18 mmHg (OS). 96
In the age of fifteen years, the patient IV:3 was found diseased with primary open angle glaucoma. She became blind in right eye because of the delayed diagnosis and after correction the VA was noticed as 6/60 in her left eye. On fundoscopic examination, the cup disc ratio was found as 1.0/0.9 (OD/OS) and the angle was assessed as open. The filtration surgery was performed in her left eye and intraocular pressure was noticed as
16 mmHg.
In the age of 47 years, the patient IV: 6 was found having primary open angle glaucoma.
Her VA was noticed as 2/60, 3/60 (OD/OS). The angle was found open on Gonioscopic examination and cup disc ratio was assessed as 0.9/0.9 (OD/OS). The filtration surgery was done bilaterally. This mutation was found to be present in all affected individuals while normal individuals did not harbor the mutation. Polyphen-2 and SIFT web tool anticipated G>T metamorphosis as possibly harmful.
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Figure No.33: Family tree of POAG-06: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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Table No. 6: Details of clinical examination of diseased persons of POAG – 06 Family
Clinical data of the patients with POAG-06.
C/D PATIENT AGE OF BCVA IOP MMHG GENDER/AGE RATIO GONIOSCOPY SURGERY ID ONSET OD/OS OD/OS OD/OS IV: 2 M/73 41 y 6/36,6/24 12,18 0.8,0.7 OA B/L Trabe
IV: 3 F/79 50 y NPL,6/60 25,16 1.0,0.9 OA L/ Trabe
IV: 6 F/75 47 y 2/60,3/60 14,20 0.9,0.9 OA B/L Trabe
BCVA: Best Corrected Visual Acuity, IOP: Intraocular pressure, C/D ratio: Cup/Disc ratio, NPL: No Perception of Light, Trabe: Trabeculectomy, OD: right eye, OS: left eye, OA: Open Angle.
A B
Figure No.34: Fundus photograph of an affected individual of POAG-06
A-right eye showing 0.9 C/D ratio and temporal crescent B-Color fundus photograph of left eye showing 0.4 C/D ratio
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Normal Diseased
Figure 35: MYOC chromatogram of normal and diseased patient.
In diseased one, the heterozygous transition of G into T is shown while normal one only indicating homozygosity only. In the result of this transition, the replacement of
Glutamine to Histidine at codon no. 48 take place. After the analysis of this substitution, it was proved pathognomic and damaging.
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3.2 FAMILIES FOUND LINKED TO CYP1B1 GENE:
POAG.02
A novel CYP1B1 mutation (p.D217N) was found in POAG-02. This massive Pakistani breed was identified from township Mola bux Karamati Baloch, taluka Ghora Bari,
Thatta, Sindh, Pakistan. There were eight patients in 2 generations belonged to Baloch ethnic group. after taking complete history, all the family members were identified with the disease in their forties. when sequenced for CYP1B1 gene, a novel heterozygous conversion c.650 G>A, replacing aspartic acid to asparagine at codon 217
(p.Asp217Asn) of cytochrome P450B1 was found. Protein prediction web tools
Polyphene2 and SIFT described this mutation as pathogenic for protein functioning.
The change was present in all affected family members and was absent in the normal individuals.
Family background revealed the discrimination of the illness as an autosomal dominant prototype in a huge consanguineous breed, having 8 affected persons in 2 creations
(Figure No. 37). All patients were alive and were declared as cases of POAG. Mean age of commencement of the illness was 43 years (range; 38 to 50 years, Table No.10).
According to their family story, one of the patients missed his vision in both eyes due to delayed identification of the illness.
Prior to the diagnosis of patient IV:1, intraocular pressure was measured as 36/40 mmHg (OD/OS). During fundoscopic examination, cup disc ratio of 0.7/0.9 was noticed and after correction the VA was recorded as 6/18 in right eye and counting finger in left eye. During Gonioscopic examination, the angle was noticed as open.
After the filtration surgery in both eyes, the intraocular pressure was measured as 18 mmHg in right eye and 25 mmHg in left eye.
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In Patient IV:3, the age of onset was 39 years and IOP was measured as 30/32 mmHg
(OD/OS). Cup disc ratio of 0.8/0.7 was observed during fundoscopic examination and
VA was recorded as 6/60 and 6/36 (OD/OS) after full correction. On Gonioscopic examination, the angle was noticed as open. The filtration surgery was carried out in left eye and intraocular pressure was normal on anti-glaucoma therapy.
In Patient IV: 8, the age of onset was 42 years and intraocular pressure was 28/35 mmHg in right and left eye prior to the treatment. During Fundoscopy, the cup disc ratio of 0,7/0.9 was noticed and VA was recorded as 6/24 in right eye and PL+ve in left eye after correction and angle was observed as open on Gonioscopic examination.
Filtration surgery was carried out in right eye whereas VA was diminished to PL+ve in left eye before surgery.
In Patient IV: 10, the age of onset was 38 years and intraocular pressure was measured as 22/26 mmHg in right and left eye. VA was recorded as 6/9 and 6/9P in right and left eye. 0.4/0.5 cup disc ratio was present on fundoscopic examination and the angle was found open on Gonioscopic examination. Filtration surgery was carried out in both eyes after diagnosis.
In Patient IV: 11, the disease was detected during age of 45 years and Prior to management, the 28/32 mmHg IOP was documented and BCVA recorded as 6/12 (OD) and 6/60 (OS). On Fundoscopic examination, the CDR of 0.6/0.7 (OD/OS) was observed. Open angle was noticed during Gonioscopic examination. Right eye
Trabeculectomy was performed and left eye IOP was controlled through medications.
Patient IV: 12 was diagnosed very late for POAG in the age of 49 years and the vision had been lost yet. The visual acuity was NPL in both the eyes. While examining the CD ratio, total cupping of the optic disc was observed. The angle of anterior chamber was found open on Gonioscopy. No any surgery was performed in both the eyes. 102
Patient IV: 19 was diagnosed for the disease in age of 43 years. Prior to treatment, the
IOP was 30/32 mmHg and VA was CF (OD) and 6/60 (OS) after correction. On
Fundoscopy, CD ratio was 0.9/0.8 (OD/OS). On Gonioscopic examination, the AC angle was found not closed. There was history of left eye Trabeculectomy.
Patient V: 7was diagnosed as a case of POAG in during 40 years of the life. Pre- treatment IOP was 30/24 mmHg (OD/OS) and BCVA recorded as 6/18 (OD) and 6/12
(OS). Cup Disc ratio of 0.7/0.6 (OD/OS) was observed during Fundoscopy. The AC angle was found not closed on Gonioscopic examination. There was the history of bilateral Trabeculectomy surgery.
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Figure No.36: Family tree of POAG-02: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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Table No. 7: Details of clinical examination of diseased persons having POAG Clinical data of the patients with POAG.
Age of BCVA Iop mmHg C/D ratio Patient id Gender/Age Gonioscopy Surgery onset Od/os od/os od/os
6/18, IV: 1 M/72 50 18/25 0.7/0.9 OA B/L Trabe CF
6/60, IV: 3 M/65 39 20/16 0.8/0.7 OA L/ Trabe 6/36
6/24, IV: 8 F/70 42 14/26 0.7/0.9 OA R/ Trabe PL+Ve
6/9, IV: 10 M/40 38 12/14 0.4/ 0.5 OA B/L Trabe 6/9P
6/12, IV:11 M/60 45 18/22 0.6/0.7 OA R/ Trabe 6/60
NPL, IV: 12 M/88 49 25/28 1.0/1.0 OA Nil NPL
CF, IV: 19 M/60 43 20/18 0.9/0.8 OA L/ Trabe 6/60
6/18, V: 7 M/ 49 40 14/16 0.7/0.6 OA B/ Trabe 6/12
BCVA: Best Corrected Visual Acuity, IOP: Intraocular pressure, C/D ratio: Cup/Disc ratio, NPL: NoBCVA: Perception Best of Corrected Light, Trabe: Visual Trabeculectomy, Acuity, IOP: IntraocularOD: right eye, pressure, OS: left C/D eye, ratio:OA: Open Cup/Disc Angle. ratio,
NPL: No Perception of Light, Trabe: Trabeculectomy, OD: right eye, OS: left eye, OA: Open
Angle.
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I II
Figure No.37: Fundus photograph of an affected individual of POAG-02
I- Right eye showing 0.9 C/D ratio. II- Left eye showing 0.7 C/D ratio.
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G>A/As
n.
A- AFFECTED
B- WILD TYPE
Figure No.38: Chromatogram of CYP1B1 sequence:
A- Heterozygous transition of G to A at codon no. 217. B- Chromatogram of a normal individual.
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3.3 FAMILIES FOUND UNLINKED TO MYOCILIN AND CYP1B1 GENE:
POAG.01
Family POAG 01 was registered from Hyderabad, Sindh, which was an Urdu speaking
“Sheikh” family, having three affected individuals in two consecutive originations.
Total six family members including three affected were enrolled. The diagnosis of the disease was made after age of 30 years.
Figure No. 39: Family tree of POAG-01: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG.03
Family POAG.03 was enrolled from Shah Bandar, Chohar Jamali, District Badin,
Sindh. This Sindhi family belonged to Khatri tribe, with four patients in two consecutive originations. Two among the diseased individuals had been died where as two were alive which were enrolled in this study. Two alive diseased individuals were detected with POAG later in 40 years.
Figure No.40: Family tree of POAG-03: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG.04
This family was enrolled from village Dobharo, TalukaTalhar, district Badin, Sindh.
This Sindhi family belongs to Mir Talpur caste, having two affected family members in two successive generations. Both the affected individuals were identified with
POAG after 35 years of age.
Figure 41: Family tree of POAG-04: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-05
This family was enrolled from a city Matli, district Badin, Sindh. This Sindhi family belongs to Soomro caste, having two affected family members in two successive generations. Aamina was identified with POAG in 43 years of the age and Ismail was diagnosed in the age of 47 years.
Figure No.42: Family tree of POAG-05: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-07
This family was enrolled from Taj Centre, Shah Faisal Colony Karachi, Sindh. This
Hindko speaking family belongs to Hindku ancestry, having 3 patients within 2 successive generations. All patients were identified with POAG after 40 years of age.
Figure No.43: Family tree of POAG-07: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-08
This consanguineous family was enrolled from village Qaiser khan Shahani, taluka
Sobhodero, district Khairpur Mir’s, Sindh. This Sindhi speaking family belongs to
Shahani Baloch tribe, with four patients in two consecutive generations. One among the patients was dead whereas three were alive. All patients were identified with POAG after 40 years of age.
Figure No.44: Family tree of POAG-08: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-09
This family was enrolled from Latifabad no.10, Hyderabad, Sindh. This Urdu speaking family belongs to Sheikh Tribe, having two patients in two consecutive generations.
One individual was identified with POAG in the age of 50 years while other was diagnosed in the 35 years of age.
Figure 45: Family tree of POAG-09: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-10
This family was registered from village Tando Soomro, District Tando Allahyar, Sindh.
This Sindhi family belongs to Nizamani caste, having 26 affected family members in four successive generations. Both the affected individuals in 1st generation were dead.
In 2nd generation, one affected individual was alive while 7 affected individuals were dead.
In 3rd generation, all 10 affected members were alive and in 4th generation, six family members were alive. All affected individuals were identified with POAG after 40 years of age.
Figure No.46: Family tree of POAG-10: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-11
This family was enrolled from village Jaffer chang, Deh Pyaro, union council Lanvari sharif, taluka and district Badin, Sindh. This Sindhi family belongs to Chang caste, having three affected individuals. All affected individuals were identified with POAG in 40 years of age.
Figure No.47: Family tree of POAG-11: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-12
This family was registered from Lajpat road, Hyderabad, Sindh. This Kachi family belongs to Khatri tribe, with five patients in two consecutive generations. One patient in 1st generation was dead, while four affected members were alive in 2nd generation.
All affected family members were identified for POAG after 40 years of age.
Figure No.48: Family tree of POAG-12: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-13
This family was enrolled from ward no 8, Abro colony, Kandiaro road, Tharushah, district NaushehroFeroze, Sindh. This Sindhi family belongs to Soomro caste, having four affected individuals in two successive generations. All patients were identified after 40 years of age, while only one affected family member was identified in 38 years of age.
Figure No.49: Family tree of POAG-13: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-14
This consanguineous family was enrolled from village Essa khan Lanjwani, taluka and district Tando Muhammad khan. This Sindhi family belongs to Lanjwani tribe, with four patients in three consecutive generations. Three among the patients were alive while one was dead. All patients were identified with POAG after 40 years of age.
Figure No.50: Family tree of POAG-14: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-15
This consanguineous family was enrolled from village Ramzan Lanjwani, Deh
Roshnai, union council Lakha at, taluka and district Tando Muhammad khan. This
Sindhi family belongs to Lanjwani tribe, with five patients in three consecutive generations. One diseased member was dead while other four were alive. All affected family members were identified with POAG after the 40 years of age.
Figure No.51: Family tree of POAG-15: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-16
This consanguineous family was enrolled from village Jam JhodhoUnar, talukaSakrand, district NawabShah. This Sindhi family belongs to Channa caste, having three affected family members in two successive generations. Two members were alive while one was dead. All individuals were identified with POAG after 40 years of age.
Figure No.52: Family tree of POAG-16: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-17
This consanguineous family was enrolled from village Liaquat Ali magsi, Tando
Muhammad khan road, Seri stop, Noraishareef road, district Tando Muhammad khan.
This Sindhi family belongs to Magsi caste, having six affected family members in three consecutive generations.
Five patients were alive while one was dead. Almost all affected family members were identified with POAG after 40 years of age.
Figure No.53: Family tree of POAG-17: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-18
This family was enrolled from Mirzapur near Halani, district NaushehroFeroze, Sindh.
This consanguineous Sindhi family belongs to Sahito tribe, with four patients in two consecutive generations. Three patients were alive while one was dead. All patients were identified with POAG after 35 years of age.
Figure No.54: Family tree of POAG-18: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-19
This Sindhi family was registered from village Abdul Majeed Qambrani, near Peer was an, Bozdar wada and village Naseer faqeer jalalani, taluka kotdigi, district Khairpur
Mir’s. Some family members were living in village Abdul Majeed Qambrani and some were living in village Naseer Fakir Jalalani. This consanguineous family belongs to
Qambrani tribe, with nine patients in three consecutive generations. Seven patients were alive while two were dead. Almost all affected individuals were identified as
POAG after 40 years of age.
Figure No.55: Family tree of POAG-19: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-20
This Urdu speaking family was enrolled from Sarferaz colony, near liberty market civil hospital Hyderabad. This family belongs to Sheerazi sheikh tribe, with five patients in three consecutive generations. Three were dead while two were alive. This family was
Rajistani in origin. Almost all family members were diagnosed after the age of 37 years.
Figure No. 56: Family tree of POAG-20: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-21
This Siraiki speaking family was enrolled from village Ghulam Hussain Haral, near village Soomar shah, Mashakh stop on Indus highway near Qazi Ahmed, Sindh. This consanguineous family belongs to Gudaro tribe, with eleven patients in three consecutive generations. Six patients were alive while five were dead. All affected individuals were identified as cases of POAG after 40 years of age.
Figure No.57: Family tree of POAG-21: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-22
This Sindhi family was registered from village Muhammad Arif Shoro, Udero city (2-
3 kilometers from Ghora Bari), Taluka Ghora Bari, District Badin. This family belongs to Shoro caste, with six patients in four consecutive generations. Four patients were alive while two were dead. All patients were identified with POAG case after 40 years of age.
Figure No.58: Family tree of POAG-22: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-23
This Sindhi family was registered from village Sundha, district Thatta. This consanguineous family belongs to Qazi tribe, with nine patients in five consecutive generations. Six patients were alive while three were dead. All patients were identified with POAG after 40 years of age.
Figure No.59: Family tree of POAG-23: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-24
This Sindhi family was registered from village Jhirk, district Thatta. This consanguineous family belongs to Memon tribe, having two patients in two consecutive generations. Both the patients were alive. Both patients were identified as case of
POAG after 45 years of age.
Figure No.60 Family tree of POAG-24: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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POAG-25
This Sindhi family was registered from village Haji Soof, District Thatta. This consanguineous family belongs to Zangyani tribe, having eight patients in two consecutive generations. Three were dead while five were alive. All patients were diagnosed as case of POAG after 40 years of age.
Figure No.61: Family tree of POAG-25: Shaded squares and circles indicating diseased male and female patients subsequently whereas crossed one are indicating dead members.
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SECTION: 02: TISSUE CULTURE PHASE
This study was approved by Board of Advance Studies & Research (BASR), Liaquat
University of medical & health sciences Jamshoro, Sindh, Pakistan. It was carried out at Cell Biology &Tissue culture laboratory at Medical Research Centre, Liaquat
University of medical & health sciences Jamshoro, Sindh, Pakistan, in collaboration with National Institute of Blood Diseases (NIBD) Karachi. Human Trabecular meshwork cells (HTMC) were used for these experiments. For this, complete Human
Trabecular meshwork cell line kit was purchased from ScienCell Research Laboratories which contained the following items in it:
Human Trabecular Meshwork Cells, 5*105 cells/vial
Trabecular Meshwork cell medium 500 ml, 0010, 0503, 6592
Dulbecco’s Phosphate-Buffered Saline, 500 ml
Trypsin/EDTA Solution, 0.25%, 100 ml
Trypsin Neutralization Solution, 100 ml
Poly-L-Lysine, 1 mg/ml
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3.4 IMPACTS OF RESVERATROL AND ASCORBIC ACID ON TM CELL METABOLISM AGAINST H2O2-INDUCED INJURY:
3.4.1 Co-incubation of Ascorbate and Resveratrol:
Cultured HTM cells were co-incubated with H2O2 in growth medium up to twenty four hours. Effects of different concentrations of H2O2 on TM cell metabolism were estimated by detecting mitochondrial enzymes functional activity with the help of MTT assay kit. The results are summarized in Table No 11. And Graph No.1 as given below:
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Co-Incubation of TM Cells with Ascorbate and Resveratrol against H2O2-Induced Metabolic Injury. Table No. 8: Co-Incubation of HTM Cells with Ascorbate and Resveratrol against H2O2-Induced Metabolic Injury 0.0 mM 0.5 mM 1.0 mM 2.0 mM 4.0 mM H2O2 H2O2 H2O2 H2O2 H2O2 Controls 100.0 98.0 61.0 32 9
Ascorbate (1mM) 100.0 100.0 99.0 65.0 31
Resveratrol (1mM) 100.0 100.0 99.0 90.0 76 MTT assay kit (Mitochondria function)
Co treatemnt MTT assay kit (Mitochondria 120 function) Controls 100 100 100 98 100 100 99 99 Ascorbate (1mM) 100 90 Resveratrol (1mM) 80 76 65 61
60 Cell Cell %
40 32 31
20 9
0 0 mM H2O2 0.5 mM H2O2 1.0 mM H2O2 2.0 mM H2O2 4.0 mM H2O2
Graph No. 1: Co-Incubation of TM Cells with Antioxidants Protects Against Peroxide-Induced Metabolic Injury. MTT assay kit (Mitochondria function).
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Highly significant differences were noticed for 1.0 mM hydrogen per oxide concentration (Chi Square test, p=0.0001). A substantial decrease in HTMC metabolism was noticed estimating sixty one percent at 1.0 mM hydrogen per oxide compared to Ascorbic Acid –ninety nine percent and Resveratrol ninety nine percent
(p=0.0001).
As shown in table No.11, the Ascorbate and Resveratrol protected against the H2O2 induced cell metabolism. Resveratrol was even more efficient than Ascorbic acid at 4.0 mM hydrogen peroxide, 76 percent of the HTMC activity was noticed. The 1mM
Resveratrol maintained the HTMC metabolism under co-treatment conditions.
Resveratrol enhanced the function of mitochondria to 4.0 mM hydrogen peroxide
(seventy six percent) as-compared to H2O2-treated TM cells; (Table-1). In order to test whether the protection of co-incubation of Ascorbate and Resveratrol was because of metabolic variation of HTMC, these were pre-treated by Ascorbate and Resveratrol for 24 hours followed by H2O2 treatment termed as “pretreat-only’’.
3.4.2 Pretreatment of TM cells with Ascorbate and Resveratrol
The results of pre-treatment of TM cells with Ascorbate and Resveratrol are shown in
Table No12. The pretreatment shows similar results in comparison to co-treatment except on 4.0 mM hydrogen peroxide. Pre-treatment HTMC metabolic activity was observed at 4.0 mM H2O2 as eleven percent, thirty one percent & forty seven percent as-compared to co-treatment at nine percent, thirty one percent & seventy six percent respectively in controls, ascorbic acid & resveratrol groups (p<0.05). The results are summarized in Table No 12 and Graph No 2 as above.
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PretreatmentTable No. 9: of Pretreatment TM Cells with of TM Ascorbate Cells with and Ascorbate Resveratrol and againstresveratrol H2O2 against-Induced H2O2-Induced MetabolicMetabolic Injury Injury.
0.0 mM 0.5 mM 1.0 mM 2.0 mM 4.0 mM H2O2 H2O2 H2O2 H2O2 H2O2
Controls 100.0 98.0 63.0 53.0 11.0
Ascorbate (1mM) 100.0 100.0 98.0 76.0 31.0
Resveratrol
(1mM) 100.0 100.0 99.0 89.0 47.0
MTT assay kit (Mitochondria function)
Pretreatement MTT assay kit (Mitochondria function)
120 Controls Ascorbate (1mM) 100 100 100 98 100 100 98 99 100 89 Resveratrol (1mM)
80 76 63 60 53 47 Cell Cell % 40 31
20 11
0 0 mM H2O2 0.5 mM H2O2 1.0 mM H2O2 2.0 mM H2O2 4.0 mM H2O2
Graph No. 2: Pretreatment of TM Cells with Ascorbate and Resveratrol against H2O2-Induced Injury (MTT) assay kit.
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3.5 Effects of Resveratrol and Ascorbate on Cell Viability against H2O2-Induced TM cell Injury:
Cultured HTMC were co-incubated by H2O2 in growth medium for twenty four hrs.
Different concentrations of H2O2 on TM cell were applied and cell viability was determined by F528 (Table No 13).
Table No. 10: ImpactsCo- Incubationon cell viability of TMof resveratrol Cells with and Antioxidants ascorbate against H2O2- inducedAlso ProtectsTM cellular Against damage H2O2 -Induced Cell Death.
0 mM 0.5 mM 1.0 mM 2.0 mM Cell viability H2O2 H2O2 H2O2 H2O2 4.0 mM H2O2
Controls 100.0 98.0 61.0 32.0 9.0
Ascorbate (1mM) 100.0 100.0 99.0 65.0 31.0
Resveratrol (1mM) 100.0 100.0 99.0 90.0 76.0
Cell Viability
Controls
120 Ascorbate (1mM)
100 100 100 98 100 100 99 99 Resveratrol (1mM) 100 90 76 80 61 65 60
Cell Cell % 40 32 31
20 9 0 0 mM H2O2 0.5 mM H2O2 1.0 mM H2O2 2.0 mM H2O2 4.0 mM H2O2
Graph No. 3: Co-Incubation of TM Cells with Antioxidants Also Protects Against H2O2-Induced Cell Death. (Determined by F528)
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The results are summarized in Table No.10 and Graph No.3 as above.
Compared to Ascorbate and Resveratrol, there was a statistically significant decrease i n viability in controls. Cell viability revealed statistically significant differences in co ntrols (P=0.0001) at 2.0 and 4.0 mM H2O2.
3.6. Reactive Oxygen Species (ROS) Effects of Resveratrol and Ascorbate in Co-Incubated TM Cells:
This experimental step determines a potential pathogenesis for the protective effect of
Ascorbate and Resveratrol against to H2O2 exposure. Intracellular ROS in TM cells were assessed by “ROS Elisa assay kit”.
“(Glory Science Co., Ltd. 2400 Veterans Blvd. Suite 16 - 101, Del Rio, TX 78840, USA)”.
HTMC were kept in incubator along with Ascorbic acid & Resveratrol up to twenty four hours. Hydrogen peroxide in concentrations of 0.0mM, 0.5 mM, 1.0 mM, 2.0mM and 4.0mM hydrogen peroxide was applied to the TM cells. Following results were found as shown in Table No 14 and Graph No 4.
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Table No. 11: Reactive Oxygen Species (ROS) Levels (IU/ml) Reactive Oxygen Species (ROS) levels (IU/ml).
0 mM 0.5 mM 1.0 mM 2.0 mM 4.0 mM ROS H2O2 H2O2 H2O2 H2O2 H2O2
Controls 21.0 115.0 321.0 657.0 789.0
Ascorbate (1mM) 20.0 98.0 295.0 534.0 711.0 Resveratrol 23.0 75.0 280.0 489.0 669.0 (1mM)
Reactive Oxygen Species
900 Controls 789 800 Ascorbate (1mM) 711 657 669 700 Resveratrol (1mM) 600 534 489 500
400 321 295 280 300
ROS levels levels ROS (IU/ml) 200 115 98 75 100 21 20 23 0 0 mM H2O2 0.5 mM H2O2 1.0 mM H2O2 2.0 mM H2O2 4.0 mM H2O2
Graph No. 4: Reactive Oxygen Species (ROS) levels (IU/ml).
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Higher levels of ROS were noted in the controls compared to Ascorbate and Resveratrol groups.
Ascorbic acid and Resveratrol decreased the ROS activity in Trabecular Meshwork cells subjected to hydrogen per oxide, as shown in Table No.11 and Graph No 4.
Resveratrol showed better results against ROS compared to Ascorbate. The results were statistically significant for Ascorbate and Resveratrol compared to controls.
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Chapter No: 04
DISCUSSION
SECTION: 01: GENETIC PHASE
This study was carried out for genetic screening of hereditary cases of POAG in people of Sindh and to observe the effect of oxygen free radicals (reactive oxygen species or oxidants) on trabecular meshwork which constitutes an important structural component of aqueous outflow pathway. This study comprises of two phases. In the first phase, role of genetic factors in pathogenesis of primary open-angle glaucoma (POAG) was determined by genetic screening of all POAG families for Myocilin and CYP1B1 genes, whereas in second phase of the study, protective effects of antioxidants on the cultured HTMC were observed.
In first phase of this study, the patients as well as normal individuals were enrolled from all over Sindh. Twenty six families of POAG with two or more alive affected members in their families were registered. Blood was drawn from all diseased as well as healthy persons as control and DNA was isolated by following a non-organic method. Direct sequencing was accomplished to look for the association of POAG with
MYOC&CYP1B1genes.
Out of twenty six POAG families, three families were found linked with
MYOC&CYP1B1 genes. The details of the families are discussed as under:
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4.1 POAG FAMILIES WITH NOVEL MUTATIONS:
POAG-02
This family was found to be linked with CYP1B1 Gene and a novel mutation
(p.D217N) was found. There were nine affected members in the family. This is a novel mutation and has not been reported earlier.
Glaucoma is a group of chronic and heterogeneous eye syndrome caused by high IOP, producing decomposition and cupping of optic nerve & manifests itself as a visual field defect and permanent sightlessness if not treated.
Glaucoma is the second major reason of blindness around the globe. Various studies have shown that the role of elevated intraocular pressure in mechanism of action of
POAG suggests that it is closely related to its advancement, but pathogenesis is unclear.
Genetic and molecular analyses have shown that genes play an important role to promote various hereditary eye diseases, involving POAG. Identifying these pathogenic genes can postulate mechanism of action of these genetic diseases (Fingert,
2011a).
Whereas it is known that many genes are involved in POAG, Myocilin is 1st gene associated to Primary Open Angle Glaucoma (Do et al., 2016).
In addition, other studies have shown that MYOC and CYP1B1 perform a complex role in the pathogenesis of POAG, though CYP1B1 has been widely considered to be involved in the etiology of PCG (Micheal et al., 2015).
In 2002, mutations in the CYP1B1 gene were described to be a significant risk factor for early onset POAG in patients without MYOC mutations (Vincent et al., 2002).
After this, CYP1B1 mutation was reported in two familial cases in 2004, the same
CYP1B1 mutation was expressed differently and affected by several people with PCG
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and POAG. In the same study, the presence of the CYP1B1 mutation was recorded in
236 sporadic and irrelevant French patients with POAG who participated in this study.
It was previously reported that all CYP1B1 mutations, in addition to one, are associated with PCG. In the homozygous state, there is only one mutation, and all other mutations are found in heterozygous or complex heterozygous conditions.
All were found POAG in both eyes with optic disc cupping and visual field defects.
The average age of patients in this group is 40 years (range from 13 to 52 years)
(Melki et al., 2004).
Two hundred POAG patients from India were reported with CYP1B1 mutations, in which the three were already found (p. W57C, p.E229K and p.R368H) but 2 were new one (p.S515L and p (p.S515L), although the mutation was novel in the homozygous state .D 530 G) was in one heterozygous state. The presence of the MYOC mutation was first investigated in all these patients. All patients had juvenile glaucoma after 20 years, and the POAG had different clinical characteristics of a mild phenotype during an ophthalmologic assessment. Therefore, this research also stresses the importance of identifying CYP1B1 mutations in patients with POAG / JOAG, who were studied to identify genes or candidate locus associated with POAG (Acharya et al., 2006).
In Iranian patients, the CYP1B1 gene mutations are also reported. 63 sporadic and 9 familial patients of POAG were assessed for CYP1B1transmutation and they all were examined for MYOC gene transmutation. In the result of this assessment, 7 CYP1B1 mutations were detected (11.1%) and 4 out of these 7 found two CYP1B1 mutated alleles.
Five patients were found having JOAG in the age of 25.5 years while 2 were found late onset POAG in the age of 62.7 years (average age). This data concludes that CYP1B1
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have not only role in PCG but it has also vital role in POAG especially in Juvenile onset form (Suri et al., 2008).
Previously, single research was carried out for the part of CYP1B1 in POAG patients in Pakistan. A study of the POAG family revealed 28 new heterozygous mutations in the family (p332V) in the CYP1B1 gene in three mutations in Pakistan and p.E229K, as previously reported. In addition, in 190 patients with sporadic GPD, three new mutant heterozygotes (K p. T 234, p. 287 P and p. 362 Q) and three heterozygous mutants (pages G61 E, s) were reported. E229K and p.R368H). The report displays genetic heterogeneity of glaucoma patients in Pakistan. Rare studies were performed on entire patients to determine if mutations are present in the Myocilin gene. Entire patients developed POAG related functions during fourth year or later, one or both elevated IOP. In our study, all the victims had glaucoma with increased intraocular pressure in the 40s (table).
Participants in this study have begun glaucoma during 38-50 years (averages = 43.3 years). Patients are common or close to IOP as soon as they receive the filtration surgery immediately following the confirmation with the POAG other than one (diabetes IV:
12). All of patients (IV: 12) before trabeculectomy and all other affected individuals have shown significant headwear at the time of the funds examination as well as the reduced VA.
The change in CYP1B1 detected in the POAG family (p.Asp217Asn) anticipated the disease through the variation of the disease such as the disease and damage caused by the SIFT web tool. According to HOPE, amino acids trans-mutated is impartial, but amino acid-wild-type has a -ve charge. This variance in the charge can cause the failure of the CYP1B1 protein to be affected by Primary Open Angle Glaucoma within this research & verification of features of POAG. Reviewing the trans-mutations in 143
CYP1B1 to patients and their relatives are very significant clinically for timely recognition of disease & management. This can be helpful in monitoring the visual acuity and quickly identify the characteristics of the POAG to those people. CYP1B1 research on these individuals can help prevent the disturbance of vision caused by the glaucoma.
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The CYP1B1 transmutation found within the glaucoma family (p. Asp217Asn) was expected to be the disease causing through SIFT web tool and mutation taster.
The mutantamino acid is impartial, while the normal amino acid has negative charge, according to the HOPE web tool. According to this research study and consequent characteristic features of glaucoma, this change in charge may cause pathogenic folding of CYP1B1protein within patients of POAG.
For early diagnosis & prognosis, screening for CYP1B1 transmutations in patients and their normal family members may be clinically helpful & also may be useful in monitoring visual acuity in such individuals and early diagnosis of POAG cases.
Screening for CYP1B1 within these persons may help prevent glaucoma-related visual impairment.
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POAG-26
This family was found to be linked with MYOC Gene and a novel mutation (p.T377R) was found. There were eight affected members in the family. This is a novel mutation and has not been reported earlier.
MYOC was the gene, initially found linked to JOAG on GLC1A1 at chromosome number 1 (Sheffield et al., 1993). Transmutations in Myocilin are resulting in adult- onset POAG & about thirteen percent cases of Juvenile Open Angle Glaucoma (Iliev et al., 2007, Vincent et al., 2002). Up to now, more than 75 pathogenic variants are reported to be present in MYOC(Young et al., 2012). More than 50% mutations are identified in particular populations. p.Gln368Stop is most commonly occurring mutation in MYOC gene in Europe. p.Gln48His and p.Cys433Arg are found in patients from India and Brazil respectively (Cabral de Vasconcellos et al., 2000). Up till now, no any MYOC related Glaucoma is reported from Pakistan. The present study reports novel variant c.1130 C>G, (p.Thr377Arg), in Myocilin gene, isolating with the disease phenotype. As most of the MYOC variants have been identified in exon 3 of the gene which signifies importance of amino acids in that region as this region encrypts a functionally significant Olfactomedin domain. In the past studies, two variants were reported at codon 377 of Myocilin, changing Threonine into Methionine and Lysine
(Alward et al., 1998, Vincent et al., 2002). p.Thr377Met is frequently occurring & is identified in people of 5 other areas of the world (Kitsos et al., 2010); while p.Thr377Lys is identified in patients from Irish and Scottish society. These two variants are reported in patients with juvenile-onset as well as adult-onset glaucoma(Alward et al., 1998, Vincent et al., 2002). It is important to establish a genotype/phenotype correlation. Studies conducted in the past have confirmed that affected individuals harboringMYOCp.Gly246ARG, p.Pro370Leu, or p.Tyr437His substitutions point out 146
firm phenotypic association with the beginning of the illness. On the other hand, the patients with Gln368Stop substitutions presented minor clinical features of illness
(Avisar et al., 2009). In the present work, heterozygous p.Thr377Arg demonstrated acute form of Juvenile-onset Glaucoma with complete penetrance even at 8 years in affected individuals. This manifestation of the glaucoma is not related to other substitutions at location 377 of MYOC protein. Diagnosis of the patient V: 5was made in 10 years of age with raised IOP in both eyes (25/25 mm Hg) and irreparable CDR of
1 in the left eye. Mostly, heterozygous p.Thr377Met results in a moderate disease with different age related penetrance (W Hewitt et al., 2007). 4 affected individuals having p.Thr377Arg allele resulted in loss of vision in both eyes. The diagnosis was made very late so they did not get medical or any form of surgical intervention.
Bioinformatics tools have suggested that threonine at 377 position of the protein is extremely conserved amino acid in various species. Furthermore, it changes the target residue of Casein Kinase 2 (CK2) motif (Shimizu et al.). GORIV software analysis of
Threonine to Arginine transversion predicted no change in their structure which confirms past reports. Though, Polyphen-2 and SIFT directed this change as probably disease causing. HOPE describes the change as damaging as wild-type and mutant residue differ from each other in their size, charge and polarity. These variations may result in protein folding problems and loss of hydrophobic interactions.
To conclude, we are first to report of MYOC-linked juvenile onset glaucoma from this part of the world i.e. Pakistan. This finding therefore suggests genetic heterogeneity of our population. These facts and figures propose that threonine at this position of the protein is essential for proper working of Myocilin protein & its replacement with arginine could result in severe consequences as far as the folding and functioning of the protein is concerned. 147
These finding conclusions may be useful in future to carry out research studies for understanding the etiology and mechanisms of Myocilin provoked POAG. Recognition of novel MYOC mutations will have positive consequences as it could help in genetic counseling of the affected family to screen out the carriers in order to stop the transmission of this form of glaucoma to off springs in next generations.
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4.2 POAG FAMILY WITH REPORTED MUTATION:
POAG-06
This family was found to be linked with MYOC Gene and a reported mutation (p.Q48H) was found. There were thirteen affected members in the family. Blood was drawn,
DNA was isolated and direct sequencing was carried out to look for association of
POAG with MYOC gene. Sequencing revealed a novel heterozygous transversion
(c.144 G> T) causing replacement of glutamine with histidine at codon 48 of the
Myocilin protein.
This transmutation was firstly reported by (Mukhopadhyay et al., 2002b); in three
POAG affected sporadic patients of Indian origin. The age of all three patients ranged between 20-70 years of age.
Subsequently, another study was carried out (Sripriya et al., 2004); in 2003, in which he enrolled 100 patients for genetic screening. On direct sequencing, this mutation
(Q48H) was observed only in two sporadic patients. Cup to disc ratio also varied considerably between 0.3-0.9 among all three patients. None of three patients reported any previous history of glaucoma in their families.
Both (Mukhopadhyay et al., 2002b)and (Sripriya et al., 2004) reported this mutation in the six sporadic cases whereas in this study, it has been found thirteen familial patients of the same family. This finding is therefore significant in this regard as it is reported firstly in familial cases from Sindhi speaking population of Pakistan.
In this family (POAG-06), thirteen affected and nine normal individuals were present in three successive generations. All patients were clinically examined and found to have characteristic features of POAG.
149
Patient IV: 2, the illness age was 41 years and intraocular pressure was measured as
28/32 mmHg (OD/OS). The C/D ratio was noticed as 0.8/0.7 (OD/OS) and VA was
6/36 & 6/24 after its correction. During Gonioscopic examination, the angle was noticed as open. Filtration surgery was carried out in both eyes and the intraocular pressure was noticed as 12/18 mmHg in both the eyes
In Patient IV: 3, age of onset of the disease was 50 years. Because of delayed diagnosis, VA of right eye was vanished and VA of her left eye was 6/60 after correction. During fundoscopic examination, cup disc ratio of 1.0/0.9 was noticed and angle was open on Gonioscopic examination. After filtration surgery in left eye, her
IOP was 16 mmHg.
Patient IV: 6 knew about the disease in age of 47 years. VA of this patient in right and left eye was recorded as 2/60, 3/60 after correction respectively. Angle was found open on Gonioscopic examination and cup disc ratio of 0.9/0.9 was observed. After filtration surgery in both eyes, intraocular pressure was measured as 14/20 mmHg.
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SECTION NO: 02: TISSUE CULTURE PHASE
4.3 RESVERATROL & ASCOBRIC ACID:
In this phase of project, human trabecular meshwork cells were cultured to detect possible preventive role of antioxidants (Resveratrol & Ascorbic acid) in prevention of
POAG.
Oxidative stress plays critical role in the pathogenesis of ocular diseases such as
Glaucoma, Cataract, Maculopathy, and Retinopathy. These patients loose the vision progressively resulting in blindness if untreated (Abu-Amero et al., 2015, Kowluru and
Mishra, 2015,Levkovitch-Verbin, 2015). Growing body of research evidence suggests that the phytochemicals exert anti-oxidant and anti-inflammatory activity and may prove beneficial in the ocular diseases such as the Glaucoma (Dib et al., 2015).
Resveratrol (3,5,41-trihydroxy-trans-stilbene) is a natural phenol termed Phytoalexin synthesized by the plants in response to injury by bacteria or fungi (Rhone and Basu,
2008). Similarly, the ascorbic acid is a time experienced anti-oxidant which is a proved biological fact (Shareck et al., 2017, Tuzcu et al., 2017). Resveratrol exerts potent anti- oxidative and anti-inflammatory activity and may function as cardio protective (Wu and Hsieh, 2011), neuroprotective (Richard et al., 2011), anti-aging effects (de la Lastra and Villegas, 2005). It is reported as chemotherapeutic agent also (Gusman et al.,
2001). This study investigated effects of Resveratrol and Ascorbate on HTMC metabolism, HTMC viability & effect of reactive oxygen species (ROS) in in-vitro TM cell experiments. Resveratrol and Ascorbic acid were used in 1 mM concentration. The
Resveratrol exhibited superior results than ascorbic acid as regards the HTMC metabolism, HTMC viability & consequences of reactive oxygen species. The results are in concurrence to past studies (Gartaganis et al., 2005, Jampel et al., 1998,Kahn et
151
al., 1983). A significant decrease in TM cell metabolism was observed in controls at approximately sixty one percent at 1.0 mM H2O2 compared to Ascorbate –ninety nine percent and Resveratrol ninety nine percent (p=0.0001). The 1mM Resveratrol retained
TM cell metabolism under conditions of co-treatment. Resveratrol enhanced function of mitochondria up to 4.0 mM hydrogen peroxide (seventy six percent) compared to
H2O2-treated TM cells, (Table-1). The findings are in conformity with previous studies
(Elisaf et al., 2001, Kahn et al., 1983,Ringvold et al., 2000). The pre-treatment demonstrates similar results in comparison to co-treatment except at 4.0 mM H2O2 .
The pre-treated metabolic activity of TM cells was present at 4.0 mM H2O2 as 11 percent, 31 percent and 47 percent compared to 9 percent co-treated, 31 percent and 76 percent respectively in control groups, Ascorbate and Resveratrol (p<0.05). These results corroborate with previous studies (Gartaganis et al., 2005, Jampel et al.,
1998,Kahn et al., 1983). Cultured HTMC were co-incubated with H2O2 in growth medium up to twenty-four hours. Different concentrations of H2O2 on TM cells were applied and cell viability was determined by F528. There was significant difference of cell viability between 2.0 and 4.0 Mm H2O2 and control groups (P=0.0001). These results corroborate with previous studies (Elisaf et al., 2001, Kahn et al., 1983, Lodovici et al., 2009,Ringvold et al., 2000). In present study, the cells were put into incubator with Ascorbate and Resveratrol for twenty-four hours. Hydrogen peroxide in concentrations of 0.0mM, 0.5 mM, 1.0 mM, 2.0mM & 4.0mM hydrogen peroxide was applied to the TM cells. Intracellular ROS in TM cells were detected by ROS Elisa assay kit. Higher levels of ROS were noticed in the controls as compared to Ascorbate and Resveratrol groups. Resveratrol showed better results against ROS compared to
Ascorbate. The results were statistically significant for Ascorbate and Resveratrol compared to controls. These results corroborate with previous studies (Elisaf et al., 152
2001, Jampel et al., 1998, Kahn et al., 1983, Lodovici et al., 2009,Ringvold et al., 2000).
From the above discussion, it suggests the protective role of Resveratrol and Ascorbate in TM cell culture. Anti ROS activity is of prime importance as the oxidative stress plays major role in the pathogenesis of age related disorders, glaucoma being one of them.
From the accumulating research evidences, it is suggested that a decrease in the ROS and oxidative stress in the HTM area of eye ball hinders the development of POAG
(Polansky et al., 1997).Oxidative stress induces glucocorticoid-response proteins which act as anti-oxidative, (Polansky et al., 1997) but these mechanisms may fall short and institution of external agent like Resveratrol and ascorbic acid may help. These observations are in agreement with previous studies (Polansky et al., 1997, Zhou et al.,
1999). A previous study reported that the short-term treatment of cultured TM cells sub-lethal hydrogen peroxide (1 mM) doses decrease the HTMC adhesion to the growth matrix. This study further reported that the repetitive exposure might possibly lead to cellular damage resulting in defective tissue integrity, & consequent compromised drainage of aqueous humor through the trabecular meshwork (Zhou et al., 1999). Other natural anti-oxidants, such as the reduced glutathione (GSH), present in large quantities in some persons, are reported with low chances of POAG as compared to age-matched controls with low circulating GSH (Gherghel et al., 2005). These findings are in agreement with the ROS studies of present study. Another previous study reported the
TM cell culture with small doses of (0.2 mM) hydrogen peroxide induced expression of inflammatory markers within trabecular meshwork in glaucoma patients. Superoxide dismutase (SOD) is one of free reactive oxygen species (ROS) scavenger agent. As the
SOD decreases with aging, it shows proportionally increased chances of age-dependent decline in the TM (De La Paz and Epstein, 1996). In present study, the TM cells were 153
put in incubator with Ascorbate and Resveratrol showed low ROS compared to higher levels of ROS in controls. These findings are consistent with previous studies (Izzotti et al., 2003, Izzotti et al., 2010,Sacca et al., 2005). These previous studies reported that the increased ROS load increases the oxidative stress of DNA in both nucleus and mitochondria of TM tissue taken form glaucoma persons compared to healthy human eyes. ROS induced oxidative stress reduces the number of TM cells in the trabecular meshwork in the normal eyes of aged persons (Alvarado et al., 1984). This proves the role of ROS in the pathogenesis of glaucoma. The findings of ROS (Masihzadeh et al.,
2011) scavenging activity of Resveratrol and Ascorbic acid support the above study. A previous study reported low NADPH (multi-photon microscopy) in cultured human
TMC exposed to hydrogen peroxide in different concentrations. Findings of above study are in support to our present research indirectly as the NADPH was not evaluated, we used different techniques of ROS Elisa assay. The observation of Resveratrol as anti ROS agent of present study is in agreement with previous study (Luna et al.,
2009);which reported that the Resveratrol phenolic compound has decreased ROS and inhibited inflammatory markers in the TM cells treated with H2O2. It is reported that the concentration of hydrogen peroxide in human aqueous humor (AH) may be very elevated 25lM (Spector and Garner, 1981), which is deleterious for the TM cells. This much H2O2 is lethal for the anatomy and physiological functions of TM cells. It is believed that the H2O2 is generated continuously in anterior chamber of eye through a light-dependent reaction within iris melanin (Wielgus and Sarna, 2008). In present study, the antioxidants were chosen to include dietary agents such as the Resveratrol and Ascorbic acid (vegetables). Findings of superior results of ROS scavenging activity of Resveratrol compared to Ascorbic acid are inconsistent to previous study of (Kahn et al., 1983), This may be due to the fact that present study used Resveratrol in 1mM 154
concentration which is double than the concentration used by Amar et al (Kahn et al.,
1983).
From the above discussion it may be resolved that Resveratrol & Ascorbic acid may be proved beneficial in avoiding and adjourning glaucoma in susceptible persons. Proper and timely intervention with these anti-oxidants may help in maintaining the normal intraocular pressure, TM cell functions and vision loss caused by glaucoma.
155
CHAPTER NO. 5:
CONCLUSION AND FUTURE DIRECTIONS
Primary open angle glaucoma, the commonest form of glaucoma, is silent mostly which may lead to irreversible blindness, if remains untreated. The exact cause of the disease is still a mystery. It has been suspected that genes may perform an important part in the pathophysiology of POAG. There are many genes which are supposed to cause glaucoma but the most common genes associated with POAG are Myocilin and
CYP1B1.
The aim of this study was to identify function of genetic factors in understanding the mechanism of action of POAG and observe the potential role of antioxidants to prevent
POAG. For this purpose, twenty-six families of POAG were enrolled from all over
Sindh region from Pakistan. In current research, all enrolled families of POAG were screened for linkage analysis with the Myocilin and CYP1B1 gene. Out of twenty six families, two families (POAG-26 and POAG-06) were found linked with the Myocilin gene in which one was found a novel mutation (p.T377R) and other was already reported mutation in Indian population (p.Q48H). Only one family (POAG-02) was found associated with CYP1B1 gene which was a novel mutation (p.D217N).
It has also been suspected that free radicals may impair the function of trabecular meshwork cells which may lead to glaucoma and antioxidants have the potential to reverse the action of free radicals. For this purpose, this study was conducted to observe the potential effect of different antioxidants (Resveratrol and Ascorbic acid). The human trabecular meshwork cells were purchased from The ScienCell Company.
These cells were exposed to the hydrogen peroxide (H2O2); then treated with the
Resveratrol and Ascorbic acid. It was observed that Resveratrol has got more protective effects than Ascorbic acid on the cultured human trabecular meshwork cells exposed to the H2O2.
156
According to findings, there is low evidence that Myocilin and CYP1B1 polymorphism are associated with POAG susceptibility with low prevalence of Myocilin (3.8%) and
CYP1B1 (7.6%) mutations among the people of Sindh region of Pakistan.
No evidence of strong linkage of POAG with CYPIB1 and Myocilin was detected in the affected families of Sindh province Pakistan. Further genetic studies are needed to detect other possible genetic linkages with POAG, which may be helpful in the prevention of the disease and finding of new treatment strategies.
157
Chapter No.06
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