CORRELATION AND ROLE OF NITRIC OXIDE (NO) AND BCL-2 IN DUCHENNE MUSCULAR DYSTROPHY (DMD) PATIENTS

Thesis Submitted to Faculty of Science, Ain Shams University In Partial Fulfillment of Master degree of Science

BY Fatma Sayed Mohamed Moawed B.Sc. Biochemistry

Under Supervision Of

Prof. Dr. Fahmy Tawfik Ali Prof. Dr. Nadia Y.S. Morcos Professor of Biochemistry Professor of Biochemistry Faculty of Science Faculty of Science Ain Shams University Ain Shams University

Prof. Dr. Soheir Saad Korraa Professor of Medical Enviromental science National Center for Radiation Research and Technology Atomic Energy Authority

2009

ﻋﻼﻗﺔ ﻭﺩﻭﺭ ﺍﻜﺴﻴﺩ ﺍﻟﻨﻴﺘﺭﻴﻙ ﻭﺒﻰ ﺴﻰ ﺍل-2 ﻓﻰ ﻤﺭﻀﻰ ﺍﻟﺤﺜل ﺍﻟﻌﻀﻠﻰ (ﺩﻭﺸﺎﻥ)

رﺳﺎﻟﺔ ﻣﻘﺪﻣــﺔ ﻣﻦ ﻓﺎﻃﻤﻪ ﺳﻴﺪ ﻣﺤﻤﺪ ﻣﻌﻮض

آﺠﺰء ﻣﺘﻤﻢ ﻟﻠﺤﺼﻮل ﻋﻠﻰ درﺟﺔ اﻟﻤﺎﺟﺴﺘﻴﺮ ﻓﻰ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ

ﺗﺤـــﺖ ﺇﺷﺮﺍﻑ

.. .. اﺳﺘﺎذ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ اﺳﺘﺎذ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ ﻗﺴﻢ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ ﻗﺴﻢ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﻋﻴﻦ ﺷﻤﺲ آﻠﻴﺔ اﻟﻌﻠﻮم – ﺟﺎﻣﻌﺔ ﻋﻴﻦ ﺷﻤﺲ ..א أﺳﺘﺎذ اﻟﻌﻠﻮم اﻟﺒﻴﺌﻴﻪ اﻟﻄﺒﻴﻪ اﻟﻤﺮآﺰ اﻟﻘﻮﻣﻲ ﻟﺒﺤﻮث وﺗﻜﻨﻮﻟﻮﺟﻴﺎ اﻹﺷﻌﺎع هﻴﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﻳﺔ

ﻛﻠﻴﺔ ﺍﻟﻌﻠﻮﻡ - ﺟﺎﻣﻌﺔ ﻋﻴﻦ ﺷﻤﺲ 2009

Acknowledgement i

Thanks to Allah first and foremost. I feel always indebted to Allah the kindest and the most beneficent and merciful.

My great thanks and gratitude extend to include Professor Dr. Fahmy Tawfik Ali, Professor of Biochemistry, Former Head of Biochemistry Department, Faculty of Science, Ain Shams University for his continuous encouragement, valuable advice and criticism. Without his generous and valuable assistance, this work would lose its value. It is an honor working under his supervision.

My deep thanks and gratitude to Professor Dr.Nadia Y. S. Morcos, Professor of Biochemistry, Biochemistry Department, Faculty of Science, Ain Shams University for her continuous encouragement, generous supervision, valuable advice, and great support and without her great efforts this work wouldn't complete.

It is great honor for me that I take this opportunity to express my sincere appreciation and my deep respect to professor Dr.Soheir Saad Korraa, Head of laboratory of toxigenomics and proteomics, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, for her precious guidance .I owe her more than I can express for all the time, she spent in revising every detail, in spit of her busy schedule. In fact all credit goes to her in bringing this study to light.

I would like to express my profound appreciation to professor Dr.Ekram Abdel-Salam ,Professor of Pediatrics Acknowledgement ii and Genetics ,Faculty of Medicine, Cairo University ,who without her I would have never accomplished this work.

Finally, I would like to thank anyone share with advice, aid in experimental analysis and in finishing this work.

I declare that this thesis has been composed by me and it has not been submitted for a degree at this or any other university.

Fatma Sayed Mohamed

CONTENTS

Items Page

Acknowledgment i Abstract. iii List of abbreviations v List of figures. viii List of tables xi Introduction. xii Aim of the work. xiii Review of Literature 1 Duchenne muscular dystrophy 1 Clinical Progression of Duchenne and Becker 1 Muscular Dystrophies Dystrophin Protein 3 Mutation in Dystrophin Gene 5 Dystrophin's Functions 8 Pathophysiology of Dystrophin Deficient 9 Muscle 1- Membrane Structure and Function 9 2- Creatine Kinase 9 3-Apoptosis 10 Morphology of apoptosis 11 Mechanism of apoptosis 12 The BCL-2 Family of Proteins 13 Necrosis and Apoptosis 16 Apoptosis and DMD 16 4-Calcium Homeostasis 17 5- Proteolysis 17

Items Page 6-Lipid Change 18 7-Oxidative stress 19 Sequalea of Free Radicals' Generation 22 Oxidative stress and Dystrophinopathies 23 Nitric Oxide (NO) 24 Biosynthesis of Nitric Oxide 24 Effector Mechanism of NO 26 Metabolism of NO in Blood 27 Role for Nitric Oxide in Muscle Repair 28 Nitric oxide and apoptosis 28 Therapeutic strategies for DMD 29 LASER 30 LASER TYPES 32 1- Semiconductor lasers 32 2-Solid crystalline and glass lasers 32 3-Liquid dye lasers 33 4- Gas lasers 33 Fields of Practice 34 Applications of laser 34 SUBJECT AND METHODS 37 A.SUBJECT 37 1. Patients 37 Inclusion criteria 37 Exclusion criteria 37

2.LASER Irradiation 38 3.Blood pecimen 38 4-Chemicals 39 B- METHODS 40 1-Detrmination of plasma Craetine Phosphokinase 40 2-Determination of Cholesterol Concentration 42 3- Determination of Triacylglycerols Concentration 44 4-Determination of Malondialdehyde Level (LP) 45 5-Determination of catalase activity (CAT) in plasma 48 6-Determination of plasma Nitrite 51 7-Determination of BCL-2 in plasma and 55 lymphocytes 8-Determination of apoptosis in circulating 58 lymphocytes 9- Statistical analysis 59 Results 60 Discussion 95 Summary and conclusion 116 References 121 Arabic summary

Figures and Tables

LIST OF FIGURES

Fig. Title Page Schematic displays of the structure of the 1 6 dystrophin gene and protein 2 The dystrophin-associated protein complex 7 (DPC) in skeletal muscle 3 The two major apoptosis signalling pathways 15 4 Standard curve of MDA 47 5 Standard curve of hydrogen peroxide 50 6 Standard curve of Nitric Oxide 54 7 Standard curve of BCL-2 57 8 CPK activity in control and patients group 61 9 Mean plot of CPK in all groups 61 10a Illustrations for the % change in cholesterol 64 and triacylglycerol levels compared to control. 10b Illustrations for the % change in cholesterol and triacylglycerol levels post-laser compared 64 to pre-laser. 11 Mean plot of cholesterol in all groups 65 12 Mean plot of triacylglycerols in all groups 65 13a Illustrations for the % change in MDA levels and catalase activity compared to control 68 group. 13b Illustrations for the % change in MDA level and catalase activity post-laser compared to 68 pre-laser group. 14a Illustrations for the % change in NO levels 69 compared to control group. Figures and Tables

Continue Contents Fig. Title Page 14b Illustrations for the % change in NO levels 69 post-laser compared to pre-laser group. 15 Mean plot of MDA in all groups 70 16 Mean plot of CAT activity in all groups 70 17 Mean plot for NO in all groups 71 18a Illustrations for % change in apoptosis percentage in the pre-laser and post-laser 74 groups compared to control group. 18b Illustrations for % change in Plasma Bcl-2 in the pre-laser and post-laser groups compared 74 to control group. 18c Illustrations for % change in lymphocyte Bcl- 75 2 compared to control. 18d Illustrations for the plasma Bcl-2, lymphocyte Bcl-2, and % apoptosis in post-laser compared 75 to pre-laser group. 19 Mean plot for plasma Bcl-2 in all groups 76 20 Mean plot for lymphocyte Bcl-2 in all groups 76 21 Mean plot for Apoptosis percentage in all 77 groups 22 Correlation between age in years and Bcl-2 in 80 circulating lymphocytes in Control group 23 Correlation between age in years and CPK in 80 DMD patients (pre-laser) 24 Correlation between NO (µmol /L) and MDA 81 (nmol/L) after laser irradiation 25 Correlation between Bcl-2 in lymphocytes (nmol/ 2 x105 Cells) and apoptosis percentage 81 after laser irradiation 26 Correlation between apoptosis percentage 82 before & after laser irradiation Figures and Tables

Fig. Title Page 27 Correlation between Bcl-2 in lymphocytes (nmol/ 2 x105 Cells) before & after laser 82 irradiation 28 ROC of MDA 84 29 ROC of NO 85 30 ROC of plasma Bcl-2 86 31 ROC of lymphocyte Bcl-2 87 32 ROC of % of apoptosis 88 33 MDA ordinariness in the different groups 91 34 NO ordinariness in the different groups 91 35 Plasma Bcl-2 ordinariness in the different 92 groups 36 Lymphocyte Bcl-2 ordinariness in the 92 different groups 37 MDA ordinariness in the different groups 93

Figures and Tables

LIST OF TABLES

Table Title Page Statistical analysis for the differences in 1 CPK activity and age difference in 60 patients as compared to controls (t-test) Statistical analysis of plasma Cholesterol (mg/dL) and Triacylglycerols (mg/dL) 2 63 levels in control and DMD patients' pre- and post-laser irradiation). Statistical analysis of plasma Malondialdhyde (MDA) nmol/mL level, 3 Catalase activity and NO (µmol/L in 67 control and DMD patients (Pre- and post- laser irradiation) Statistical analysis of plasma Bcl-2 nmol/mL, lymphocyte BCL-2 nmol/ 2 x10 4 5 cell and apoptosis percentage in control 73 and DMD patients (Pre-and post-laser irradiation) Significant correlations between some 5 studied parameters in DMD patients and 79 control group (Pearson correlation). Cross tabulation showing statistical analysis (Chi-Square Tests “x2”) of all the 6 89 parameters ordinariness in the different groups (22 patients/group)

Introduction xii

Introduction

Duchenne muscular dystrophy (DMD) is a form of muscular dystrophy characterized by decreasing muscle mass and progressive loss of muscle function in male children. DMD occurs in approximately 1 out of 4,000 live born males and can either be inherited or occur spontaneously. A family history of Duchenne muscular dystrophy is a significant risk factor (Faulkner et al., 2008).

DMD is named after the French neurologist Guillaume Benjamin Amand Duchenne (1806-1875), who first described the disease in the 1860s (Grounds, 2008). The disease is usually diagnosed based on gait abnormalities at the age of 4-5 years. In some patients, neurological and cardiac symptoms may also appear by the early teens (Thrush et al., 2008). Further progression of muscle degeneration eventually leads to death in the early twenties as a result of respiratory or cardiac failure (Bertoni, 2008).

Duchenne muscular dystrophy is caused by a mutation of the dystrophin gene located at Xp21. Females can be carriers but generally do not experience the symptoms of the condition (Jazedje et al., 2009). Introduction xiii

The dystrophin protein is located beneath the cell membrane (sarcolemma) of the muscle cell (myofiber) and serves to link the contractile machinery (sarcomeres) and associated cytoskeleton to the extracellular matrix where collagens transmit the muscle force (Grounds, 2008). Absent or defective dystrophin results in myofiber fragility leading to breakdown (necrosis) that is repeated over time until formation of new muscle (regeneration) fails and the damaged skeletal muscle is replaced by fibrous or fatty connective tissue (Cyrulnik and Hinton, 2008; Matsumura et al., 2009).

Skeletal muscle is capable of complete regeneration due to stem cells that reside in skeletal muscle and non-muscle (circulating) stem cell populations (Narciso et al., 2007). However, in severe myopathic diseases such as DMD, this regenerative capacity is exhausted (Shi and Garry, 2006). This exhaustion could be explained by two plausible theories: oxidative stress (Sato et al., 2008) and replicative aging, that lead to increased rate of myofiber death (Abdel et al., 2007).

If a physician suspects DMD after examining the boy, he will use the creatine phosphokinase (CPK) test to determine Introduction xiv if the muscles are damaged. This test measures the amount of CPK in the blood. In DMD patients, CPK leaks out of the muscle cell into the bloodstream, so a high level (nearly 50 to 100 times more) confirms that there is muscle damage (Burdi et al., 2009).

During the 1990s and through the early years of the 21st century, many promising, sophisticated genetic techniques have been designed to ameliorate the devastating impact of muscular dystrophy on the structure and function of skeletal muscles. There is no known cure for Duchenne muscular dystrophy, although recent stem-cell research is showing promising vectors that may replace damaged muscle tissue (Faulkner et al., 2008). Meanwhile, the use of low energy laser irradiation, is a promising means to enhance both the survival and functionality of primary myogenic cells, by promoting the cell cycle in lymphocytes and inhibiting cell apoptosis (Shefer et al., 2008). A i m o f t h e w o r k

Aim of the work

The goal of this study was to evaluate some biochemical parameters in plasma of Duchenne Muscular Dystrophy patients as a diagnostic marker of the disease, such as creatine phosphokinase, nitric oxide, malondialdhyde, BCL- 2, percent of apoptosis in circulating lymphocytes, catalase, total cholesterol and triacylglycerol. In addition, elucidation of the ameliorative effect of laser irradiation on disorder associated with the disease. Review of literature 1

REVIEW OF LITERATURE Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease affecting all world populations equally, with an incidence of 1 in every 3,500 live male births (Emery, 1993; Friedrich et al., 2008). DMD was named in recognition of Dr. G. Duchenne de Boulogne from France, who was the first to attribute the signs and symptoms to a distinct familial disease entity about 150 years ago, originally describing the condition pseudo- hypertrophic muscular paralysis (Bogdanovich et al., 2004).

Clinical Progression of Duchenne and Becker Muscular Dystrophies: Initial physical signs such as muscle weakness are not generally observed until the child reaches 2–5 years of age and are typified by a waddling gait, difficulties in running and climbing stairs. In retrospect of the diagnosis, parents often also provide a history of delays in achievement of motor milestone. Subsequent onset of pseudo-hypertrophy of the calf muscles, proximal limb muscle weakness, a positive Gowers sign, or the child s use of arms to climb from a lying to standing position clinically suggest DMD. Review of literature 2

Progressive muscle wasting continues throughout life, initially affecting proximal muscles, with a decrease in lower limb muscle strength resulting in loss of ambulation by about 12 years of age. In the latter stages almost all skeletal muscles are severely involved, which in turn leads to additional problems such as joint contractures and progressive kyphoscoliosis (Blake et al., 2002; Bertoni, 2008).

The overall clinical course is unfortunately relentless. While supportive measures such as cord lengthening, spinal stabilization, orthoses, and mechanical long-term ventilation improve the quality of life of patients, death usually occurs in the late teens or early twenties, commonly from cardiac or respiratory causes (Bogdanovich et al., 2004, Jazedje et al., 2009).

Becker muscular dystrophy (BMD) is an allelic disorder that follows a similar, albeit milder DMD course distinguished by a later age at onset and slower rate of progression. A clinical continuum seems to exist between a mildly affected BMD patient and a severely affected DMD patient. Historically, the varied clinical course and milder phenotype led to reluctance in classifying these patients with Review of literature 3

DMD, until an elegant hypothesis concerning its allelic nature of the disorder was proposed by Becker and Kiener from Germany. More than 90% of BMD patients are still alive in their 20s, with some remaining mobile until old age. Both BMD and DMD patients can present with varying degrees of cognitive impairment, indicating that brain function is abnormal in these disorders (Emery, 1993; Bogdanovich et al., 2004).

At the cellular level both disorders involve the loss of skeletal muscle fibers, with marked degeneration. Eventually the regenerative capacity of the muscles is lost, and muscle fibers are gradually replaced by adipose and fibrous connective tissue, giving rise to the clinical appearance of pseudo-hypertrophy followed by atrophy (Emery, 1993; Bogdanovich et al., 2004).

Dystrophin Protein: Both DMD and BMD are known to be caused by mutations in the gene encoding dystrophin, resulting in the absence or expression of mutant forms of the protein. Dystrophin is the largest human gene located in chromosome Xp21 and spans up to 2.4 mega base pairs of DNA in length. The full length human dystrophin protein is composed of Review of literature 4

3685 amino acid residues with a molecular weight of 427 kD and is a subsarcolemmal component of the cytoskeleton

(Mital et al., 1998; Cyrulnik and Hinton, 2008; Matsumura et al., 2009).

Dystrophin shows structural homology with spectrin and α-actinin and contains four distinct domains; amino-terminal domain, helical-rod domain, C-terminus and a cysteine-rich domain (Figure 1). Dystrophin binds F-actin filaments at its amino-terminal domain and parts of the helical-rod domain. The C-terminus and a cysteine-rich domain interact with integral membrane proteins, including sarcoglycan, dystroglycans, syntrophin, and dystrobrevin, which are assembled together to form the dystrophin- associated protein complex (DAPC) (Figure 2). The DAPC provides a crucial structural and signaling link between the extracellular matrix (ECM) and the intracellular actin cystoskeleton across the sarcolemma (Rando, 2001; Guang-qian et al., 2006).

Deficiency of dystrophin expression affects formation of the DAPC and causes a disruption of the molecular bridge (Blake et al., 2002; Grounds, 2008). Review of literature 5

Mutation in Dystrophin Gene: The dystrophin gene is comprised of 79 exons, and the fully processed transcript is 14 kb (Nobile et al., 1997; Guang-qian et al., 2006). The dystrophin gene is expressed in a cell-specific and developmentally regulated manner controlled by seven independent promoters. Three of these regulate the expression of full-length isoforms present in brain, muscle and Purkinje cerebellar neurons respectively, whereas four intragenic promoters regulate expression of different short isoforms in various tissues (Figure 1) (Mital et al., 1998; Guang-qian et al., 2006).

In around 30% of cases the mutation arises spontaneously, and the remaining cases are inherited in an X-linked recessive manner. Therefore the disease affects the male gender, though in rare instances females may also show a similar phenotype if they carry X-autosomal translocations that disrupt the dystrophin gene (Mital et al., 1998; Guang- qian et al., 2006). Deletions of single or multiple exons are responsible for about 65% - 70% of DMD cases; the remaining has point mutations resulting from nonsense or frame-shift mutations (30%) or duplications (6%) (Nishino and Ozawa, 2002). Review of literature 6

Figure 1: Schematic displays of the structure of the dystrophin gene and protein. The domain composition of the various dystrophin proteins is indicated. The amino- terminal domain is followed by the spectrin-like rod domain, the cysteine rich (Cys), and the carboxy-terminal domain (CT). H: representing hinge regions (lower). Genomic organization of the dystrophin gene. The vertical lines representing the 79 exons. The arrows indicating the various promoters: in particular are brain, muscle, and Purkinje promoters and promoters for short isoforms (upper) (Blake et al., 2002).

Review of literature 7

Figure 2: The dystrophin-associated protein complex (DPC) in skeletal muscle. Dystrophin binds to cytoskeletal actin at its NH2 terminus. At its COOH terminus, dystrophin is associated with a number of integral and peripheral membrane proteins (Rybakova and Ervasti., 1997).

Review of literature 8

Dystrophin's Functions : Dystrophin deficiency has been related to the degeneration and insufficient regeneration of muscle fibers and extensive proliferation of connective tissue, mainly in limb and trunk muscles. The function of dystrophin in

-muscle is mystery (Niebrَj-Dobosz and Hausmanowa Petrusewicz, 2005). It has been suggested that: 1. Dystrophin supports sarcolemma against mechanical stress and stabilizes it in course of the contraction– relaxation cycle (Reynolds et al., 2008). 2. Dystrophin takes part in the regulation of intracellular calcium and the further cascade of calcium –related events (Boittin et al., 2006). 3. Dystrophin works in force and signal transduction (Gee et ,al., 1998; Niebrَj-Dobosz and Hausmanowa-Petrusewicz 2005). 4. Dystrophin influences the aggregation of neurotransmitter receptors (Kong and Anderson, 1999; Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005). 5. Dystrophin prevents excessive generation of reactive oxygen free radical species (Brown, 1995; Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005). Review of literature 9

Pathophysiology of Dystrophin Deficient Muscle:

1- Membrane Structure and Function: There is good evidence that dystrophin-deficient muscle is characterized by increased permeability to macromolecules flowing in and out of the cell and that this abnormal permeability is made worse by mechanical stress (Straub et al., 1997).

Mokri and Engel (1975), used electron microscopy to describe the ultrastructural features of DMD muscle. They noted absent or disrupted sections of sarcolemma overlying wedge-shaped areas of abnormal cytoplasm, the so-called delta lesions. This observation, subsequently confirmed, together with the high levels of several cytosolic proteins in the blood of patients with DMD, gave rise to the theory that the primary pathology of DMD muscle might be an abnormal fragility and leakiness of the cell membrane (Blake et al., 2002).

2- Creatine Kinase: Creatine kinase (CK) isoenzymes are found in cells with intermittently high-energy requirement. The tissues of mammals and birds have four distinct types of CK subunits Review of literature 10 and are expressed species specifically, developmental stage specifically, and tissue specifically. Isoenzymes of CK are found in different tissues, including the brain, but not in hepatocytes. The cytosolic M-CK (M for muscle) and B-CK (B for brain) subunits form dimeric molecules and thus give rise to the MM-, MB-, and BB-CK isoenzymes. All CK isoenzymes catalyze the reversible transfer of the γ-phosphate group of ATP to the guanidino group of creatine to yield ADP and Phosphocreatin (PCr) (Wyss and Kaddurah- Daouk, 2000). Typically, DMD patients are clinically normal at birth although serum levels of the muscle isoform of creatine kinase are elevated as a consequence of muscle fiber degeneration (Bogdanovich et al., 2004; Burdi et al., 2009).

3-Apoptosis: Apoptosis is a physiological cell suicide program that is critical for the development and maintenance of healthy tissues. Regulation of programmed cell death allows the organism to control the cell number and the tissue size and to protect itself from rogue cells that threaten homeostasis. Apoptosis requires coordinated action and fine tuning of a set of proteins that are either regulators or executors of the process. Cancer, autoimmune diseases, immunodeficiency Review of literature 11 disease, reperfusion injury and neurodegenerative disorders are characterized by disregulation of apoptosis (Barisic et al., 2003).

Morphology of apoptosis: During the early process of apoptosis, cell shrinkage and pyknosis are visible by light microscopy.With cell shrinkage, the cells are smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis (Kerr et al., 1972; Elmore, 2007).

Extensive plasma membrane blebbing occurs followed by separation of cell fragments into apoptotic bodies during a process called “budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. The organelle integrity is still maintained and all of this is enclosed within an intact plasma membrane. These bodies are subsequently phagocytosed by macrophages, parenchymal cells, or neoplastic cells and degraded within phagolysosomes. Macrophages that engulf and digest apoptotic cells are called “tingible body macrophages (Savill and Fadok, 2000; Kurosaka et al., 2003). Review of literature 12

Mechanism of apoptosis: The mechanisms of apoptosis are highly complex and sophisticated, involving an energy dependent cascade of molecular events (Figure 3). Research indicates that there are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. However, there is evidence that the two pathways are linked and that molecules in one pathway can influence the other (Igney and Krammer, 2002; Elmore, 2007).

There is an additional pathway that involves T-cell mediated cytotoxicity and perforin-granzyme dependent killing of the cell. The perforin/granzyme pathway can induce apoptosis via either granzyme B or granzyme A. The extrinsic, intrinsic, and granzyme B pathways converge on the same terminal, or execution pathway. This pathway is initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells. The granzyme A pathway activates a parallel, caspase-independent cell death Review of literature 13 pathway via single stranded DNA damage (Martinvalet et al., 2005).

The BCL-2 Family of Proteins: Bcl-2 family members are important regulators of the mitochondrial apoptotic pathway. These members include both anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, Bcl-w, A- 1) and pro-apoptotic proteins (Bax, Bak, Bid, Bad, Bik). The family was named after its discoverer isolated as a gene involved in B-cell lymphoma (Bcl). Bcl-2 family consists of three functional groups (Petros et al., 2001).

Members of the first group (Bcl-2, Bcl-xL, Bcl-w, etc.) are characterized by four conserved Bcl-2 homology (BH) domains (BH1–BH4). They have a C-terminal hydrophobic tail, which localizes the proteins to the outer surface of the mitochondrial, endoplasmic reticulum (ER) and nuclear membrane, with the bulk of the protein facing cytosol. They possess anti-apoptotic activity (Petros et al., 2001).

The second group consists of pro-apoptotic Bcl-2 family members having hydrophobic tails and BH1-BH3 domains (Bax, Bak, Bok, and Bcl-xS). The third group is represented by the Bcl-2 family members (Bik, Blk, BimL, Bid, Bad, Review of literature 14 etc.) having pro-apoptotic activity and sharing sequence homology only in the BH3 domain (Petros et al., 2001).

Several models have been proposed for the involvement of the Bcl-2 family members in apoptosis regulation. Bcl-2 family members exert their apoptosis-modulating effects at least by controlling the release of cytochrome c from the mitochondrial intermembrane space into cytosol (Elmore, 2007). Review of literature 15

Figure 3: The two major apoptosis signaling pathways converge at the level of the effector caspases. Moreover, there can be cross-talk between the extrinsic and intrinsic pathways. Caspase 8 cleaves the pro-apoptotic BCL2 family member BID, which in turn engages the mitochondria through BAX and BAK, leading to activation of caspase 9 and further activation of caspases 3, 6, and 7. The effector caspases also can activate caspase 8 and 10, forming a positive amplification loop. Where APAF1 is , apoptotic protease activating factor-1; BAK, BCL2 homologous antagonist/killer; BAX, BCL2- associated protein; BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; BCLXL, BCL2-like 1; BID, BH3- interacting domain death agonist; DR, death receptor; FADD, Fas- associated death domain; FLIP, FLICE (FADD-like interleukin 1β- converting enzyme) inhibitory protein; IAP, inhibitor of apoptosis protein; MCL1, myeloid cell leukemia sequence 1 (BCL2-related); PUMA, p53-upregulated modulator of apoptosis; SMAC/DIABLO: second mitochondria-derived activator of caspase/direct IAP binding protein (Elmore, 2007).

Review of literature 16

Necrosis and Apoptosis: Necrosis accounts for numerous destructive changes associated with pathological conditions such as energy deprivation, trauma and inflammation. Apoptosis was originally distinguished from necrosis on the basis of its morphology with a random DNA degradation (Kerr, 1972; Sandri et al., 1999).

Necrosis, the traditional description for cells that have ceased to demonstrate viability, is no longer believed to be the only term synonymous with a non-functional and non- viable cell. Necrosis has been associated with conditions that lead to cell membrane damage, osmotic imbalances, ion fluxes, cell swelling and death. The passive influx of Ca2+ is the hallmark mechanism of necrosis and expression of new RNA is not required for the necrotic process (Schwartz and Osborne, 1993).

Apoptosis and DMD: Degeneration of dystrophic skeletal muscle has generally been accepted to occur by necrotic pathways, but several studies suggest that during the phase of acute muscle degeneration in the mdx mouse, apoptosis precedes necrosis. After pioneering observation of single-strand DNA breaks in Review of literature 17 nuclei of a sub-population of cells within regenerating skeletal muscle of the dystrophic mdx mice, several authors have shown the occurrence of apoptosis in skeletal muscle of adult mdx mice. The presence of apoptosis in mdx mice is estimated between 0.5 and 2% of total nuclei (Sandri et al., 1999).

4-Calcium Homeostasis: Calcium homeostasis is critical to many aspects of muscle function (Berchtold et al., 2000), and early suggestions that it might be perturbed in dystrophin-deficient muscle stemmed from several observations. Hypercontracted fibers are the earliest morphological abnormality of DMD and were ascribed to persistently raise intracellular [Ca2+] (Cullen and Fulthorpe, 1975; Blake et al., 2002).

5- Proteolysis: Abnormal levels of several proteases are a feature of a wide variety of muscle diseases (Helliwell et al., 1998). Changes in protease expression or activity in DMD or muscle of mdx mice may therefore be nonspecific features, causally far removed from the primary pathological process

(Kumamoto et al., 2000). The consequences of elevated cytosolic calcium may include activation of proteases, such Review of literature 18 as calpain, which have an important role in the pathophysiology of dystrophin deficiency (Whitehead et al., 2006).

Turner et al (1988) observed a significant increase of Ca2+ in mdx myofibers. Proteinolysis occurred 80 % faster than in normal muscle, but this difference could be abolished by lower extracellular calcium concentrations (and perhaps 2+ therefore normalizing [Ca ]i). This result was subsequently confirmed, and the effect was shown to be blocked by leupeptin (a thiol protease inhibitor) (Maclennan et al., 1991; Blake et al., 2002).

6-Lipid Change: Abnormalities of lipids in muscles, non - muscle cell and serum have been investigated in various pathological conditions including myopathies. Intracellular droplets were detected in skeletal muscles by light microscopic technique and were clearly distinguished histochemically from the interstitial granules (mitochondria) by their staining properties (Ansari ,1984).

Kalofoutis et al (1977) reported that the concentrations of the major phospholipids and cholesterol were within normal Review of literature 19 limits in DMD red cells but there were alterations in the concentrations of individual phospholipids. These findings related to various abnormalities of lipids in muscular dystrophies clearly indicate a disturbance in the metabolism of lipids, their organization and accumulation in muscles (Ansari, 1984).

7-Oxidative stress:

Reactive oxygen / nitrogen species (ROS/RNS) and free radicals are constantly formed in the human body and removed by an antioxidant defense system. A certain amount of ROS / RNS production is, in fact, necessary for proper health; for example ROS / RNS help the immune system to eliminate microorganisms. In healthy individuals, the generation of ROS / RNS appears to be approximately in balance with antioxidant defense. An imbalance between ROS / RNS and antioxidant defenses in favor of the former via excessive production of ROS / NOS, loss of antioxidant defenses, or both, has been described as oxidative / nitrosative stress (Dalle-Donne et al., 2006).

Oxidative stress in human biology comes from a variety of sources. In aerobic cells, incomplete reduction of oxygen Review of literature 20

• in the mitochondrial electron transport chain releases O 2⎯ into the cytosol; 5% of molecular oxygen consumed may be • converted to O 2⎯by this way. Free transition metal ions like iron and copper are strong catatalysts for oxidatation reactions in the presence of hydroperoxides. The superoxide anion interacts with these free transition metal ions to produce the highly reactive hydroxyl radical (OH•).Macrophages and neutrophils produce also ROS such as Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) as a means of bacterial killing (Yücel et al., 2006).

Myeloperoxidase (MPO) is a heme containing protein and the only human enzyme known to generate HOCl. Phagocytes, including neutrophils, monocytes and macrophages contain membrane-bound NAD (P) H oxidases • generating O 2⎯.upon activation. Like phagocytes, endothelial cells, vascular smooth muscle cells and advential fibroblasts contain a membrane - bound NAD(P)H oxidase which generates O•⎯2 via reduction of molecular oxygen (Heymes et al.,2003).

Human body controls and survives the continuous ROS production due to a delicate balance between cellular systems generating the various oxidants and those maintaining the Review of literature 21 antioxidant defense mechanisms. There are three antioxidant defense mechanisms existing inside (intracellular) and outside (extracellular) the cells and in membranes. Intracellular defense mechanisms include the enzymes superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (Gpx) (Yücel et al., 2006).

These enzymes eliminate toxic reduction intermediates of oxygen inside the cell, prevent radical formation and so • restrict oxygen toxicity. SOD removes O2 ⎯ catalytically by • promoting the dismutation of O2 ⎯ to H2O2 and O2; catalase removes H2O2 when present in high concentrations; Gpx removes H2O2 when present in low concentrations and can remove organic hydroperoxides by conversion of reduced glutathione (GSH) to oxidized form (GSSG); and GR substitutes the GSSG for the reaction of Gpx. Extracellular defense mechanisms include plasma antioxidants such as albumin, bilirubin, urate, ceruloplasmin and ascorbic acid, and extracellular forms of SOD and Gpx (Yücel et al., 2006).

Defense mechanisms in membranes include vitamin E, betacarotene and coenzyme Q. Vitamin E (α-tocopherol) is a very effective, lipid-soluble, chain-breaking antioxidant. Beta-carotene is also a lipid-soluble antioxidant and plays a Review of literature 22 role as a radical scavenger and singlet oxygen quencher. These lipid-soluble antioxidants have also antioxidant effects in lipoproteins. Coenzyme Q is another membrane antioxidant in addition to its major role in cellular energy metabolism (Yücel et al., 2006).

Sequalea of Free Radicals' Generation: Lipid peroxidation Lipids are most susceptible macromolecules and are present in the plasma membrane in the form of polyunsaturated fatty acids (PUPA). ROS attack PUPA in the cell membrane leading to a chain of chemical reactions called lipid peroxidation. The reaction occurs in three distinct steps- initiation, propagation and termination. During initiation, the free radicals react with fatty acid chain (LH) and releases lipid free radical. This lipid radical further reacts with molecular oxygen to form lipid peroxyl radical. Peroxyl radicals again react with fatty acid to produce lipid free radical and this reaction is propagated. During termination, the two radicals react with each other, and the process comes to an end. This process of fatty acid breakdown produces hydrocarbon gases (ethane or pentane) and aldehydes (Agarwal and Prabakaran, 2005). Review of literature 23

Oxidative stress and Dystrophinopathies:

Studies strongly support the notion of the importance of

oxidative stress in dystrophinopathies (Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005; Tidball and Wehling- Henricks, 2007). There is increasing evidence that the degenerative processes in dystrophic muscles may be due to oxidative stress which can contribute to the necrotic process (Mendell et al., 1971). The significance and precise extent of the oxidative stress contribution is poorly understood

;Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005) Tidball and Wehling-Henricks, 2007). The increased action of oxidative stress in Duchenne muscular dystrophy is indicated by: 1. Increased excretion of 8-hydroxy-2’-deoxyguanosine indicating oxidative damage of DNA (Rodriquez and Tarnopolsky, 2003). 2. Changes in proteins, enhanced lipid peroxidation and

induction of antioxidant enzymes (Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005). 3. Similarities between changes in Duchenne muscular dystrophy and such conditions as ischemia, exhaustive Review of literature 24

exercise, and vitamin E deficiency (Niebrَj-Dobosz and Hausmanowa-Petrusewicz, 2005) 4. Increased sensitivity of dystrophin-deficient cells to injury from oxidative stress (Degl’Innocenti et al., 1999). 5. Lipofuscin accumulation in dystrophic muscle (Nakae et al., 2004).

Nitric Oxide (NO):

NO is a small gaseous molecule rather soluble in hydrophobic phases. It is a free radical with an odd number of electrons. It is colorless and paramagnetic and unstable. In the presence of oxygen and water it oxidizes to nitrites and nitrates. Its half–life in blood is very short (seconds) and cannot be transported away from its source of synthesis (Gustavo et al., 2005).

Biosynthesis of Nitric Oxide:

NO is produced by enzymes known as NO synthase (NOS), of which there are three recognized isoforms (Alderton et al., 2001). Endothelial and neuronal NOS (eNOS or NOS3 and nNOS or NOS1) are expressed constitutively in endothelial cells and neurons, respectively, and are sometimes collectively referred to as constitutive or Review of literature 25 cNOS. eNOS is membrane bound calcium dependent and associated with membrane structures known as caveolae (Kashiwagi et al., 2002).

eNOS is activated by shear stress at the endothelial cell/blood interface and produces NO, which diffuses away from the vessel lumen and acts to relax vascular smooth muscle or into the vessel lumen where it acts to inhibit platelet aggregation and leukocyte adhesion to the vessel wall. Inhibition of eNOS causes an increase in blood pressure, increased platelet aggregation and increased leukocyte adhesion in venules. eNOS has now been found in other tissues including lymphocytes. nNOS is largely found in neurons, where its role is producing NO as a neurotransmitter. It is also found in mast cells and most probably in neutrophils (Kashiwagi et al., 2002).

The third isoform (iNOS or NOS2) is induced rather than constitutive and was initially identified in macrophages, but has now been found in neutrophils and platelets (Sethi and Dikshit, 2000). iNOS synthesis is induced by various signals, particularly those related to inflammation. All isoforms of NOS metabolize arginine to citrulline and NO in the presence of adequate levels of a cofactor, Review of literature 26 tetrahydrobiopterin. It has been suggested that NOS may also produce NO– or even superoxides in the absence of appropriate cofactor or substrate (Gilchrist et al., 2003).

There are many systems to produce NO, while it is more difficult to scavenge it. Many classes of NO donors are described. For example Nitrosothiols are supposed to be important in the storage, transport and delivery of NO. Since nitrosothiols are important NO releasing molecules, there is a great interest to synthesize novel molecules with good pharmacokinetic properties. In particular nitrosothiols connected to a glucose molecule have promising proprieties (Wang et al., 2002).

Effector Mechanism of NO:

NO cause vasodilatation in vascular smooth muscle and inhibits platelet aggregation by binding to a haem-iron associated with cytoplasmic guanylate cyclase, increasing production and intracellular levels of cyclic-GMP (Ignarro, 2002). In skeletal muscle, NO increases both blood flow (Clark et al., 2003) and glucose uptake (Higaki et al., 2001). NO may also alter the activity of proteins by binding to cysteine residues to form S-nitrosothiols (Sun et al., 2003). Review of literature 27

Circulating nitrosothiols may act similarly following release of NO from the carrier thiol either at the cell membrane or within the cell, also may act directly by trans- nitrosation of NO from the free nitrosothiol to the target protein. A possible third effector mechanism is the formation of peroxynitrite (OONO–) from NO and superoxides, which may then cause nitrosation or oxidation of other molecules such as lipids or tyrosine residues (Wallis, 2005).

NO has disparate roles during infection. NO is toxic to infectious organisms but can also react with superoxide to – form NO intermediates, such as peroxynitrite (NO3 ), that activate the host stress response and may cause tissue injury (Nabeyrat et al .,2003).

Metabolism of NO in Blood:

NO may be present in the blood in at least two active forms. Firstly, in aqueous form as a dissolved gas, as produced directly by endothelial cells. Secondly, in combination with sulfydryl (thiol) groups either of single amino acids such as cysteine of small cysteine-containing peptides such as glutathione, or of cysteine-containing proteins such as albumin (Stamler et al., 1992). These forms Review of literature 28 are collectively called nitrosothiols or RSNOs, where R represents the carrier molecule. Other forms of NO, such as OONO– or the nitroxyl anion (NO–), may also have biological activity (Crawford et al., 2003).

Role for Nitric Oxide in Muscle Repair: Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence has been presented that nitric oxide (NO) and hepatocyte growth factor (HGF) mediates satellite cell activation. (Wozniak and Anderson, 2006).

Nitric oxide and apoptosis: Among signaling qualities, NO affects cellular decisions of life and death either by turning on apoptotic pathways or by shutting them off (Brune, 2003).

First indications on NO-evoked apoptosis appeared in 1993 (Albina et al., 1993). Apparently, the threshold for a proapoptotic triggering event of NO varies considerably from one cell to another. Mitochondria comprise a target for NO and there is accumulating evidence that inhibition of Review of literature 29 respiration may contribute to the proapoptotic effect of NO by membrane potential reduction, transition pore opening and release of cytochrome c (Moncada and Erusalimsky, 2002). There is consolidation that a caspase-3-like group (caspase- 3 and -7), caspase-2 and caspase-9 are central components in a cascade triggered by NO (Moriya et al., 2000).

The antiapoptotic role of NO was reported by Boyd and Cadenas (2002). Some studies stressed over caspase inhibition by NO as a mechanism to block cell death. There is unquestionable evidence that the active site of caspases can be S-nitrosated in cell free studies, which results in loss of enzyme function, while activity can be regained by an excess of reducing equivalents (Daiber et al ., 2002).

Therapeutic strategies for DMD:

In general, therapeutic strategies for DMD can be classified into three groups based on their approach: (a) gene therapy, including viral, plasmid, and oligonucleotide-based approaches, (b) cell therapy (myoblast and stem cell), and (c) pharmacological (Faulkner et al., 2008). The first two approaches have the advantage that they would correct both Review of literature 30 the primary and secondary problems associated with DMD, especially if initiated early. The pharmacological approaches, with few exceptions use a variety of means to target specific components of the pathology (Noguchi et al., 2003).

The vast majority of pharmacological strategies for DMD do not endeavor to correct the underlying genetic defect that causes DMD. Instead, these approaches involve the use of drugs/molecules to improve the disease phenotype by targeting specific components of the pathophysiology such as increasing muscle progenitor commitment or differentiation, maintenance of calcium homeostasis, or upregulation of genes encoding compensatory proteins such as utrophin (Bogdanovich et al., 2004).

LASER

The word laser is an acronym derived from the phrase “light amplification by stimulated emission of radiation.” A laser-equipped device can generate a high-intensity light that is monochromatic, unidirectional, and parallel. These unique characteristics make the laser useful for commercial and medical applications .The basic laser device consists of 3 components: (1) an active medium, or lasing medium; (2) an Review of literature 31 optical cavity, or resonator; and (3) an energizing source, or pump. The active medium in lasers may be a solid, liquid, or gas. Different active media emit different energies or wavelengths of light. However, they all operate with the same basic principles (Synsitizer and Young, 1968).

The beam of laser energy is highly coherent, polarized, focused and monochromatic. Its selectively destructive quality was first used in ophthalmology in the early 1960s, although the principle on which lasers are based was postulated by Einstein in 1917. Non-invasive or soft lasers were introduced into medicine in the 1980s and, since then, have gained wide application in many areas of health care. Non-invasive, low-power lasers with an output up to 500 mW have been reported to have stimulatory, anti- inflammatory and analgesic effects (Koutna et al., 2003).

The transmission of laser radiation in tissues is related to its wavelength. Infrared laser radiation shows a higher penetration into tissues then the laser light of the red region of the visible spectrum. Therefore, the latter has proved useful in treatment of skin and mucosal disorders (Koutna et al., 2003).

Review of literature 32

LASER TYPES:

Lasers may be classified according to the type of active medium, excitation mechanism, or duration of laser out put.Classification by active medium is utilized here (Wright and Fisher, 1993).

1- Semiconductor lasers: The active medium of a semiconductor (injection) laser is the junction between two types of semiconductor materials. A semiconductor is a material whose electrical conductivity is greater than that of an insulator, such as glass or plastic, but is less than that of a good conductor, such as silver or copper. Gallium arsenide is an example of a material used in the manufacture of a semiconductor laser (Fan et al., 1996).

2-Solid crystalline and glass lasers:

Another important family of lasers contains solid crystalline or glass material as an active medium. Ruby and neodymium are two common examples of solid lasers with widespread industrial applications. Ruby is crystalline aluminum oxide in which some of the aluminum ions in crystal lattice have been replaced by chromium ions. These chromium ions are the active elements in the ruby laser Review of literature 33

(Coleman, 1995).

3-Liquid dye lasers:

In this case a liquid is the active medium. Complex organic dyes, such as rhodamine 6G and disodium fluorescein, dissolved in an alcohol solution, are specific examples. The dye solution is circulated by a pump through a glass or quartz tube. Liquid lasers frequently are excited by means of a powerful pulse of light. (Hamlin et al., 1991).

4- Gas lasers:

A large and important family of lasers utilizes a gas or gas mixture as the active medium. Excitation usually is achieved by current flow through the gas. The most popular type of gas laser contains a mixture of helium (He) and neon (Ne) gases (Coleman, 1995).

Fields of Practice:

Laser technology is widely used in medical research, diagnostics, and treatment. The clinical efficacy of laser therapy is well known, and lasers are used in many fields and settings, e.g. Dentistry (Ishikawa et al., 2004), Dermatology (Tanzi et al .,2003), Oncology (Dima et al .,2002), Review of literature 34

Ophthalmology (Arevalo, 2004) and Plastic surgery (Jacobson et al .,2000).

Applications of laser:

Laser scanning cytometry: Lasers have an application in the analysis of cells and their compartments. Laser scanning cytometry (LSC) is a new technique that has a significant advantage over other cytometric methods because of its use of the minimal sample volume and its ability to connect fluorescence and morphologic data (Da Silveira et al., 2003)

Laser tissue welding: Laser tissue welding is an alternative method for closure. This technique is becoming important in many surgical specialties, including urology, cardiothoracic surgery, plastic surgery, and neurosurgery (Poppas et al., 1998).

Low-level laser therapy: Review of literature 35

The low-intensity laser, with energy of less than 10-100 W, has been scrutinized as a possible therapeutic tool (Posten et al., 2005).

Helium-Neon (He-Ne) laser (632.8 nm) is a low-energy laser emitting radiation at visible light spectrum. As the thermal effects associated with low-energy, laser irradiation was extremely minute (Babapour et al., 1995).

Low-power laser therapy is used by physiotherapists (to treat a wide variety of acute and chronic musculoskeletal aches and pains), by dentists (to treat inflamed oral tissues and to heal diverse ulcerations), by dermatologists (to treat edema, indolent ulcers, burns, and dermatitis), by rheumatologists (to relieve pain and treat chronic inflammations and autoimmune diseases), and by other specialists, as well as general practitioners. Laser therapy is also widely used in veterinary medicine (especially in racehorse-training centers) and in sports-medicine and rehabilitation clinics (to reduce swelling and hematoma, relieve pain, improve mobility, and treat acute soft-tissue injuries). Lasers (coherent-light sources) and noncoherent light (light-emitting diodes (LEDs)) are applied directly to the respective areas (e.g., wounds, sites of injuries) or to Review of literature 36 various points on the body (acupuncture points, muscle- trigger points) (Karu, 2003).

The physiologic effects imparted by low-energy lasers were attributed to their direct biostimulation on exposed cells. In vitro studies have revealed that He-Ne laser irradiation stimulates the release of various growth factors and results in the proliferation of fibroblasts and keratinocytes (Yu et al., 1996; Shefer et al., 2008).

Subject and Method 37

SUBJECTS AND METHODS

A-Subjects: 1. Patients: Participants for this study were recruited at the outdoor clinic of Pediatric Hospital, Faculty of Medicine, Cairo University. All families gave their written informed consent after a thorough explanation of the study aim to both the parents and the children. Group of patients with Duchenne Muscular Dystrophy were selected on the basis of the following eligibility criteria:

Inclusion criteria: • Male sex aged 7–15 years. • Diagnosis of muscular dystrophy was based on clinical onset of progressive weakness before 5 years of age. • Muscle biopsy that was deficient in dystrophin and compatible with DMD

Exclusion criteria: • Children with acute pathology • Cardiac or respiratory insufficiency • Children who were taking corticosteroids Subject and Method 38

After selection, 22 patients were classified into two subgroups. Group I: Irradiated blood with He: Ne laser

Group II: Non-irradiated blood

Control: 22 children were selected to be age and socioeconomic matchers, who were carrying out ordinary blood checkup.

2. LASER Irradiation:

Whole blood DMD samples were irradiated with 2.5 J/cm2 by He-Ne laser at wave length 632.8 nm and power output 10 mW provided by the National Center for Radiation and Technology (NCRRT) (El Batanouny and Korraa, 2003).

3. Blood Specimen:

-Whole blood: 10 ml of venous blood were collected in tubes containing 4µl of 1.2mg/ml of potassium ethylene diamine tetracetic acid (EDTA), then mixed well by inversing the tubes up and down several times, shaking was avoided. -Lymphocytes: Whole blood was allowed to stand for one hour to separate the plasma from packed cells. Then the Subject and Method 39 plasma was transferred into dry tube for centrifugation at 3000rpm for 15 minutes. Two layers formed after centrifugation, plasma portion and lymphocytes .The plasma was separated from lymphocytes in dry Ependorff tube.

-Blood irradiation: 2 ml of whole blood was distributed in 96 well tissue culture plates. To minimize cross-irradiation between cells, at least two empty wells separated each experimentally irradiated well. Every treatment was investigated in duplicate. Then the plate was incubated at 37cº for 2 hour after the incubation the plasma was separated to extract the lymphocytes.

4-Chemicals:

Chemicals used in the present study were of high analytical grade and purchased from: Biosource (Germeny), Spinreact (Germeny), Sclavo (Italy), Biocon (Germany), Merk (Germeny) and Sigma (USA).

Subject and Method 40

The following determinations were carried out:

• Plasma Creatine Phosphokinase • Cholesterol Concentration • Triacylglycerols Concentration • Malondialdehyde Level • Catalase activity (CAT) in plasma • plasma nitric oxide • BCL-2 in plasma and lymphocytes • Apoptosis percentage in circulating lymphocytes

B- Methods:

1-Detrmination of Creatine Phosphokinase (CPK) activity:

This method is carried out by Meiattini et al., 1978a using Creatine Phosphokinase Kit from Sclavo Company.

Reagents: 1-Buffer: It contains 10 mM imidazole acetate pH 6.75, 10mM magnesium acetate, 20mM D-glucose, 6mM potassium fluoride and 0.9g/L sodium azide. Subject and Method 41

2-Enzymes: 30mM creatine phosphate, 2mM ADP, 2mM NADP (yeast), 2mM AMP, 2U/ml hexokinase, 2U/mlG-6- PDH, 20mM N-acetylcysteine and 20mM EDTA .

Procedure: 1- 25 ml of buffer was added to enzymes (one vial) .Mixed gently and waited for 2 minutes before using reconstituted reagent is stable for 24 hours at 2-80c 2- 1ml of reconstituted reagent was added to 20 µl of serum then the tubes was mixed gently and incubated at 370c for 2 minutes. 3- After the incubation the absorbance was read at 340nm. The reading was repeated at 30 seconds or 1 minute intervals then the mean ∆A /min was determined.

Calculation: The formula for the calculation is ∆A / min X 8200. Explanation of the formula Vt X 1000 ∆A / min x = U/L M.E.C X L.P x Vs Where: U/L= International unit per unit ∆A / min =Change in absorbance per minute Subject and Method 42

Vt = Total volume of reaction (ml) 1000= Convert concentration to liter M.E.C= micromolar extinction coefficient of NADPH 6.22cm2µmole at 340 L.P =light path Vs= volume sample in final reaction mixture (ml).

2-Determination of Cholesterol Level

Cholesterol concentration was determined according to the method of Meiattini et al., 1978b. Using cholesterol Kit from Spinreact Company.

Reagents: R1: PIPES pH 6.9 90mmol/L Buffer Phenol 26mmol/L R2: Cholesterol Esterase (CHE) 300 U/L Enzymes: Cholesterol Oxidase (CHOD) 300U/L Peroxidase (POD) 250U/L 4- Aminophenazone 0.4mmol/L

Cholesterol aqueous primary standard 200mg / dl

Subject and Method 43

Procedure: Preparation of working reagent (WR): The content of one vial R2 enzymes was dissolved in one bottle of R1 buffer. The contents were dissolved by capping and mixing. 1- Appropriate set of test tubes was labeled 3 tubes for each sample, the standard, the blank and the test. 2- 1ml of the working solution was added in all the tubes. 3-10 µl of the standard was added in the standard tube. 4-10 µl of the sample was added in the sample tube. 5-The tubes was mixed and incubated for 5 min. At 37 °c or 10 min. at room temperature 6-The absorbance (A) of the samples and standard was read against the Blank at 505 nm. The color is stable for at least 60 minutes.

Calculation:

A sample × Standard conc. = cholesterol conc. (mg/dl)

A standard

Subject and Method 44

3- Determination of Triacylglycerols level:

Triacylglycerols concentration was determined according to the method of Bucolo and David, 1973.Using Triacylglycerols Kit from BIOCON Diagnostics.

Reagents:

R1: pipes Buffer pH 7.2 50mmol/L p- Cholesterol 2mmol/L

R2: Lipoprotein lipase 150000U/L Glycerokinase 800U/L Glycerol- 3- p- oxidase 4000U/L Peroxidase 440U/l 4- Aminoantipyrin 0.7mmol/L ATP 0.3mmol/L

R3: Glycerol equivalent to a concentration of 200mg/dl Triacylglycerols. Preparation of working solution: The contents of enzyme reagent R2 was dissolved with the corresponding volume of buffer R1.

Procedure: 1- Appropriate set of test tubes was labeled 3 tubes for each sample, the standard, the blank and the test. Subject and Method 45

2- 10 µl of the standard was added in the standard tube. 3- 10 µl of the sample was added in the sample tube. 4- 1000 µl of the working solution was added to all tubes. 5- The mixture was measured after incubating at 37°C for 5 minutes or 10 minutes at 20° C to 25°C within 60 minutes read absorbance of sample against reagent blank was used at 546 nm.

Calculation:

A sample × Standard conc. = Triacylglycerols conc. (mg/dl)

A standard

4-Determination of Malondialdehyde (MDA) Level:

The determination of lipid peroxidation was carried out according to Yoshioka et al., (1979).

Principle: This method is based on the measurement of malondialdehyde (MDA) as the main end product of lipid peroxidation by the use of thiobarbituric acid.

Subject and Method 46

Reagents: 1. Trichloroacetic acid 20% 2. Thiobarbituric acid 0.67% 3. n-Butyl alcohol (Analar) 4. 1, 1, 3, 3-tetra-ethoxypropane (Analar)

Procedure: 1. 0.5 ml of plasma was taken with 2.5 ml of 20 % trichloroacetic acid in 10 ml centrifuge tubes. 2. 1 ml of 0.67% thiobarbituric acid (T.B.A) was added, tubes were shaken. The tubes were put for 30 minutes in boiling water bath followed by rapid cooling. 4ml of n- butyl alcohol were added to each tube and shaken; the mixture was centrifuged at 3000 r.p.m. For 10 minute. 3. The n-butyl alcohol layer that contains malondialdehyde (MDA) was taken into separate tube and its color intensity was measured by Biometer spectrophotometer at 535 nm. 4. (1, 1, 3, 3 tetraethoxy propane), serial dilutions of standard MDA were prepared to set up a standard curve for lipid peroxidation (5- 30 nmol / ml).

Subject and Method 47

Calculation: Malondialdehyde concentration was determined by nmol of malondialdehyde / mL

0.7

0.6

y t

i 0.5 s n

e 0.4

d 0.3 cal i t p 0.2 O 0.1

0

0 5 10 15 20

Conc. of standard MDA

Figure 4: Standard curve of MDA

Subject and Method 48

5-Determination of catalase activity (CAT) in plasma:

The assay is based roughly on the method of Sinha, (1972).

Principle

The dichromate/acetic acid reagent can be thought of as a stop bath for catalase activity .As soon as enzyme reaction mixture hits the acetic acid, its activity was destroyed; any hydrogen peroxide which hasn’t been split by the catalase will react with the dichromate to give a blue precipitate of perchromic acid .This unstable precipitate was then decomposed by heating to give the green solution .This green color was measured by spectrophotometer at 570nm.

Reagent • 5% of aqueous solution of potassium dichromate • 0.2 M hydrogen peroxide • Glacial acetic acid

• 0.1M phosphate buffer (Na2HPO4)

Procedure 1. 150 ml of glacial acetic acid was added to 50 ml of 5% aqueous solution of potassium dichromate slowly. 2. 0.2 M of hydrogen peroxide was prepared in 250 ml of 0.1 M phosphate buffer. PH 7.0 (Enzymes are Subject and Method 49

generally the most stable in a solution that buffered near that of their original environment). 3. 5 ml of hydrogen peroxide and phosphate buffer was added to 1ml of plasma. 1ml of mixture was taken. 2ml of dichromate/acetic acid was added to the mixture. When a blue color formed, tubes were heated for 10 minutes in a boiling water bath to decompose the blue precipitate .This will leave a green a solution of chromic acetate. 4. Solution was cooled to room temp and centrifuged for 10 minute, and the absorbance was measured at 570 nm in spectrophotometer. 5. Preparation of Standard Curve The standard curve is carried out by using 6 different test

tubes containing increasing amount of H2O2 (10 up to 160 micromoles). To each of these 2ml of the dichromate/acetic acid was added. When a blue color formed in each, the following steps were done: a. Each test tube was heated for 10 minutes in a boiling water bath to decompose the blue precipitate .This will leave a green solution of chromic acetate. b. Solution was cooled to room temp. Enough H2O was added to each tube to make the volume up to 3ml. Subject and Method 50 c. 3ml of the test tubes contents were transferred to a cuvette and was measured the absorbance at 570nm in spectrophotometer. using a cleaned cuvette for each tube.

From the standard curve the reminder of H2O2 was determined. Catalase activity was determined according to the following equation.

Ab-At 1 1 catalase activity = x Conc.of standard x x

Ab t n Where: At= Absorbance of the test sample

Ab= Absorbance of the blank sample n= Dilution factor t = time of incubation Catalase activity is in term of unite of catalase activity that consumed 1µmole of H2O2/min.

Figure 5: Standard curve of hydrogen peroxide Subject and Method 51

6-Determination of Plasma Nitrite:

Nitric oxide was determined in plasma according to the method described by Miranda et al., 2001

Principle Nitric oxide is relatively unstable in the presence of molecular oxygen ,with an apparent half life of approximately 3-5 seconds and is rapidly oxidized to nitrate and nitrite totally designated as NOx .A high correlation between endogenous nitric oxide production and nitrite /nitrate (NOx) levels has been established .The measurement of these levels provides a reliable and quantitative estimate of nitric oxide output in vivo .The assay determines total nitrite/nitrate level based on the reduction of any nitrate level to nitrite by vanadium followed by the detection of total nitrite (intrinsic + nitrite obtained from reduction of nitrate by Griess reagent) .the Griess reaction entails formation of chromophore from the diazotization of sulphanilamide by acidic nitrite followed by coupling with bicycle amines such as N-1-naphthylethylenediamine .The chromophoric azo derivative can be measured calorimetrically at 540 nm .

Subject and Method 52

Reagents 1. Absolute ethanol: used for de-proteinization of the sample.

2. Vanadium trichloride (VCL3): saturated solution of VCL3 was prepared by dissolving 0.4 gm in 50 ml of 1M HCL .the blue solution was stored in the dark at 40c for less than 2 weeks, development of lighter blue color indicated oxidation, after which the solution was discarded. 3. Griess reagent: i) N-1-naphthyl ethylenediamine dihydrochloride (NEDD) 0.1 %( w/v) in distilled water. ii) Sulfanilamide: 2% (w/v) in 5% HCL (v/v) complete dissolution of each solution may require stirring and heating after which each solution was filtered to remove trace particulates .Both solutions are stable for several months when stored in dark at 40c and must be discarded if become colored .Both solutions used to prepare the Griess reagent can be combined but prepared as separate solutions it is preferable also that they can be added as separate solutions in the reaction medium or they may be mixed immediately before addition . Subject and Method 53

4. Standard nitrite solution: sodium nitrite standard was used to prepare serial dilutions ranging between 1.6-100 µM .A standard curve was constructed using serial dilutions of nitrite.

Procedure: 1-In Eppendorf tube, 0.5 ml cold absolute ethanol was added to 250 µL plasma then left for 48 hours in the refrigerator to attain complete protein precipitation followed by centrifugation at 15,000 rpm for 1 hours using cooling centrifuge. To 250 µL of the obtained supernatant, 250 µL

VCL3 was added followed by rapid addition of 125 µL Sulfanilamide and 125 µL (NEDD). 2- The mixture was incubated at 37 0c for 30-45 minutes then cooled and the absorbance of the pink colored chromophore was measured at 540 nm using double beam spectrophotometer against a blank treated in similar manner as to the test but using 250 µL distilled water instead of the sample. 3-The standard was treated exactly as supernatant and measured against a blank reagent containing 250 µL distilled water.

Subject and Method 54

Calculation The level of total nitrite /nitrate (NOx) in the plasma samples was expressed as µM and was calculated using the following standard curve:

1.6 1.4

1.2 y t i s 1 n e d

l 0.8 a c i

t 0.6

Op 0.4 0.2 0 0 102030405060 Concentration of sodium nitrite

Figure 6: Standard curve of sodium nitrite

Subject and Method 55

7-Determination of plasma and lymphocytes

Bcl-2:

This method is carried out according to Giuliani et al., 2006 .Using Biosource kit.

Reagent: • Bcl-2 standard: Lyophilized. • Standard dilutent buffer: Contains 15mM sodium azide; 25 ml per bottle. • Bcl-2 antibody coated wells, 96 wells per plate. • Rabbit anti Bcl-2 (detection antibody) contains 15 mM sodium azides 11ml bottle. • Antirabbit IgG–Horseradish Peroxidase (HRP) concentrate (100x) contains 3.3mM thymol.. • wash buffer concentrate 25x; 100 ml per bottle • Stabilized chromogen, tetramethylbenzidine (TMB); 25 ml per bottle. • HRP dilutent : contains 3.3mM thymol; 25 per bottle • Stop solution; 25ml per bottle. • Plate covers, adhesive strips.

Subject and Method 56

Procedure: 1. 100 µ L of a monoclonal antibody specific for Bcl-2 has been coated onto the wells of the microtiter strips provided. 2. 100 µ L of samples, including a standard containing Bcl-2, control specimens and unknowns were piptted into these wells then incubated for 1 hour .During the first incubation, the Bcl-2 antigen binds to immobilized (capture) antibody. 3. The well was aspirated and washed four times. 4. 100 µ L of rabbit antibody, specific for BCL-2, was added to the wells then incubated for 1 hour, during the second incubation this antibody serves as a detector by binding to the immobilized Bcl-2 protein captured during the first incubation. 5. The well was aspirated and washed four times. 6. After removal of excess detection antibody, 100 µ L of Horseradish Peroxidase labeled Anti- rabbit IgG (anti-rabbit IgG-HRP) , was added and incubated for 30 minutes This binds to the detection antibody to complete the four member sandwich .Then The well was aspirated and washed four times to remove all the excess anti–rabbit IgG HRP. Subject and Method 57

7. 100 µ L of a stabilized chromogen was added and incubated for 30 minutes at room temperature. 8. 100 µ L of stop solution was added then read at 450 nm by ELISA.

Calculation: From standard curve the concentration was calculate.

3

2.5

y 2 t i s

den 1.5 cal i t p

O 1

0.5

0 0246810 Conc.of BCL-2 standard nmole/mL

Figure 7: Standard curve of Bcl-2

Subject and Method 58

8-Determination of apoptosis in circulating lymphocytes:

This is carried out according to Ioannou and Chen, (1996).

Principle: Viable cells take up diacetyl fluorescein and hydrolyze it to fluorescein to which the cell membrane of live cells is impermeable but dead cell unable to hydrolyze it. Non viable cells may be stained with ethidium bromide or propeidium iodide (PIO4) and will fluorescein red. Viability is expressed as the percentage of cells fluorescing green.

Reagents: Phosphate buffer saline (PBS): 8 gm sodium chloride (NaCl), 0.2 gm potassium chloride (KCl), 1.44 sodium dibasic phosphate (Na2HPO4) and 0.24 gm potassium dihydrogen phosphate (KH2PO4) were dissolved in 900 ml distilled water. PH was adjusted to 7.4 with 1M HCl, and water was added to a final volume of 1L Stock propidium iodide (PIO4): 0.05gm in 10 ml distilled water. Working PIO4:100 µl stock + 1ml phosphate buffer salin (PBS). Subject and Method 59

Stock RNase: 0.025 gm in 10 ml distilled water Working RNase: 100 µ stock + 1ml PBS Procedure: 1. Lymphocytes were subjected to histopaque separation, incubated with 1% fluorescene diacetate for 15 minutes at 370c. 2. The lymphocytes were fixed with 70% ethanol for 1hour. 3. The pellet with 50 µl PBS containing RNase and 50 µl of propidium iodide was incubated for 15 minutes in the dark at room temperature 4. The cell was inspected by fluorescent microscope.

9- Statistical analysis

The significance of the results was calculated by the aid of a digital computer, using SPSS version 13.0 program according to the technique described by (Bailey, 1994).

Results 60

Results

The goal of this study was to evaluate some biochemical parameters in plasma of Duchenne Muscular Dystrophy patients as a diagnostic marker of the disease, such as creatine phosphokinase, nitric oxide, malondialdehyde, Bcl- 2, percent of apoptosis in circulating lymphocytes, catalase, total cholesterol and triacylglycerol. Also, to elucidate the ameliorative in vitro effect of laser irradiation of lymphocytes on the disorders associated with the disease.

The activity of Creatine Phosphokinase (CPK) was determined in plasma of control and patients. The results are given in table (1) and Figure (8&9).

Results in Table (1) indicate that there is a high significant increase in CPK activity in DMD patients than control. Due to the wide range of data in patients’ group the median values were given. This wide range in the results, did not affect the significance of the consequences (p<0.001).

Results 61

Table 1: Statistical analysis for the differences in CPK activity and age difference in patients as compared to controls (t-test)

Group Control Patients Parameter Pre-Laser

Number 22 22 Age (Years) Mean ± SD 9.07±1.906 9.32± 2.0

Range 5-13 4-12 % Change a p NS CPK (U/L)

Mean ± SD 100.5±4.5 7680 b

Range 80-164 877-9910 % Change 7542% p 0.001

NS: non- significant. b: Median value

Results 62

20000

15000

10000 K U/L CP

5000

0

Control Patients Groups

Figure 8: CPK activity in control and patients group

Figure 9: Mean plot of CPK in all groups Results 63

The levels of cholesterol (mg/dL) and triacylglycerols (mg/dL) in plasma are given in the Table (2) and Figures (10, 11& 12).

Data presented in Table (2) showed that there was a significant increase in cholesterol level in DMD patients (24.5%; p<0.05), which was still within the normal range (<150 mg/dL), and a non significant increase in triacyl- glycerols level compared to control. These levels were not altered after laser irradiation (as calculated by paired t-test).

Results 64

Table 2: Statistical analysis of plasma Cholesterol (mg/dL) and Triacylglycerols (mg/dL) levels in control and DMD patients' pre- and post-laser irradiation).

Group Control Patients

Parameter Pre-Laser Post-Laser Number 22 22 22

Cholesterol mg /dL

Mean ± SD 91.5±13.9 114±9.15 119±10.8

Range 75-104 104-129 104-134 % Changea 24.5 % 30% Pa 0.05 NS Paired t-testb NS Triacylglycerols mg/dL

Mean ± SD 57.5±15.0 68.8±4.14 64.6±14.0 Range 40-73 66-76 49-83 % Changea 20.1 % 12.73% Pa NS N.S Paired t-testb N.S a, compared to control. b; compared to pre-laser.

Results 65

l

o 35 r

t 30

n 30 o 24.5*

C 25 20.1 om

r 20 f

15 12.7 ge

an 10 h

C 5 % 0 Cholesterol mg /dL Triacylglycerols mg/dL

pre-laser post-laser

* Significantly different from control (p<0.05).

Figure 10a: illustrations for the % change in cholesterol and triacylglycerol levels compared to control.

r 6

e 4 s

a 4 L - e

r 2 P 0 om r

f -2 ge

an -4 h

C -6

% -6 -8 Cholesterol mg /dL Triacylglycerols mg/dL

Figure 10b: illustrations for the % change in cholesterol and triacylglycerol levels post-laser compared to pre-laser.

Results 66

Figure 11: Mean plot of cholesterol in all groups

Figure 12: Mean plot of triacylglycerols in all groups

Results 67

Malondialdehyde (MDA nmol/mL), Catalase activity

(1unit of catalase that consumed 1µmole of H2O2/min) and Nitric Oxide (NO µmole/L) were determined in the plasma of the controls and patients (pre-laser and post-laser). The results are given in Table (3) and Figures (13, 14, 15, 16& 17).

From the results in Table (3) we find that MDA level in patients before laser was significantly higher from that of control group (335%; p<0.001), while NO was significantly lower (-32%; p<0.001). Treating DMD blood in vitro with laser caused a significant decrease in MDA (-127%; p<0.001) when compared to pre-laser level, but was still significantly higher than control (208%; p<0.001). Radiation caused significant increase in NO (+88%; p<0.001) compared to pre-laser level (paired t-test), this elevation caused NO to be higher than control levels (by 56.8%; p<0.001, using ANOVA). On the other hand catalase activity among all groups did not show significant differences.

Results 68

Table 3: Statistical analysis of plasma Malondialdehyde (MDA) nmol/mL level, Catalase activity and NO (µmol/L in control and DMDpatients (Pre- and post-laser irradiation)

Group Control Patients Parameter Pre-Laser Post-Laser Number 22 22 22 MDA(nmol/mL) Mean ± SD 2.41 ± 0.76 9.53± 1.58 6.69 ± 1.16

Range 1.4 - 4.0 6.1 - 12.2 5.3 - 9.2 % Changea 335% 208% Pa 0.001 0.001 Paired t-testb 0.001 NO (µmol/L) Mean ± SD 1.53 ± 0.63 1.04± 0.32 2.4 ± 0.79 Range 1.2 - 2.5 0.3 - 1.4 4 - 12

% Changea -32% 56.8% Pa 0.001 0.001 Paired t-testb 0.001 Catalase (U of catalase /1µmol of H2O2/min) Mean ± SD 0.033 ± 0.012 0.025±0.025 0.031± 0.018 Range 0.020 - 0.044 0.011-0.088 0.007 - 0.059

% Changea -24.2 % -6.06 % Pa NS NS Paired t-testb NS a, compared to control. b; compared to pre-laser. Results 69

400 l

o 335**

r 350 t

n 300

co 250 208** m

o 200 r

f 150 e

g 100

an -24.2 50 -6.06

ch 0 % -50 Catalase MDA Pre-laser post-laser

**Highly significant (p<0.001)

Figure 13a: Illustrations for the % change in MDA levels and catalase activity compared to control group.

r

e 30

s 18 a

l 20 - e 10 pr 0 om r

f -10 -20 nge a

h -30 -28 c -40 % Catalase MDA

** Highly significant difference from control

Figure 13b: illustrations for the % change in MDA level and catalase activity post-laser compared to pre-laser group. Results 70

l o

r 450 388**

nt 400 o 350

C 300

om 250 r 200 f 150 100 ange -29**

h 50 C

0 -50 % Pre-Laser Post-Laser NO

** Highly significant (p<0.001) Figure 14a: Illustrations for the % change in NO levels compared to control group.

NO 670**

r 700 e s

a 600 L - e

r 500 P

m 400 o r

f 300

200 ange h 100 C

% 0

** Highly significant (p<0.001)

Figure 14b: Illustrations for the % change in NO levels post-laser compared to pre-laser group. Results 71

Figure 15: Mean plot of MDA in all groups

Catalase

0.034 f o

l 0.032 ) mo

n 0.03 i µ 1 m /

/ 0.028 2

(U 0.026 e s a H2O l 0.024 ta

a 0.022 C 0.02 Control Pre-Laser Post-Laser

Figure16: Mean plot of CAT activity in all groups

Results 72

Figure17: Mean plot for NO in all groups

Results 73

Bcl-2 was determined in plasma and in circulating lymphocytes, while the percentage of apoptosis was determined in circulating lymphocytes. The results are given in Table (4) and Figures (18, 19, 20 & 21).

From data in Table (4) we find that there was a significant increase in plasma Bcl-2 (1100%; p<0.001) compared to controls. On the other hand the Bcl-2protein inside the lymphocytes showed a significant decrease (-36%; p<0.001). Apoptosis percentage in the patients circulating lymphocytes was significantly higher in DMD (600%; p<0.001) compared to controls. Post-laser levels were also significantly different from the control ones with +1500 % in plasma Bcl-2; -21% change in lymphocytes Bcl-2; and + 407% in Apoptosis percentage.

Meanwhile, post-laser data also revealed significant changes from the pre-laser levels. A significant increase in plasma Bcl-2 level (p<0.001), and lymphocytes Bcl-2 (p<0.001), accompanied by a significant decrease in apoptosis percentage (p < 0.001) were observed (by paired t- test).

Results 74

Table 4: Statistical analysis of plasma Bcl-2nmol/mL, lymphocyte Bcl-2 nmol/ 2 x10 5 cell and apoptosis percentage in control and DMD patients (Pre- and post-laser irradiation).

Group Control Patients Parameter Pre-laser Post-Laser Number 22 22 22 Plasma BCL--2 (nmol/mL) Mean ± SD 0.1 ± 0.02 1.2 ± 0.6 1.6 ± 0.4 Range 0 - 1 1.3 - 1.5 1.38 - 2 % Changea 1100 1500 Pa 0.01 0.01 Paired t-testb 0.001 Lymphocyte BCL-2 (nmol/2 x10 5 cell) Mean ± SD 10 ± 2.8 6.4 ± 1.6 7.9 ± 2.3 Range 5 - 14 5.3 - 8.5 6.2 - 9.2 % Changea -36 -21 Pa 0.005 0.001 Paired t-testb 0.001 Apoptosis percentage Mean ± SD 1.4 ± 0.6 9.8 ± 2.3 7.1 ± 3.2 Range 0 - 2 6 - 15 4 - 10 % Changea 600 407 Pa 0.001 0.001 Paired t-testb 0.001 a, compared to control. b; compared to pre-laser. Results 75

% Apoptosis

800 688**

l 700 o r t

n 600 o

c 470** 500 m o

r 400 f e 300 ng

ha 200 c

% 100 0 Pre-laser Post-laser

**Highly significant (p<0.001)

Figure 18a: Illustrations for % change in apoptosis percentage in the pre-laser and post-laser groups compared to control group.

Plasma BCL-2

2600 2555** l o r 2500

cont 2400 m o

r 2300 2226** 2200

hange f 2100 C

% 2000 Pre-laser Post-laser

**Highly significant (p<0.01)

Figure 18b: Illustrations for % change in Plasma Bcl-2in the pre-laser and post-laser groups compared to control group.

Results 76

Lymphocyte BCL-2 0

l -5 o r

ont -10 c -15 om r f -20

nge -20** -25 Cha

% -30 --33** -35 Pre-lase Post -laser

**Highly significant (p<0.001)

Figure 18c: Illustrations for % change in lymphocyte Bcl-2compared to control.

30 23**

r 19**

e 20 s a l -

e 10

pr 0 om r

f -10

nge -20 a h

C -30 -28** % -40 % Apoptosis Plasma BCL-2 Lymphocyte BCL-2

**Highly significant (p<0.001)

Figure 18d: Illustrations for the plasma Bcl-2, lymphocyte Bcl-2and % apoptosis in post-laser compared to pre-laser group.

Results 77

Figure 19: Mean plot for plasma Bcl-2in all groups

Figure 20: Mean plot for lymphocyte Bcl-2in all groups

Results 78

Figure 21: Mean plot for Apoptosis percentage in all groups

Results 79

Significant correlations between the estimated parameters: By computing the correlation matrix of the studied parameters using Pearson correlation coefficient analysis it was found that (Table 5 and Figures 22-27): 1- In control group: negative correlation between age and lymphocyte Bcl-2. 2- In DMD patients pre-laser: positive correlation between age and CPK 3- In DMD patient post-laser: a. Positive correlation between MDA and NO. b. Negative correlation between apoptosis % and lymphocyte Bcl-2. 4- In DMD patients pre- and post-laser irradiation: a. Positive correlation between Apoptosis %. b. Positive correlation between lymphocyte Bcl-2. 5-Non-signficant correlation between NO & Lymphocyte BCL-2 in all groups.

Results 80

Table (5): Significant correlations between some studied parameters in DMD patients and control group (Pearson correlation).

Group Parameters r-value P< Control Age & lymphocyte Bcl-2 -0.640 0.001 DMD Age & CPK 0.458 0.05 DMD post- NO &MDA 0.523 0.01 laser DMD Apoptosis% & lymphocyte post- -0.425 0.05 Bcl-2 laser apoptosis % pre-laser & DMD 0.885 0.001 apoptosis % post- laser Lymphocyte Bcl-2 pre- laser & DMD 0.603 0.01 Lymphocyte Bcl-2post- laser Results 81

Figure 22`: Correlation between age in years and Bcl-2in circulating lymphocytes in Control group.

Figure 23: Correlation between age in years and CPK in DMD patients (pre- laser)

Results 82

Figure 24: Correlation between NO (µmol /L) and MDA (nmol/L) after laser irradiation

Figure 25: Correlation between Bcl-2in lymphocytes (nmol/ 2 x105 Cells) and apoptosis percentage after laser irradiation.

Results 83

Figure 26: Correlation between apoptosis percentage before & after laser irradiation.

Figure 27: Correlation between Bcl-2 in lymphocytes (nmol/ 2 x105 Cells) before & after laser irradiation.

Results 84

In the following section statistical analysis were carried out in order to evaluate the ameliorative effect of laser irradiation that caused normalization in any of the studied parameters.

In order to define in each parameter the discriminating cutoff values; which separates normal from abnormal values, analysis by receiver operating characteristic (ROC) curves were carried out by using the DMD pre-laser levels vs. the control ones. From these curves the cut-off value of each parameter was calculated (according to the highest sensitivity and specificity). The data were expressed as “area under curve” (AUC), significance (p), sensitivity% and specificity % (Figures 28 - 32).

Chi-square test (or X2-test) analysis was then carried out in order to calculate the % of samples that reverted to the normal level for each parameter. The results are given as cross tabulation, and illustrated in bars showing the % of irradiated samples that gave normal values (Table 6 and Figures 33 -37).

Results 85

Figure 28: Area Under the Curve for MDA nmol/L Area E ut-Off % Sensitivity % Specificity .000 .000 .001 .050 00 00

From Figure (28), we found that the MDA cut-off value was 5.05 nmol/L, with 100% sensitivity and 100% specificity, i.e., that 100% of the control samples had values <5.05 nmol/L, while 100% of the pre-laser DMD samples showed MDA ≥ 5.05 nmol/L.

Results 86

Figure 29: Area Under the Curve for NO µmol/L Area SE p Cut-Off % Sensitivity %Specificity 0.911 0.043 0.001 1.350 68 95

From Figure (29), we found that the NO cut-off value was 1.35 µmol/L, with 68% sensitivity and 95% specificity, i.e., that 68% of the control samples had values >1.35 µmol/L, while 95% of the pre-laser DMD samples showed NO ≤ 1.35 µmol/L.

Results 87

Figure 30: Area Under the Curve for Plasma BCL-2 (nmol/mL) Area SE p Cut-Off % Sensitivity % Specificity 1.000 0.00 0.001 1.150 100 100

From Figure (30), we found that the Plasma BCL-2 cut- off value was 1.15 nmol/L, with 100% sensitivity and 100% specificity, i.e., that 100% of the control samples had values <1.15 nmol/L, while 100% of the pre-laser DMD samples showed Plasma BCL-2 ≥ 1.15 nmol/L.

Results 88

Figure 31: Area Under the Curve for Lymphocyte BCL-2 (nmol/2 x10 5 cell) Area SE p Cut-Off % Sensitivity % Specificity 0.895 0.060 0.001 8.900 82 100

From Figure (31), we found that the Lymphocyte BCL-2 cut-off value was 8.9 nmol/2 x 105cell, with 82% sensitivity and 100% specificity, i.e., that 82% of the control samples had values >8.9 nmol/2 x 105cell, while 100% of the pre- laser DMD samples showed Lymphocyte Bcl-2 ≤ 8.9 nmol/2 x 105cell.

Results 89

Figure 32: Area Under the Curve for Apoptosis % Area SE p Cut-Off % Sensitivity % Specificity 1.000 0.000 0.001 4.000 100 100

From Figure (33), we found that the Apoptosis percentage cut-off value was 4%, with 100% sensitivity and 100% specificity, i.e., that 100% of the control samples had values <4%, while 100% of the pre-laser DMD samples showed lymphocytic apoptosis percentage ≥ 4%. Results 90

Table 6: Cross tabulation showing statistical analysis (Chi-Square Tests “x2”) of all the parameters ordinariness in the different groups (22 patients/group)

Parameter Cut off N* Group X2 Control Patients Pre-laser Post-laser <5.05 N 22 0 0 Normal MDA 0.001 (nmol/L) ≥5.05 N 0 22 22 Abnormal NO >1.35 (µmol/L) N 15 1 22 Normal 0.001 ≤1.35 N 7 19 0 Abnormal Plasma <1.15 BCL-2 N 22 0 0 Normal (nmol/mL) 0.001 ≥1.15 N 0 22 22 Abnormal Lymphocyte >8.9 BCL-2 N 18 0 4 Normal (nmol/2 0.001 x10 5 cell) ≤8.9 N 4 22 18 Abnormal Apoptosis <4% Percentage N 22 0 3 Normal 0.001 ≥4% N 0 22 19 Abnormal *N= number of patients in each group

Results 91

As shown in Table 6 the He:Ne laser in vitro irradiation caused a significant improvement in the NO level, in all the samples with 100% normalization. A slight improvement was observed in the lymphocytes’ Bcl-2and apoptosis percentage. On the other hand, the laser ameliorative effect was not potent enough to normalize the plasma MDA or Bcl- 2in any of the samples. These results are illustrated in Figures 33-37.

Results 92

MDA (cut off 5.05nmol/L)

100% s 80% ect j b

u 60% S 40% % 20% 0% Control (n=22) Patients Pre- Patients Post- ≥5.05 Abnormal 02222 <5.05 Normal 22 0 0

Figure 33: MDA ordinariness in the different groups

NO (cut off 1.35µmol/L

100% s t

c 80% e j b

u 60% 40% % S 20% 0% Control (n=22) Patients Pre- Patients Post- ≤1.35 Abnormal 7190 >1.35 Normal 15 1 22

Figure 34: NO ordinariness in the different groups

Results 93

Plasma BCL-2 (cut off 1.15 nmol/mL)

100% s t

c 80% e j b 60% 40% % Su 20% 0% Control (n=22) Patients Pre- Patients Post- ≥1.15 Abnormal 02222 <1.15 Normal 22 0 0

Figure 35: Plasma Bcl-2 ordinariness in the different groups

Lymphocyte BCL-2 (cut off 8.9 nmol/2 x10 5 cell) 100% s t 80% c e 60% ubj

S 40% % 20% 0% Control (n=22) Patients Pre- Patients Post- ≤8.9 Abnormal 42218 >8.9 Normal 18 0 4

Figure 36: Lymphocyte Bcl-2 ordinariness in the different groups

Results 94

Apoptosis % (cut off 4%)

100%

s 80% ect j

b 60% u

S 40% % 20% 0% Control (n=22) Patients Pre- Patients Post- ≥4% Abnormal 02219 <4% Normal 22 0 3

Figure 37: % Apoptosis ordinariness in the different groups

Discussion 95

Discussion

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease caused by a genetic mutation that leads to the complete absence of the cytoskeletal protein dystrophin in muscle fibers. DMD exhibits a spectrum of muscle disease, and clinical variability is observed regarding age of onset, patterns of skeletal muscle involvement, heart damage, and rate of progression. Despite the fact that various prognostic serum and cellular markers have been identified for different diseases, such as cardiovascular diseases and tumor pathologies, in the field of muscular dystrophies no specific indicators have been identified thus far (Marchesi et al,. 2008).

The development of reliable prognostic markers for the progression of the disease or response to treatment would not only be advantageous for patient treatment, but also help facilitate the evaluation of new experimental approaches in DMD. The main aim of the present study was to determine whether the level of some biochemical parameters was related to the disease status; which may predict its Discussion 96 progression, and also to study the possible ameliorative in vitro effect of He:Ne laser on these parameters.

Dystrophin-deficiency results in degeneration of most, but not all skeletal muscles (Emery, 1993). The pathology of DMD involves a broad and not easily related set of defects that includes skeletal muscle weakness, inflammation, wasting and fibrosis, increased fatigability, vascular dysfunction, cardiomyopathy, lower IQ, muscle metabolic defects, and synaptic dysfunction (Tidball and Wehling- Henricks, 2004).

Several lines of evidence suggest that circulating somatic stem cell populations participate in the development and regeneration of their host tissues. Skeletal muscle is capable of complete regeneration due to stem cells that reside in skeletal muscle and non-muscle (circulating) stem cell populations (Austin et al., 1992). However, in severe myopathic diseases such as DMD, this regenerative capacity is exhausted (Shi and Garry, 2006). This exhaustion is explained by two plausible theories: oxidative stress and replicative aging (Abdel et al., 2007).

Discussion 97

The oxidative stress theory indicates failure of muscle regeneration to keep up with the ongoing apoptosis and necrosis following oxidative stress that normally associates muscular exercise (Shi and Garry, 2006). The theory suggests that the interactions between the primary genetic defect and disruptions in the production of free radicals contribute to DMD (Rodriquez and Trarnopolsky, 2003). Replicative aging theory indicates aging in the replicative myogenic cells (satellite cells) (Stolzing and Scutt, 2006).

In the present study oxidative stress theory and Replicative aging theory were tested to verify which is more pertinent. Accordingly, markers of oxidative stress were measured in terms of lipid peroxidation, and plasma nitric oxide in blood of DMD patients compared to controls. Markers of replicative aging were measured in terms of BCL-2 protein levels, and apoptosis percentage in circulating lymphocytes.

Plasma Creatine Kinase (CK): Is one of the most valid and reliable methods for assessing muscular damage, since a high percentage of the body's CK is present in skeletal muscle tissue and located in the sarcolemma and Discussion 98 mitochondrial intermembrane space of healthy muscle cells (Epstein, 1995).

In the current study, there was a significant elevation (75 times the normal) in the level of plasma creatine kinase, this increase was positively correlated with age of patients (r=0.46; p<0.05). This agrees with the results of Iwanczak et al. (2000) and Biggar (2006) who recorded that a marked increase in plasma CK (50 to 100 times the normal) occurs in DMD. CK is elevated at birth before there is clinical evidence of muscle weakness due to mechanical damage of sarcolemma which causes an increase in the permeability of cell membrane to several proteins (Biggar, 2006).

Oxidative Stress: Various properties of skeletal muscle, including a high rate of oxygen metabolism, render it particularly susceptible to free radical injury. Indeed, many muscle disorders have been attributed to toxic actions of reactive oxygen species (ROS) (Narciso et al., 2007) including DMD (Messina et al., 2007).

Evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, Discussion 99 constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to DMD (Tidball and Wehling-Henricks, 2007).

Oxidative stress could be determined through lipid peroxidation, a term commonly used to indicate oxidative decomposition of the polyunsaturated fatty acids of membrane phospholipids. This process, which is very common in pathological conditions, is usually initiated by the interaction of a reactive oxygen species (ROS) or other free radical with polyunsaturated fatty acids and exacerbated by the presence of divalent metal ions. This reaction leads to the generation of other radical species that further propagate lipid peroxidation such as malonldialdehyde (MDA) (Messina et al., 2007).

Discussion 100

Skeletal muscles of DMD patients and mdx mice have been shown to experience recurrent bouts of functional ischemia during muscle contraction (Thomas et al., 1998). Reperfusion after ischemia is a well-established cause for oxidative stress (Grisotto et al., 2000; Carmo-Araüjo et al., 2007).

Results in the present study showed that the level of malondialdehyde; an indicator of lipid peroxidation, was significantly higher (335%) among DMD patients compared to controls. Similar results on lipid peroxidation, in humans and other species, were reported in the literature with an assortment of markers and different interpretations Ragusa et al. (1997); Disatnik et al. (2000);Rodriquez and Trarnopolsky (2003); Williams and Allen (2007).

In humans, Rodriquez and Trarnopolsky (2003) showed a significant increase in the 24 hours excretion level of 8- hydroxy-2'-deoxyguanosine, which is a marker of oxidative stress, in urine of DMD patients. While Haycock et al. (1998) found that the protein carbonyls, another marker of oxidative stress, was significantly increased in the quadriceps femoris muscle biopsy samples obtained from patients with DMD as compared to control subjects. Discussion 101

In mice, Ragusa et al. (1997) measured lipid peroxidation products in terms of thiobarbituric acid reactive substances (TBARS) in muscles of mdx and control mice. TBARS content was consistently higher in both affected and spared muscles of mdx mice. In their study when they exposed the control strain mice to hyperoxia, muscle TBARS was similar to that of normoxic mdx mice.

Disatnik et al. (2000) also investigated muscle cells derived from mdx mice, which express no dystrophin, and mdx-transgenic strains that express full-length dystrophin or truncated forms of dystrophin, to explore further the relationship between dystrophin expression and susceptibility of muscle to oxidative injury. Their conclusion supported the hypothesis that the dystrophin protein complex may have important regulatory or signaling properties in terms of cell survival and antioxidant defense mechanisms, which was assumed to represent physiological compensation for increased oxidative stress.

Likewise, Williams and Allen (2007) pointed out that mdx hearts had increased NADPH oxidase activity, suggesting it could be a possible source of increased reactive oxygen species in mdx mice. Discussion 102

Another marker for oxidative stress is plasma nitric oxide (NO). Alterations in the neuronal nitric oxide synthase (nNOS) pathway in DMD have been known for some time (Reynolds et al., 2008). The discovery that nNOS is a member of the dystrophin protein complex (DPC) and is lost in dystrophin deficiency, provided the first link between dystrophin deficiency and inherent defects in free radical production in dystrophic muscle (Brenman et al., 1995).Because NO is a versatile and rapid reactant with other free radicals, the extreme disruptions of its production in muscle could cause major shifts in the redox environment.

Furthermore, NO functions as a pluripotent signaling molecule in muscles and other tissues, and perturbations in its production could have broad effects on muscle homeostasis (Stamler and Meissner, 2001).

Moreover, it is also known that there is a potential role of NO in protecting cells from damage by the reactive oxygen intermediate superoxide (Miles et al., 1996). Experimental evidence demonstrated the importance of nNOS deficiency in the pathology of mdx dystrophy. Expression of a muscle- specific nNOS transgene in dystrophic muscle prevented the majority of histologically noticeable pathology in mdx mice, Discussion 103 including reducing sarcolemmal lesions by -80% (Wehling et al., 2001).

In the present study plasma nitric oxide was measured and was found to be significantly lower in blood of DMD patients (-29%) compared to controls. Similarly Kasai et al. (2004) reported that plasma nitric oxide (NO) levels in DMD patients were significantly lower than those observed in both healthy controls and in patients with other neuromuscular disorders.

Previous reports performed to evaluate alterations and functions of nitric oxide synthase (NOS) in DMD depended on studies carried out on animal models of dystrophic (mdx) mice (Zhou et al., 2006). There is a controversy regarding the expression of induced NOS (iNOS) mRNA in DMD models. Some studies indicated that iNOS mRNA is increasingly expressed in (mdx) mice compared to controls (Buchwalow et al., 2006). Others indicated that iNOS is activated in the smooth muscle cells of normal mouse and defective in young adult (2-month-old) mdx mice (Vannuchi et al., 2001). Another study demonstrated the presence of protein inhibitor of nNOS (PIN) mRNA, which is Discussion 104 significantly higher in dystrophic muscles compared with normal muscles of mdx mouse (Guo et al., 2001).

It is worth mentioning that most of the oxidative stress studies in humans with DMD were performed on tissue biopsies taken from patient's muscles. Here in the present study lipid peroxidation and NO were investigated in blood indicating that markers of oxidative stress in DMD patients could be detected in blood, and thus the abrasive technique of biopsy sampling can be spared during such abrasive technique.

Effect of He:Ne laser on the oxidative stress: There is a substantial need for finding new avenues to promote muscle recovery when acute skeletal muscle loss extends beyond the natural capacity of the muscle to recover. A low-energy laser has been found to modulate various biological processes in cell cultures and animal models. At the cellular level, it can generate significant biological effects including cellular proliferation, collagen synthesis, and release of growth factors from cells. Low-energy lasers produce biologic alterations due to their direct irradiation effects but not thermal effect (Hu et al., 2007). Discussion 105

Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. However, according to recent studies the muscle environment has a profound effect on the regenerative capacity of resident and implanted cells, and muscle regeneration may be promoted by modifying the host muscle environment (Ehrhardt and Morgan, 2005; Nagata et al., 2006; Shefer et al., 2008).

Indeed, studies have shown that low-energy laser irradiation (LELI) increased the level of superoxide dismutase enzyme and lowered the increase in lipid peroxidation associated with experimental ischemia and reperfusion (Volotovskaia et al., 2003). These findings together with the finding that He:Ne laser treatment reduces the incidence and the severity of chemotherapy-induced mucotitis in bone marrow transplantation patients suggest that He:Ne laser has an anti-inflammatory effect and that it Discussion 106 exerts this function via the stimulation of endogenous antioxidant enzymes (Barasch et al. 1995).

He:Ne laser has been demonstrated to cause marked increases in the arteriolar blood flow mediated by the production of NO (Maegawa et al 2000 & Shefer et al., 2002) and induces the expression of iNOS mRNA (El Batanouny and Korraa, 2003; Klebanov et al., 2006). Ben- Dov et al. (1999) speculated that the shift in oxidant- antioxidant balance induced by He:Ne laser leading to increased levels of NO stimulatory functions could be the cause to a decrease in markers of oxidative stress.

Since nitric oxide generation is considered to be low in DMD patients (Vannuccchi et al., 2004) and oxidative stress is significantly high, the present study examined whether He:Ne laser in vitro can ameliorate the oxidative stress and enhance NO generation in circulating blood of DMD patients.

The present study clearly indicating that treating DMD blood in vitro with laser caused a significant decrease in MDA (from 9.5 to 6.7 nmol/L; p<0.001) and a significant increase in NO (from 1.04 to 2.40 µmol/l; p<0.001) (paired t- Discussion 107 test). It was also noticed that the changes in both MDA and NO were not correlated to the pre-laser levels, and that catalase activity, total cholesterol, and triacylglycerol levels were not significantly altered before or after laser treatment.

From the ROC curve and cross tabulation analyses, it was found that the effect of laser on MDA was not potent enough to revert it to the normal levels, where none of the samples studied showed normalization of its MDA. On the other hand, all of the irradiated samples had normal NO levels. Discussion 108

From the aforementioned results, it could be concluded that oxidative stress is a prime cause for muscle degeneration in DMD, and that there is a possible ameliorative effect of He:Ne laser on this stress. Moreover, it was found that the assessment of oxidative stress in plasma is a reliable non- invasive technique.

Replicative Aging Theory: Every day, a healthy human loses billions of cells by design. Cell death processes, such as apoptosis and other tightly regulated mechanisms that kill old, damaged, or unwanted cells, are critically important for normal embryonic development and for the maintenance of physiological functions in multicellular organisms (Narciso et al., 2007). Insufficient or excessive cell death underlies most human pathologies, including cancer, autoimmunity, infectious diseases, and degenerative disorders (Basañez and Hardwick, 2008). Thus, it is not surprising that efforts at identifying the factors responsible for cell death and elucidating their detailed mechanisms of action have received considerable research attention.

Much of this attention has focused on the Bcl-2 protein family, which has been recognized as the principal regulatory component of the intracellular apoptotic machinery for over Discussion 109 two decades (Chipuk and Green, 2008). Bcl-2 family proteins can either inhibit or promote apoptosis, largely depending on the number of Bcl-2 homology (BH) domains they share. For example, anti-apoptotic family members, such as Bcl-2 and Bcl-XL, have four BH domains, while pro- apoptotic members such as Bax and Bak contain three, and a third subset, the BH3-only proteins, contains just one (Basañez and Hardwick, 2008).

Though not universally accepted, it is widely thought that Bcl-2 family proteins regulate commitment to apoptosis primarily through their capacity to control the permeability of the outer mitochondrial membrane (OMM): permeabilization triggers the release of multiple apoptogenic factors into the cytosol and/or leads to mitochondrial dysfunction (Narciso et al., 2007; Chipuk and Green, 2008). Various Bcl-2 family members affect this key event of the apoptotic cascade in different ways, determining their pro- or anti-apoptotic status (Kim, 2005). The Bcl-2-type proteins inhibit OMM permeabilization, thereby preserving cell viability. In contrast, Bax-type proteins and the diverse group of BH3-only proteins facilitate OMM permeabilization and thus promote cell death. Yet despite intense effort, the Discussion 110 question of exactly how different types of Bcl-2 family proteins fulfill these tasks remains actively debated (Basset et al., 2006; Antignani and Youle, 2007).

In muscle cells after injury, satellite cells are stimulated to replace dead cells by replicating, but depletion of this reservoir has been reported during aging and pathological conditions (Narciso et al., 2007). During the aging process, ROS production may drastically increase due to the altered function of the respiratory chain and insufficient levels of the cellular antioxidant defenses. Such an oxidative insult, combined with a less efficient base excision repair (BER) pathway system and the lack of postmitotic repair processes, might play a key role in the age-related decrease of muscle performance and mass (Narciso et al., 2007).

Previous data showed that apoptotic morphology is increased in dystrophic (mdx) muscle and in cultured muscle cells (Gutteridge, 1995). Increased expression of death factor Fas and decreased expression of the anti-apoptotic proteins; including BCL-2, were shown to be expressed in muscles of DMD patients compared to normal (Smith et al., 1995; Marchesi et al., 2008). Discussion 111

Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers leading to apoptosis (Reynolds et al., 2008). Concurrently, aging of mesenchymal progenitor cells in DMD may not purely be due to a decline in progenitor cells numbers (satellites) but also to a loss of progenitor functionality due to the accumulation of oxidative damage (Haycock et al., 1998).

In the present study the levels of BCL-2 in plasma and lymphocytes were determined to investigate their association with DMD.

A significant increase in plasma BCL-2 protein was observed in the DMD patients however this increase was not related to any of the studied parameters. Our results corroborate the few previous studies on plasma BCL-2 (Alireza et al., 2008), where it could be implicated that this increase might be due to leaking of the protein from the distorted mitochondria of the muscular tissue, since impaired muscle mitochondrial membrane is documented in DMD (Chipuk and Green, 2008). Thus the use of plasma BCL-2 levels would presumably be very limited for clinical purposes. Discussion 112

Conversely, a significant decrease in lymphocyte BCL-2 level (-36%; p<0.01) was observed in the DMD patients as compared to controls. These results concur with those of Cereda et al. (2006) and Cova et al, (2006), who explained this decrease in BCL-2 to be related to the increase in oxidative stress (Cova et al., 2006) , and increased nitrosative stress (Cereda et al., 2006); two abnormalities found in DMD patients.

Similar conclusions were given in the literature in studies carried out on the animal model of dystrophic (mdx) mice (Dominov et al., 2005; Basset et al., 2006; Whitehead et al., 2006; Williams and Allen, 2007; Basañez and Hardwick, 2008).

Lymphocytes of the DMD patients revealed a 600% increase in the apoptosis percentage compared to the normal values, an observation expected from the results of the present study, where an increase in the oxidative stress; expressed as elevated MDA and decreased NO, together with a decrease in lymphocyte BCL-2 are strong stimulators for apoptosis (Basset et al., 2006; Narciso et al., 2007; Basañez and Hardwick, 2008). Discussion 113

The use of low energy laser irradiation a non-surgical tool is a promising means to enhance both the survival and functionality of primary myogenic cells (Shefer et al., 2008).

In the present study, He:Ne laser in vitro treatment caused a significant increase in the lymphocyte BCL-2 (19%; p<0.001)) accompanied by a significant decrease (-28%; p<0.001)) in apoptosis percentage (r = -0.425; p<0.05). This negative correlation signifies the ameliorative effect of He:Ne laser treatment of DMD patients. Similar observations were found in the literature (Shefer et al., 2002).

Also El Batanouny et al. (2002) stated that low doses of He:Ne laser promotes the cell cycle in lymphocytes and satellite cells around fibers, and also inhibits cell apoptosis. According to Avni et al. (2005) this inhibiting effect was accompanied with an increase in BCL-2 in both the fibers and myogenic cultured cells.

It is worth mentioning that the effect of laser on the lymphocyte BCL-2 content or apoptotic percentage was found to be significantly (p<0.01) correlated to their pre-laser levels. Discussion 114

The stimulatory effects of lasers on cell proliferation have been studied by many authors. Shefer et al. (2002) found that Low-energy laser irradiation (LELI) enhanced the activation and cell cycle progression of quiescent satellite cells on freshly isolated muscle fibers. LELI potentiates the entrance of quiescent satellite cells into the cell cycle, acting synergistically with serum elements to enhance their proliferation and survival immediately after muscle injury.

The effect of low energy laser light on cell proliferation has been accounted for by the involvement of photoreceptors in cellular processes leading to enhancement of cell growth (Khullar et al., 1996). Karu (1988) proposed a theory that laser light is absorbed by components of the respiratory chain, such as flavin dehydrogenase, and cytochrome oxidase, which results in respiratory chain activation ,and subsequently, in changes in the redox status of both mitochondria and cytoplasm.

This was later confirmed by Koutna et al. (2003) who suggested that cytochrome oxidase is a target for the absorption of light from the 800 to 830 nm region of the spectrum and, therefore, the respiratory chain is the starting point of any photo-induction effects elicited by infrared laser Discussion 115 light. Consequently, changes in the redox status affects membrane permeability and the Ca2+ flux, which is involved in the production of cyclic nucleotides that modulate DNA and RNA synthesis and, eventually, cell proliferation (Koutna et al., 2003).

From aforementioned results, it can be concluded that: • Oxidative stress is a prime cause for muscle degeneration in DMD, and that there is a possible ameliorative effect of He:Ne laser on this stress. Moreover, it was found that the assessment of oxidative stress in plasma is a reliable non-invasive technique. • Lymphocyte BCL-2 level and apoptosis percentage are reliable markers for the DMD, and that the use of He:Ne laser radiation on circulating lymphocytes is a reliable non-invasive technique .

Summary and conclusion 116

SUMMARY

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease caused by a genetic mutation that leads to the complete absence of the cytoskeletal protein dystrophin in muscle fibers. DMD exhibits a spectrum of muscle disease, and clinical variability is observed regarding age of onset, patterns of skeletal muscle involvement, heart damage, and rate of progression. Despite the fact that various prognostic serum and cellular markers have been identified for different diseases, such as cardiovascular diseases and tumor pathologies, in the field of muscular dystrophies no indicators have been identified thus far (Marchesi et al,. 2008).

The development of reliable prognostic markers for the progression of the disease or response to treatment would not only be advantageous for patient treatment, but also help to facilitate the evaluation of new experimental approaches in DMD. The main aim of the present study was to determine whether the level of some biochemical parameters was related to the disease status; which may predict its Summary and conclusion 117 progression, and also to study the possible ameliorative in vitro effect of He:Ne laser on these parameters.

This study includes 22 patients of DMD, they were chosen from those attending the outdoor clinic of pediatric Hospital, Faculty of Medicine, Cairo University, together with 22 healthy control subjects. Group of patients with Duchenne muscular dystrophy were selected on the basis of the following eligibility criteria: Male sex aged 7–15 years, diagnosis of muscular dystrophy was based on clinical onset of progressive weakness before 5 years of age and muscle biopsy that was deficient in dystrophin and compatible with DMD. Children with acute pathology, cardiac or respiratory insufficiency and children who were taking corticosteroids were excluded.

After blood was withdrew, patients blood were divided into 2 group Group I: Irradiated blood with He: Ne laser at 2.5 J/cm2 Group II: Non-irradiated blood Control group: Includes 22 control subjects with age matching.

Summary and conclusion 118

In this study malondialdehyde, nitric oxide, catalase, cholesterol and triacylglycerols were determined as marker of oxidative stress. While Bcl-2 protein and apoptosis percentage in circulating monocular cells as a marker of replicative aging

Results of the present study showed that: • High significant increase in plasma creatine kinase among DMD patients compared to control indicating that patients in the present study were typical DMD patients.

• High significant increase in plasma MDA among DMD patients compared to control. Laser irradiation significantly decreases level of MDA after 2 hours. • Non - significant change in the level in catalase activity, triacylglycerols and cholesterol pre and post laser irradiation. • High significant decrease in plasma NO among DMD patients. 2 hours of laser irradiation significantly increase levels of plasma nitric oxide. • Apoptosis percentage was significantly higher among DMD patients. Laser irradiation significantly Summary and conclusion 119

decreased apoptosis percentage. 2 hours post laser irradiation.

• Bcl-2 in plasma was high significant elevated while there was a high significant decrease in lymphocyte

Bcl-2. Laser irradiation significantly increases levels of

Bcl-2 in plasma and mononuclear cells 2 hours post irradiation. • Positive correlation between ages and CPK, in pre- laser group. • Positive correlation between NO and MDA post laser. • Positive correlation between apoptosis percentage and

lymphocyte Bcl-2 pre and post laser irradiation. • Negative correlation between age and lymphocyte Bcl-

-2 in control group. • Negative correlation between apoptosis percentage and lymphocyte Bcl-2.

From aforementioned results, it can be concluded that: • Oxidative stress is a prime cause for muscle degeneration in DMD, and that there is a possible ameliorative effect of He:Ne laser on this stress. Moreover, it was found that the assessment of oxidative stress in plasma is a reliable non- invasive technique. Summary and conclusion 120

• Lymphocyte BCL-2 level and apoptosis percentage are reliable markers for the DMD, and that the use of He:Ne laser radiation on circulating lymphocytes is a reliable non-invasive technique .

References 121

Abdel, S.E., Abdel-Meguid, I., Korraa, S. (2007): Markers of oxidative stress and aging in Duchene muscular dystrophy patients and the possible ameliorating effect of He:Ne laser. J.Acta Myol.;26(1):14-21.

Agarwal, A. and Prabakaran, A. S. (2005): Mechanism measurement and prevention of oxidative stress in male reproductive physiology. Indian J. Experimental Biology, 43: 963-974.

Alireza,A., Raheleh, S., Abbass, R., Mojgan, M., Mohamadreza, M., Gholamreza, M., Shadi, B. (2008): An immunohistochemistry study of tissue bcl- 2 expression and its serum levels in breast cancer patients. J.Ann. N Y Acad Sci.;1138:114-20.

Albina, J. E., Cui, S., Mateo, R. B. and Reichner J. S. (1993): Nitric Oxide-mediated apoptosis in murine peritoneal macrophages, J. Immunol., 150: 5080- 5085.

Alderton, W. K., Cooper, C. E. and Knowles, R. G. (2001): Nitric oxide synthases: structure, function and inhibition. J. Biochemical., 357: 593–615.

Ansari, N. (1984): Studiess on some biochemical changes associated with Duchenne muscular dystrophy. Thesis physiology department, Karachi University, Pakistan.

Antignani, A., Youle, R.J. (2007): The cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), can deliver Bcl-XL as an extracellular fusion protein to protect cells from apoptosis and References 122

retain differentiation induction. J. Biol. Chem.; 282(15):11246-54.

Arevalo, J. F. (2004): Retinal complications after laser- assisted in situ keratomileusis (LASIK). J. Curr. Opin. Ophthalmol., 15:184-191.

Austin, L., de Niese, M., McGregor, A., Arthur, H., Gurusinghe, A. and Gould, M. K. (1992): Potential oxy-radical damage and energy status in individual muscle fibres from degenerating muscle diseases. J. Neuromuscul. Disord., 2: 27-33.

Avni, D., Levkovit, S., Maltz, L. and Oron, U. (2005): Protection of skeletal muscles from ischemic injury: low-level laser therapy increases antioxidant activity. Photmed. J. Laset Surge., 23(3):273-277.

Babapour, R., Glassberg, E. and Lask, G. P. (1995): Low-energy systems. J. Clin. Dermatol., 13: 87–90.

Bailey, N. T. J. (1994): In statical methods in biology, 3rd.ed.cambridge University press (UK).

Barisić, K., Petrik, J. and Rumora, L. (2003): Biochemistry of apoptotic cell death. J. Acta. Pharm., 53(3):151-164.

Barasch, A., Peterson, D. E., Tanzer, J. M., D’Ambrosio, J. A., Nuki, K., Schubert, M., Franquin, J. C., Clive, J. and Tutschka, P. (1995): Helium-neon laser effects on conditioning-induced oral mucotitis in bone marrow transplantation patients. J. Cancer, 76: 2550-2556. References 123

Basañez, G., Hardwick, J.M. (2008): Unravelling the bcl- 2 apoptosis code with a simple model system. J.PLoS. Biol.; 6(6): e154.

Basset, O., Boittin,F.X., Cognard, C., Constantin, B. and Ruegg, U.T.(2006):Bcl-2 overexpression prevents calcium overload and subsequent apoptosis in dystrophic myotubes. Biochem. J.395 (2):267-276.

Ben-Dov, N., Shefer, G., Irintchev, A., Wernig, A., Oron ,U. and Halevy, O. (1999): Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.J.Biochim Biophys Acta. 11; 1448(3):372-80.

Bertoni C. (2008): Clinical approaches in the treatment of Duchenne muscular dystrophy (DMD) using oligonucleotides. Front Biosci.;13:517-27.

Berchtold, M. W., Brinkmeier, H. and Muntener, M. (2000): Calcium ion in skeletal muscle: its crucial role for muscle function, plasticity, and disease. J. Physiol. Rev., 80: 1215-1265.

Biggar, W.D. (2006): Duchenne muscular dystrophy. Pediatr. Rev. 27(3):83-8.

Blake, J. D., Weir, A., Sarah, E., Newey, and Davies, E. K. (2002): Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle. J. Physiological. Reviews, 82 :( 2): 291-329.

References 124

Boyd, C. S. and Cadenas, E. (2002): Nitric Oxide and cell signaling pathways in mitochondrial–dependent apoptosis. J. Biol. Chem., 383:411-423.

Boittin, F. X., Petermann, O., Hirn, C., Mittaud, P., Dorchies, O. M. Roulet, E. and Ruegg, U. T. (2006): Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers. J. Cell Sci., 119: 3733-3742.

Bogdanovich, S., Perkins, K. J., Krag, T.O., Khurana, T. S. (2004): Therapeutics for Duchenne muscular dystrophy: current approaches and future directions. J. Molecular Medicine, 82(2): 1432-1440.

Brenman, J.E., Chao, D.S., Xia, H., Aldape, K. and Bredt, D.S.(1995): Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.J. Cell 82: 743–752.

Brown, R. H. (1995): Free radicals, programmed death and muscular dystrophy. J. Curr. Opin. Neurol., 8: 373- 378.

Brune, B. (2003): Nitric Oxide; no apoptosis or turning it on. J. Cell Death and Differentiation, 10:864-869.

Bucolo, G. and David H. (1973): Quantitative determination of serum triglyceride by the use of enzyme. J. Clin. Chem., 19: 476.

Buchwalow, I. B., Minin, E. A., Muller, F. U., et al (2006): oxide synthase in muscular dystrophies: a re- References 125

evaluation. J. Acta Neuropathol., (Berl). 111(6):579- 588.

Burdi R, Rolland JF, Fraysse B, Litvinova K, Cozzoli A, Giannuzzi V, Liantonio A, Camerino GM, Sblendorio V, Capogrosso RF, Palmieri B, Andreetta F, Confalonieri P, De Benedictis L, Montagnani M, De Luca A. (2009): Multiple pathological events in exercised dystrophic mdx mice are targeted by pentoxifylline: outcome of a large array of in vivo and ex vivo tests. J Appl Physiol 8.

Carmo-Araüjo, E., Dal-Pai-Silva, V., Cecchini, R. and Ferreira A. A. (2007): Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies. Int. J. Exp. Pathol., 88(3): 147- 154.

Cereda, C., Cova, E., Di Poto, C., Galli, A., Mazzini, G., Corato, M.and Ceroni, M.(2006):Effect of nitric oxide on lymphocytes from sporadic amyotrophic lateral sclerosis patients: toxic or protective role.J.Neurol. Sci. 27(5):312-316.

Chipuk, J.E, Green, D.R. (2008): How do BCL-2 proteins induce mitochondrial outer membrane permeabilization. J.Trends Cell Biol.;18(4):157-64.

References 126

Clark, M. G., Wallis, M.G., Barrett, E. J., Vincent, M. A., Richards, S. M., Clerk, L. H. and Rattigan, S. (2003): Blood flow and muscle metabolism: a focus on insulin action.Am J. Physiol. Endocrinol. Metab., 284(2): E241-58.

Cova, E., Cereda, C., Galli, A., Curti, D., Finotti, C., Di Poto, C., Corato, M., Mazzini, G. and Ceroni, M.(2006):Modified expression of Bcl-2 and SOD1 proteins in lymphocytes from sporadic ALS patients.J.Neurosci. Lett. 22;399(3):186-190.

Coleman, J. (1995): Quamtum well heterogenous lasers in Semiconductor Laser, parst, present and future. G.P. Agrawal, ed. Woodprey, N.Y.

Crawford,J.H., White, C.R and Patel, R.P.(2003): Vasoactivity of S- nitrosohemoglobin:role of oxygen,heme, and NO oxidation states. J. Blood, 101: 4408-4415.

Cullen, M. J. and Fulthorpe, J. J. (1975): Stages in fiber breakdown in Duchenne muscular dystrophy. An electron- microscopic study. J. Neurol. Sci., 24: 179- 200.

Cyrulnik, S.E. and Hinton, V.J. (2008): Duchenne muscular dystrophy: a cerebellar disorder? Neurosci Biobehav Rev.;32(3):486-96.

Daiber, A., Frein, D., Namgaladze, D. and Ullrich, V. (2002): Oxidation and nitrosation in the nitrogen monoxide/superoxide system. J. Biol. Chem., 277:11882-11888. References 127

Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D. and Milzani, A. (2006): Biomarkers of oxidative damage in human disease. J. Clin. Chem., 52: 601- 623.

Da Silveira, A. C., Daw, J. L., Jr, Kusnoto, B., Evans, C. and Cohen, M. (2003): Craniofacial applications of three-dimensional laser surface scanning. J. Craniofac Surg., 14:449-456.

Degl’Innocenti, D., Pieri, A., Rosati, F. and Ramponi G. (1999): Oxidative stress and calcium homeostasis in skin fibroblasts. J. IUBMB Life 48: 391-396.

Dima, V. F., Ionescu, M. D., Balotescu, C. and Dima, S. F. (2002): Photodynamic therapy and some clinical applications in oncology. J. Roum Arch Microbiol. Immunol., 61:159-205.

Disatnik, M. H., Chamberlain, J. S. and Rando, T. A. (2000): Dystrophin mutations predict cellular susceptibility to oxidative stress. J. Muscle Nerve, 23: 784–792.

Dominov, J.A., Kravetz, A.J., Ardelt, M., Kostek, C.A., Beermann, M.L., Miller, J.B.(2005): Muscle- specific BCL2 expression ameliorates muscle disease in laminin 2-deficient, but not in dystrophin- deficient, mice. J. Human Molecular Genetics; 14(8):1029-1040.

References 128

Ehrhardt, J. and Morgan, J. (2005): Regenerative capacity of skeletal muscle. J.Curr Opin Neurol.; 18(5):548-53.

El Batanouny, M., Korraa, S. S. and Fekry, O. (2002): Mitogenic potential inducable by He: Ne laser irradiation on human lymphocytes.B. J. Photochem. Photobiol., 68(1): 1-7.

El Batanouny, M. and Korraa, S. (2003): Effect of low intensity laser on the activity and expression of nitric oxide synthase enzyme in human polymorphonuclear leucocytes in vitro. Arab J. Lab. Med., 28: 289–297.

Elmore, S. (2007): Apoptosis: A Review of Programmed Cell Death. J. Toxicol. Pathol., 35(4): 495–516.

Emery, A. E. H. (1993): Duchenne Muscular Dystrophy. Oxford Monographs on Medical Genetics (2nd ed.). Oxford, UK: Oxford Univ. Press, vol. xv, p. 392.

Epstein,Y.(1995). Clinical significance of serum creatine phosphokinase activity levels following exercise. J. Med. Sci. 31(11):657-659.

Fan T. (1996): Diode pumped solid state lasers, In Laser Sources and applications, (A. Millard and D.L. Finlayson eds.) Institute of Physics, Bristol. PP 162.

Faulkner, J.A., Ng, R., Davis, C.S., Li, S, Chamberlain, J.S,. (2008): Diaphragm muscle strip preparation for evaluation of gene therapies in mdx mice. Clin Exp Pharmacol Physiol; 35(7):725-9.

References 129

Friedrich, O., Von Wegner,F., Chamberlain S.J., Fink,A.H.R., Rohrbach,P., (2008): L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle. J.plos one 3(3). e1762.

Gee, S. H., Madhavan, R., Levinson, S. R., Caldwell, J. H., Sealock, R. and Froehner, S. C. (1998): Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins. J. Neurosci., 18: 128- 137.

Gilchrist, M., Hesslinger, C. and Befus, A. D. (2003): Tetrahydrobiopterin: critical factor in the production and role of nitric oxide in mast cells. J. Biological. Chemistry, 278: 50607–50614.

Giuliani, D., Mioni, C., Altavilla, D., Leone, S., Bazzani, C., Minutoli, L., Bitto, A., Cainazzo, M. M., Marini, H., Zaffe, D., Botticelli, A. R., Pizzala, R., Savio, M., Necchi, D., Schiöth, H. B., Bertolini, A., Squadrito, F. and Guarini, S. (2006): Both early and delayed treatment with melanocortin 4 receptor- stimulating melanocortins produces neuroprotection in cerebral ischemia. J. Endocrinology. 147(3):1126- 1135.

Grisotto, P., dos Santos, A., Coutinho-Netto, J., Cherri, J. and Piccinato C. (2000): Indicators of oxidative injury and alterations of the cell membrane in the skeletal muscle of rats submitted to ischemia and reperfusion. J. Surg. Res., 92(1):1-6.

References 130

Grounds, M.D. (2008): Two-tiered hypotheses for Duchenne muscular dystrophy. J.Cell Mol Life Sci.;65(11):1621-5.

Guang-qian, Z., Hui-qi, X., Su-zhen, Z. and Zhi-ming, Y. (2006): Current understanding of dystrophin- related muscular dystrophy and therapeutic challenges ahead. J. Chinese Medical., 119 (16):1381-1391 Review article

Guo, Y., Petrof, B. J. and Hussain, S. N. (2001): Expression and localization of protein inhibitor of neuronal nitric oxide synthase in Duchenne muscular. J. Muscle Nerve., 24(11):1468-1475.

Gustavo, R., Stefania, M. and Paolo, G. (2005): Nitric Oxide and its Antithrombotic Action in the Cardiovascular System. J. Current Drug Targets - Cardiovascular & Haematological Disorders, 5: 65- 74.

Gutteridge, J.M. (1995): Lipid peroxidation and antioxidants as biomarkers of tissue damage. J.Clin Chem. 41:1819-1828.

Hamlin S., Myers J. and M. Myrs, (1991): High repetition rate, Q-switched erbium glass laser: Component system and application; (A. Galson ed.) S.pie 141- 100.

Haycock, J. W., Mac, N. S. and Mantle, D. (1998): Differential protein oxidation in Duchenne and Becker muscular dystrophy. J. Neuroreport, 9: 2201– 2207. References 131

Helliwell, T. R., Wilkinson, A., Griffiths, R. D., McClelland, P., Palmer, T. E. and Bone, J. M. (1998): Muscle fiber atrophy in critically ill patients is associated with the loss of myosin filaments and the presence of lysosomal enzymes and ubiquitin. J. Neuropathol. Appl. Neurobiol., 24: 507-517.

Heymes, C., Bendall, J. K., Ratajczak, P., Cave, A. C., Samuel, J. L., Hasenfuss, G. and Shah, A. M. (2003): Increased myocardial NADPH oxidase activity in human heart failure. Am. J. Coll. Cardiol., 41: 2164-2171.

Higaki, Y., Hirshman, M. F., Fujii, N. and Goodyear, L. J. (2001): Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. J. Diabetes. 50(2):241-247.

Hu, W.P., Wang, J.J., Yu, C.L., Lan, C.C., Chen, G.S. and Yu, H.S. (2007): Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria. J.Invest. Dermatol.; 127(8):2048-57.

Ignarro, L. J. (2002): Nitric oxide in the regulation of vascular function: an historical overview. J. Cardiac Surgery, 17: 301–306.

Igney, F. H. and Krammer, P. H. (2002): Death and anti- death: tumour resistance to apoptosis. Nat. J. Rev. Cancer, 2:277–288.

References 132

Ioannou, Y. and Chen, F. (1996): Quantification of DNA fragmentation in apoptosis. J. Nucleic Acid Research, 24:992-993.

Ishikawa, I., Aoki, A. and Takasaki, A. A. (2004): Potential applications of erbium: YAG laser in periodontics. J. Periodontal Res., 39:275-285.

Iwańczak, F., Stawarski, A., Potyrała, M., Siedlecka- Dawidko, J. and Agrawal, G.S.(2000): Early symptoms of Duchenne muscular dystrophy-- description of cases of an 18-month-old and an 8- year-old patient.J.Med. Sci. Monit. 6(3):592-595.

Jacobson, D., Bass, L. S., VanderKam, V. and Achauer, B. M. (2000): Carbon dioxide and ER:YAG laser resurfacing. Results. J. Clin. Plast Surg., 27:241-250.

Jazedje, T., Secco, M., Vieira, N.M., Zucconi, E., Gollop, T.R., Vainzof, M., Zatz, M. (2009): Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro. J Transl Med.;7(1):6.

Kalofoutis, A., Jullien, G. and Spanos, V. (1977) : Erythrocyte phospholipids in Duchenne muscular dystrophy. J.Clin. Chim. Acta. 3, 74(1):85-87.

Karu T. (1988): Molecular mechanism of the therapeutic effect of low-intensity laser radiation. J.Laser Life Sci., 2:53–74.

Karu,T.(2003): Low-Power Laser Therapy. Institute of Laser and Information Technologies,Russian References 133

Academy of Sciences,Troitsk, Moscow Region,Russian Federation.

Kashiwagi, S., Kajiura, M., Yoshimura, Y. and Suematsu, M. (2002): Nonendothelial source of nitric oxide in arterioles and methemoglobin with nitrogen monoxide: but not in venules. J.Circulation Research, 91: e55–e64.

Kasai, T., Abeyama, K., Hashiguchi, T., Fukunaga, H., Osame, M. and Maruyama, I. (2004): Decreased total nitric oxide production in patients with Duchenne muscular dystrophy J. Biomedical Science, 11:534-537.

Kerr, J. F., Wyllie, A. H. and Currie, A. R. (1972): Apoptosis: a basic biological phenomenon with wide- ranging implications in tissue kinetics. Br. J.Cancer, 26:239–257.

Khullar, S.M., Brodin, P., Barkvoll, P. and Haanaes, H.R., (1996): Preliminary study of low-level laser for treatment of longstanding sensory alterations in the inferior alveolar nerve. J. Oral Maxillofac. Surg., 54:2–7.

Kim, R. (2005): Unknotting the roles of Bcl-2 and Bcl-xL in cell death. J.Biochem. Biophys.Res. Commun.; 333(2):336-43.

Klebanov, G.I., Shuraeva, N.Iu., Chichuk, T.V., Osipov, A.N. and Vladimirov, Iu.A. (2006): A comparison of the effects of laser and light-emitting diodes on superoxide dismutase activity and nitric oxide References 134

production in rat wound fluid. J.Biofizika.; 51(1):116- 122.

Kong, J. and Anderson, J. E. (1999): Dystrophin is required for organizing large acetylocholine receptor aggregates. J. Brain Res., 839: 298-304.

Koutna,M., Janisch, R. and Veselskar. (2003): effects of low power laser irradiation on cell proliferation .J. Scripta Medica (bron) – 76 (3): 163–172.

Kumamoto, T., Fujimoto, S., Ito T., Horinouchi, H., Ueyama, H. and Tsuda, T. (2000): Proteasome expression in the skeletal muscles of patients with muscular dystrophy. J. Acta. Neuropathol., 100: 595- 602.

Kurosaka, K., Takahashi, M., Watanabe, N. and Kobayashi, Y. (2003): Silent cleanup of very early apoptotic cells by macrophages. J. Immunol, 171:4672–4679.

MacLennan, P. A., McArdle, A. and Edwards, R. H. (1991): Effects of calcium on protein turnover of incubated muscles from mdx mice. Am. J. Physiol. Endocrinol. Metab., 260: E594-E598.

Maegawa, Y., Itoh, T., Hosokawa, T., Yaegashi, K. and Nishi, M. (2000): Effects of near-infrared low-level laser irradiation on microcirculation. J. Lasers Surg. Med., 27(5):427-437.

References 135

Marchesi, C., Belicchi, M., Meregalli, M., Farini, A., Cattaneo, A., Parolini, D., Gavina, M., Porretti, L., D'Angelo, M.G., Bresolin, N., Cossu, G. and Torrente, Y. (2008): Correlation of circulating CD133+ progenitor subclasses with a mild phenotype in Duchenne muscular dystrophy patients. PLoS ONE.;3(5):e2218.

Matsumura, C.Y., Pertille, A., Albuquerque, T.C., Santo Neto, H., Marques, M.J. (2009): Diltiazem and verapamil protect dystrophin-deficient muscle fibers of MDX mice from degeneration: A potential role in calcium buffering and sarcolemmal stability. J.Muscle Nerve.;39(2):167-176.

Martinvalet, D., Zhu, P. and Lieberman, J. (2005): Granzyme A induces caspase- independent mitochondrial damage, a required first step for apoptosis. J. Immunity, 22:355–370.

Meiattini, F., Prencipe, L., Bardelli, F., Giannini, G. and Tarli, P. (1978b): the 4- hydroxybenzoate / 4- amino phenazone chromogentic system used in the enzymic determination of serum cholesterol. J. Clin. chem., 24 (12):2161-2165.

Meiattini, F., Giannini, G. and Tarli, P. (1978a): Adenylate kinase inhibition by adenosine 5'- monophosphate and fluoride in the determination of creatine kinase activity. J. Clin. Chem., 24(3):498-501.

Mendell, J. R., Engel, W. K. and Derrer, E. C. (1971): Duchenne muscular dystrophy: Functional ischemia References 136

reproduces characteristic lesions. J. Science, 172: 1143-1145.

Messina S, Mazzeo A, Bitto A, Aguennouz M, Migliorato A, De Pasquale MG, Minutoli L, Altavilla D, Zentilin L, Giacca M, Squadrito F, Vita G.(2007): VEGF overexpression via adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle function in mdx mice. FASEB J. 21(13):3737-46.

Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., Grisham, M. B. (1996): Modulation of superoxide-dependent oxidation and hydroxylation reactions by nitric oxide. J. Biol. Chem., 271:40±47.

Miranda, K. M., Espey, M. G. and Wink, D. A. (2001): A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. J. Nitric Oxide, 5(1):62-71.

Mital, A., Kumari, D., Gupta, M. and Goyle, S. (1998): Molecular characterization of Duchenne muscular dystrophy and phenotypic correlation. J. Neurol. Sci., 157: 179-186.

Moncada, S. and Erusalimsky, J. D. (2002): Dose nitric oxide modulates mitochondrial energy generation and apoptosis. Nat. J. Rev. Mol. Cell. Biol., 3:214-220.

Mokri, B. and Engel, A. G. (1975): Duchenne dystrophy: electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fiber. J. Neurology, 25: 1111-1120. References 137

Moriya, R., Uehara,T.and Nomura,Y(2000): Mechanism of nitric oxide –induced apoptosis in human neuroblastoma SH-SY5Y cells .FEBS Lett.484:253- 260.

Nabeyrat, E., Jones, G.E., Fenwick ,P.S., Barnes, P.J. and Donnelly LE (2003): Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells.Am J. Physiol Lung Cell Mol Physiol,284(6):L1112-20.

Nagata, Y., Partridge, T.A., Matsuda, R. and Zammit, P.S. (2006): Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling. J. Cell Biol. 17; 174(2):245-253.

Nakae, Y., Stoward, P. J., Kashiyama, T., Shono, M., Akagi, A., Matsuzaki, T. and Nonaka, I. (2004): Early onset of lipofuscin accumulation in dystrophin- deficient skeletal muscles of DMD patients and mdx mice. J. Mol. Histol., 35: 489-499.

Narciso, L., Fortini, P., Pajalunga, D., Franchitto, A., Liu, P., Degan, P., Frechet, M., Demple, B., Crescenzi, M. and Dogliotti E. (2007): Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury. PNAS.; 104: 17010-17015.

.Niebrَj-Dobosz, I. and Hausmanowa-Petrusewicz, I (2005): The involvement of oxidative stress in determining the severity and progress of pathological processes in dystrophin-de.cient muscles. J. Acta. Biochemica. Polonica, 52: 449–452.

References 138

Nishino, I. and Ozawa, E. (2002): Muscular dystrophies. J. Curr. Opin. Neurol., 15: 539-544.

Nobile, C., Marchi, J., Nigro, V., Roberts, R. G. and Danieli, G. A. (1997): Exon-intron organization of the human dystrophin gene. J. Genomics, 45: 421- 424.

Noguchi, S., Tsukahara, T., Fujita, M., Kurokawa, R., Tachikawa , M., Toda, T., Tsujimoto, A., Arahata, K. and Nishino, I. (2003): cDNA microarray analysis of individual Duchenne muscular dystrophy patients. J. Hum. Mol. Genet., 12:595–600.

Petros, A. M., Medek, A., Nettesheim, D. G., Kim, D. H., Yoon, H. S., Swift, K., Matayoshi, E. D., Oltersdorf, T. and Fesik, S. W. (2001): Solution structure of the antiapoptotic protein bcl-2. J. Proc. Natl. Acad. Sci., 98(6):3012-3017.

Poppas, D. P. and Scherr, D. S. (1998): Laser tissue welding: a urological surgeon's perspective. J. Haemophilia, 4:456-462.

Posten, W., Wrone, D. A., Dover, J. S., Arndt, K. A., Silapunt, S. and Alam, M. (2005): Low-level laser therapy for wound healing: mechanism and efficacy. J. Dermatol. Surg., 31:334-340.

Ragusa, R. J., Chow, C. K., Porter, J. D., (1997): Oxidative stress as a potential pathogenic mechanism in an animal model of Duchenne muscular dystrophy. J. Neuromuscul. Disord., 7: 379–386.

References 139

Rando, T. A. (2001): The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. J. Muscle Nerve, 24: 1575-1594.

Rodriquez, M. C. and Tarnopolsky, M. A. (2003): Patients with dystrophinopathy show evidence of increased oxidative stress. J. Free Radic. Biol. Med., 34:1217-1220.

Rybakova, I. N. and Ervasti, J. M. (1997): Dystrophin- glycoprotein complex is monomeric and stabilizes actin filaments in vitro through a lateral association. J. Biol. Chem., 272: 28771-28778.

Reynolds, J. G., McCalmon, S. A., Donaghey, J. A. and Naya, F. J. (2008): Deregulated PKA signaling and myospryn expression in muscular dystrophy. J. Biol. Chem., 28(283):8070-8074.

Sandri, M., Rossini, K., Podhorska-Okolow, M. and Carraaro, U. (1999): Role of apoptosis in muscle disorder. J. basic Appl. Myol., 9(6):301-310.

Sato K, Yokota T, Ichioka S, Shibata M, Takeda S. (2008): Vasodilation of intramuscular arterioles under shear stress in dystrophin-deficient skeletal muscle is impaired through decreased nNOS expression. Acta Myol.; 27:30-6.

Savill, J. and Fadok, (2000): Corpse clearance defines the meaning of cell death. J. Nature, 407:784–788.

References 140

Schwartz, L. M. and Osborne, B. A. (1993): Programmed cell death, apoptosis and killer genes. J. Immunol. Today, 14(12):582-590.

Sethi, S. and Dikshit, M. (2000): Modulation of polymorphonuclear leukocytes function by nitric oxide. J. Thrombosis Research, 100, 223–247.

Shefer, G., Partridge, T., Heslop, L., Gross, J., Oron, U. and Halevy, O. (2002): Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells. J. Cell Sci., 115:1461-1469.

Shefer, G., Ben-Dov, N., Halevy, O. and Oron, U.(2008): Primary myogenic cells see the light: improved survival of transplanted myogenic cells following low energy laser irradiation. : J.Lasers Surg Med.40(1):38- 45. Shi, X. and Garry, D. J. (2006): Muscle stem cells in development, regeneration, and disease. J. Genes Dev., 1; 20(13):1692-1708.

Sinha, A.K.(1972): Colorimetric assay of catalase. J.Anal Biochem.47(2):389-94.

Smith, J., Fowkes, G and Schofield, P. (1995): Programmed Cell death in dystrophic (mdx) muscle is inhibited by IGF-II. J. Cell Death Differ, 2(4):243- 251.

Stamler, J. S., Jaraki, O., Obsorone, J. A., Simon, D. I., Keaney, J., Vita, J., Singel, D. J., Valeri, C. R. and Loscalzo, J. (1992) :Nitric Oxide circulates in mammalian plasma as an S-nitroso adduct of serum References 141

albumin. Proceedings of the National Acdamy of Science of the United States of America, 89:7674- 7677.

Stolzing, A. and Scutt, A. (2006): Age-related impairment of mesenchymal progenitor cell function. J. Aging Cell, 5(3):213-224.

Stamler, J.S. and Meissner, G. (2001): Physiology of nitric oxide in skeletal muscle. Physiol.Rev. 81: 209– 237.

Straub, V., Rafael, J. A., Chamberlain, J. S. and Campbell, K. P. (1997): Animal models for muscular dystrophy show different patterns of sarcolemmal disruption. J. Cell Biol., 139: 375-385.

Sun, J., Xu, L., Eu, J.P., Stamler, J.S. and Meissner, G. (2003): Nitric oxide, NOC-12, and S- nitrosoglutathione modulate the skeletal muscle calcium release channel/ryanodine receptor by different mechanisms. An allosteric function for O2 in S-nitrosylation of the channel. J. Biological. Chemistry, 278: 8184–8189.

Synsitizer, E. and Young, G. (1968): Glass laser. In Laser vol 2. (A.K. Levin, ed.). Marcel-Dekker N.Y.A.

Tanzi, E. L., Lupton, J, R. and Alster, T. S. (2003): Lasers in dermatology: four decades of progress. Am J. Acad Dermatol., 49: 1-31

Thomas, G. D., Sander, M., Lau, K. S., Huang, P. L., Stull, J. T. and Victor, R. G. (1998): Impaired References 142

metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle. J. Proc. Natl. Acad. Sci., USA 95: 15090– 15095.

Thrush, P.T., Allen,H,D., Viollet, L., Mendell, J.R. (2008): Re-examination of the electrocardiogram in boys with Duchenne muscular dystrophy and correlation with its dilated cardiomyopathy. Am J Cardiol.;103(2):262-5.

Tidball, J. G. and Wehling-Henricks, M. (2004): Evolving therapeutic strategies for Duchenne muscular dystrophy: targeting downstream events. J. Pediatr. Res., 56: 831–841.

Tidball, J. G. and Wehling-Henricks, M. (2007): The role of free radicals in the pathophysiology of muscular dystrophy J. Appl. Physiol., 102(4):1677-1686.

Turner, P. R., Westwood, T., Regen, C. M. and Steinhardt, R. A. (1988): Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice. J. Nature, 335: 735- 738.

Vannucchi, M. G., Corsani, L., Serio, R. and Faussone- Pellegrini, M. S. (2001): Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice. Am. J. Physiol. Gastrointest. Liver Physiol., 281(5):G1264-1270.

Vannucchi, M. G., Corsani, L., Azzena, G. B., Faussone- Pellegrini, M. S. and Mancinelli, R. (2004): References 143

Functional activity and expression of inducible nitric oxide synthase (iNOS) in muscle of the isolated distal colon of mdx mice. J. Muscle Nerve, 29(6):795-803.

Volotovskaia, A.V., Ulashchik, V.S. and Filipovich , V.N. (2003): Antioxidant action and therapeutic efficacy of laser irradiation of blood in patients with ischemic heart disease. J.Vopr. Kurortol. Fizioter. Lech. Fiz. Kult.(3):22-5

Wallis, J. P. (2005): Nitric oxide and blood: a review J. Transfusion Medicine, 15: 1–11

Wang, P.G., Xiang, M., Tang, X., Wu, X., Wen, Z., Cai, T. and Janczuk, A. J. (2002): NO donors: Chemical activities and biological applications.J. Chem. Rev., 102: 1091.

Whitehead, N.P., Yeung, E.W. and Allen, D.G. (2006): Muscle damage in mdx (dystrophic) mice: role of calcium and reactive oxygen species. J. Clin. Exp. Pharmacol. Physiol., 33(7):657-662.

Wehling, M., Spencer, M. J. and Tidball, J. G. (2001): A nitric oxide synthase transgene ameliorate muscular dystrophy in mdx. J. cell Biol., 155:123-131.

Williams, I. A. and Allen, D. G. (2007): The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice. Am. J. Physiol. Heart Circ. Physiol., 293(3):H1969-1977.

References 144

Wozniak, C. A. and Anderson, E. J. (2006): Nitric Oxide- Dependence of Satellite Stem Cell Activation and Quiescence on Normal Skeletal Muscle Fibers. J. Developmental Dynamics, 236:240–250.

Wright A. and S. Fisher, (1993): Laser surgery in Gynecology, W.B. Saunders.

Wyss, M. and Kaddurah-Daouk, R. (2000): Creatine and Creatinine Metabolism. Physiological Reviews 80(3): 1107-1213.

Yoshioka, T., Kawada, K., Shimada, T. and Mori, M. (1979): lipid peroxidation inmaternal and cord blood and protective mechanism against activated oxygen toxicity in the blood. Am. J. Obstet. Gynecol., 135: 372-376.

Yu, H. S., Chang, K. L., Yu, C. L., Chen, J. W. and Chen, G. S. (1996): Low-energy helium-neon laser irradiation stimulates interleukin 1 alpha and interkeukin-8 release from cultured human keratinocytes. J. Invest Dermatol., 107:593–596.

Yücel, D., Şeneş, M., Topkaya, B. C. and Zengi, O. (2006): Oxidative/Nitrosative Stress in Chronic Heart Failure: A Critical Review. Turkish J. Biochemistry, 31 (2): 86–95.

Zhou, G.Q., Xie, H.Q., Zhang, S.Z. and Yang, Z.M. (2006): Current understanding of dystrophin related muscular dystrophy and therapeutic challenges ahead. Chin. Med. J. (Engl); 119(16):1381-1391.

اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ

ﻣﺮض ﺿﻤﻮر اﻟﻌﻀﻼت (دوﺷﺎن) ﻳﻌﺪ ﻣﺮض ﻣﻤﻴﺖ وﻳﺮﺟﻊ اﻟﻰ ﻃﻔﺮة ﺟﻴﻨﻴﺔ أدت اﻟﻰ ﻏﻴﺎب ﺑﺮوﺗﻴﻦ اﻻﻟﻴﺎف اﻟﻌﻀﻠﻴﺔ (دﻳﺴﺘﺮوﻓﻴﻦ) وﻋﻠﻰ اﻟﺮﻏﻢ ﻣﻦ وﺟﻮد دﻻﻻت ﺗﺸﺨﻴﺼﻴﺔ ﻟﻠﻌﺪﻳﺪ ﻣﻦ اﻻﻣﺮاض ﻣﺜﻞ اﻣﺮاض اﻟﻘﻠﺐ و اﻻورام اﻻ اﻧﻪ ﻻﻳﻮﺟﺪ دﻻﻻت ﺧﺎﺻﻪ ﺑﺎﻣﺮاض اﻟﻀﻤﻮر اﻟﻌﻀﻠﻰ ﺣﺘﻰ اﻵن.

وﻣﻦ هﻨﺎ ﻓﺎن اﻳﺠﺎد دﻻﻻت ﺗﺸﺨﻴﺼﻴﺔ ﻣﻮﺛﻮق ﺑﻬﺎ ﻟﺘﺤﺪﻳﺪ ﻣﺪى ﺗﻘﺪم اﻟﻤﺮض أو اﺳﺘﺠﺎﺑﺘﻪ ﻟﻠﻌﻼج ﻟﻦ ﺗﻜﻮن ﻣﻴﺰة أو ﻓﺮﺻﺔ ﻟﻌﻼج اﻟﻤﺮﻳﺾ ﻓﺤﺴﺐ وﻟﻜﻦ ﺳﻮف ﺗﺴﺎﻋﺪ ﻓﻰ ﺗﻘﻴﻴﻢ اﻟﺘﺠﺎرب اﻟﻨﻈﺮﻳﺔ اﻟﺠﺪﻳﺪة ﻋﻦ هﺬا اﻟﻤﺮض و ﻟﺬﻟﻚ ﺗﻬﺪف هﺬﻩ اﻟﺪراﺳﺔ اﻟﻰ ﺗﻘﻴﻴﻢ ﻣﺪى ﻋﻼﻗﺔ ﺑﻌﺾ اﻟﻘﻴﺎﺳﺎت اﻟﻜﻴﻤﻴﺎﺋﻴﺔ اﻟﺤﻴﻮﻳﺔ ﺑﺎﻟﺤﺎﻟﺔ اﻟﻤﺮﺿﻴﺔ واﻟﺘﻰ ﺑﺪورهﺎ ﻗﺪ ﺗﻘﻴﻢ ﻣﺪى ﺗﻄﻮرهﺎ أو ﺗﻘﺪﻣﻬﺎ وأﻳﻀﺎ دراﺳﺔ اﻟﺘﺎﺛﻴﺮ اﻟﻤﺤﺘﻤﻞ ﻟﻠﻬﻠﻴﻮم- ﻧﻴﻮن ﻟﻴﺰر ﻋﻠﻰ ﺗﺤﺴﻴﻦ هﺬﻩ اﻟﻘﻴﺎﺳﺎت.

وﺗﻀﻤﻨﺖ هﺬﻩ اﻟﺪراﺳﺔ 22 ﻣﺮﻳﺾ ﺑﻤﺮﺿﻰ ﺿﻤﻮر اﻟﻌﻀﻼت (دوﺷﺎن) ة وﻗﺪ ﺗﻢ اﺧﺘﻴﺎر اﻟﻤﺮﺿﻰ ﻋﻠﻰ اﺳﺎس اﻧﻬﻢ ذآﻮر، ﺗﺘﺮواح اﻋﻤﺎرهﻢ ﺑﻴﻦ 7-15 ﺳﻨﺔ ، ﺑﺪاﻳﺔ ﺗﺪهﻮر اﻟﻌﻀﻼت ﻗﺒﻞ ﺳﻦ اﻟﺨﺎﻣﺴﺔ، ﻧﻘﺺ ﻓﻰ ﺑﺮوﺗﺒﻦ اﻟﺪوﺳﺘﺮوﻓﻴﻦ ﻓﻰ ﻋﻀﻼت اﻟﻤﺮﺿﻰ ﺑﻌﺪ ﻋﻤﻠﻴﺔ اﻟﺒﺬل اﻟﻌﻀﻠﻰ ﺑﻤﺎ ﻳﺘﻨﺎﺳﺐ ﻣﻊ ﺗﻘﺪم اﻟﻤﺮض وﻗﺪ ﺗﻢ اﺳﺘﺒﻌﺎد اﻟﺤﺎﻻت اﻟﺘﻰ ﺗﻌﺎﻧﻰ ﻣﻦ اﻣﺮاض اﻟﻘﻠﺐ واﻟﺠﻬﺎز اﻟﺘﻨﻔﺴﻰ أو اﻟﺬﻳﻦ ﻳﺘﻌﺎﻃﻮن أدوﻳﺔ ﺳﺘﻴﺮودﻳﺔ وﺑﻌﺪ ﺳﺤﺐ اﻟﺪم ﻣﻦ اﻻﻃﻔﺎل ﺗﻢ ﺗﻘﺴﻴﻢ هﺬا اﻟﺪم اﻟﻰ ﻣﺠﻤﻮﻋﺘﻴﻦ:

اﻟﻤﺠﻮﻋﺔ اﻻوﻟﻰ: دم ﻣﻌﺮض ﻷﺷﻌﺔ اﻟﻠﻴﺰر. اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ: دم ﻏﻴﺮ ﻣﻌﺮض ﻷﺷﻌﺔ اﻟﻠﻴﺰر (اﻟﻬﻠﻴﻮم- ﻧﻴﻮن ). اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﻪ: و ﺗﻀﻢ 22 ﻃﻔﻞ ﻣﻦ اﻷﻃﻔﺎل اﻷﺻﺤﺎء ﻣﻦ ﻧﻔﺲ ﻋﻤﺮ اﻟﻤﺮﺿﻰ.

وﻓﻰ هﺬﻩ اﻟﺪراﺳﺔ ، ﺗﻢ ﺗﻌﻴﻴﻦ آﻞ ﻣﻦ ﻧﻮاﺗﺞ أآﺴﺪة اﻟﺪهﻮن ، أآﺴﻴﺪ اﻟﻨﻴﺘﺮﻳﻚ ، اﻧﺰﻳﻢ catalase ،اﻟﻜﻮﻟﻴﺴﺘﺮول و اﻟﺪهﻮن اﻟﺜﻼﺛﻴﺔ آﺪﻻﻻت ﻟﻔﺮط اﻻآﺴﺪة اﻣﺎ ﺑﺮوﺗﻴﻦ Bcl-2 وﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ ﻓﻰ اﻟﺨﻼﻳﺎ اﺣﺎدﻳﺔ اﻟﻨﻮاة آﺪﻻﻻت ﻟﻠﺸﻴﺨﻮﺧﺔ اﻟﻨﺎﺗﺠﺔ ﻣﻦ ﺳﺮﻋﺔ ﺗﻜﺎﺛﺮ اﻟﺨﻼﻳﺎ.

وﻗﺪ أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ اﻵﺗﻰ:

• زﻳﺎدة ﻣﻌﻨﻮﻳﺔ ﻓﻰ ﻧﺸﺎط اﻧﺰﻳﻢ CPK ﻓﻰ ﺑﻼزﻣﺎ اﻟﻤﺮﺿﻰ ﻣﻘﺎرﻧﺔ ﺑﺎﻻﺻﺤﺎء. • زﻳﺎدة ﻣﻌﻨﻮﻳﺔ ﻟﻨﻮاﺗﺞ اآﺴﺪة اﻟﺪهﻮن ﻓﻰ ﺑﻼزﻣﺎ اﻟﻤﺮﺿﻰ ﻣﻘﺎرﻧﺔ ﺑﺎﻷﺻﺤﺎء وﻗﺪ أدى اﻟﺘﻌﺮض ﻷﺷﻌﺔ اﻟﻠﻴﺰر اﻟﻰ ﻧﻘﺺ ﻣﻌﻨﻮى ﻓﻰ ﻧﻮاﺗﺞ أآﺴﺪة اﻟﺪهﻮن ﺑﻌﺪ ﺳﺎﻋﺘﻴﻦ ﻣﻦ اﻟﺘﻌﺮض ﻟﻼﺷﻌﺎع. وﻟﻜﻦ ﻟﻢ ﻳﺤﺪث أى ﺗﻐﻴﺮ ﻣﻌﻨﻮى ﻓﻰ ﻣﺴﺘﻮى اﻧﺰﻳﻢ Catalase ، آﻮﻟﻴﺴﺘﺮول و ﻧﺴﺒﺔ اﻟﺪهﻮن اﻟﺜﻼﺛﻴﺔ ﻗﺒﻞ و ﺑﻌﺪ ﺳﺎﻋﺘﻴﻦ ﻣﻦ اﻟﺘﻌﺮض ﻟﻼﺷﻌﺎع. • وﻋﻠﻰ اﻟﻨﻘﻴﺾ ، وﺟﺪ أن ﺗﺮآﻴﺰ اآﺴﻴﺪ اﻟﻨﻴﺘﺮﻳﻚ ﻓﻰ اﻟﺒﻼزﻣﺎ أﻗﻞ ﻣﻌﻨﻮﻳﺎ ﻓﻰ اﻟﻤﺮﺿﻰ ﻣﻘﺎرﻧﺔ ﺑﺎﻷﺻﺤﺎء وﻗﺪ زاد ﺗﺮآﻴﺰ اآﺴﻴﺪ اﻟﻨﻴﺘﺮﻳﻚ ﺑﻌﺪ ﺳﺎﻋﺘﻴﻦ ﻣﻦ اﻟﺘﻌﺮض ﻟﻼﺷﻌﺎع. • ﻧﻘﺺ ﻣﻌﻨﻮى ﻓﻰ ﺗﺮآﻴﺰ ﺑﺮوﺗﻴﻦ Bcl-2 ﻣﻘﺪرة ﺑﺎﻟﻤﻮل ﻟﻜﻞ 510x2ﺧﻠﻴﺔ ﻓﻰ اﻟﺨﻼﻳﺎ اﺣﺎدﻳﻪ اﻟﻨﻮاﻩ ﻟﻠﻤﺮﺿﻰ ﺑﺎﻟﻨﺴﺒﺔ ﻟﻼﺻﺤﺎء ﺑﻴﻨﻤﺎ آﺎن ﺗﺮآﻴﺰ ﺑﺮوﺗﻴﻦ Bcl-2 ﻣﻘﺪرة ﺑﺎﻟﻤﻮل ﻟﻜﻞ ﻣﻠﻠﻴﻠﻤﺘﺮ ﻓﻰ اﻟﺒﻼزﻣﺎ أﻋﻠﻰ ﻣﻌﻨﻮﻳﺎ ﻓﻰ اﻟﻤﺮﺿﻰ ﻣﻘﺎرﻧﺔ ﺑﺎﻻﺻﺤﺎء.وﻗﺪ أدى ﺗﻌﺮض اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮأة اﻟﻰ أﺷﻌﺔ اﻟﻠﻴﺰر اﻟﻰ زﻳﺎدة ﻣﻌﻨﻮﻳﺔ ﻓﻰ ﺑﺮوﺗﻴﻦ Bcl-2 داﺧﻞ اﻟﺨﻼﻳﺎ. • زﻳﺎدة ﻣﻌﻨﻮﻳﺔ ﻓﻰ ﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ ﻟﻠﻤﺮﺿﻰ ﻣﻘﺎرﻧﺔ ﺑﺎﻻﺻﺤﺎء وﻗﺪ أدى اﻟﺘﻌﺮض ﻟﻼﺷﻌﺎع اﻟﻰ ﻧﻘﺺ ﻣﻌﻨﻮى ﻟﻬﺬﻩ اﻟﻨﺴﺒﺔ. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻋﻤﺮ اﻟﻤﺮﻳﺾ واﻧﺰﻳﻢ CPK ﻓﻰ اﻟﻤﺮﺿﻰ. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ أآﺴﻴﺪ اﻟﻨﻴﺘﺮﻳﻚ و أآﺴﺪة اﻟﺪهﻮن ﺑﻌﺪ ﺗﻌﺮض دم اﻟﻤﺮﺿﻰ ﻟﻠﻴﺰر. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ و ﺑﺮوﺗﻴﻦ Bcl-2 ﻓﻰ اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮاة ﻗﺒﻞ وﺑﻌﺪ اﻟﺘﻌﺮض ﻟﻠﻴﺰر. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻋﻤﺮ اﻟﻄﻔﻞ و ﺑﺮوﺗﻴﻦ Bcl-2 ﻓﻰ اﻷﺻﺤﺎء. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﺗﺮآﻴﺰ آﻼ ﻣﻦ أآﺴﻴﺪ اﻟﻨﻴﺘﺮﻳﻚ و أآﺴﺪة اﻟﺪهﻮن ﺑﻌﺪ ﺗﻌﺮض اﻟﺪم ﻷﺷﻌﺔ اﻟﻠﻴﺰر. • ﻋﻼﻗﺔ ﻋﻜﺴﻴﺔ ﺑﻴﻦ ﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ و ﺑﺮوﺗﻴﻦ Bcl-2 ﻓﻰ اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮاة ﺑﻌﺪ اﻟﺘﻌﺮض ﻷﺷﻌﺔ اﻟﻠﻴﺰر. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻧﺴﺒﺔ اﻟﺨﻼﻳﺎ اﻟﻤﻴﺘﺔ ﻗﺒﻞ و ﺑﻌﺪ اﻟﺘﻌﺮض ﻟﻼﺷﻌﺎع. • ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﺑﺮوﺗﻴﻦ Bcl-2 ﻓﻰ اﻟﺨﻼﻳﺎ أﺣﺎدﻳﺔ اﻟﻨﻮاة ﻗﺒﻞ و ﺑﻌﺪ ﺗﻌﺮض دم اﻟﻤﺮﺿﻰ ﻟﻠﻴﺰر.

اﻷﺳﺘﻨﺘﺎج:- ﻣﻦ هﺬﻩ اﻟﺪراﺳﺔ ﻳﻤﻜﻦ اﺳﺘﻨﺘﺎج أن: 1 - ﻓﺮط اﻷآﺴﺪة هﻮ اﻟﺴﺒﺐ اﻟﺮﺋﻴﺴﻰ ﻻﻧﻬﻴﺎر اﻟﻌﻀﻼت ﻓﻰ ﻣﺮﺿﻰ ﺿﻤﻮر اﻟﻌﻀﻼت (دوﺷﺎن) ﻣﻊ اﻷﺷﺎرة اﻟﻰ ﺗﺄﺛﻴﺮ (اﻟﻬﻠﻴﻮم- ﻧﻴﻮن) ﻟﻴﺰر آﻌﻼج ﻣﺤﺘﻤﻞ ﻟﻬﺬا اﻟﻔﺮط. 2- ﻣﺴﺘﻮى ﺑﺮوﺗﻴﻦ Bcl-2 ﻓﻰ أﺣﺎدﻳﺔ اﻟﻨﻮاة وﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ ﻣﻦ اﻟﺪﻻﻻت اﻟﻤﻮﺛﻮق ﻓﻴﻬﺎ ﻟﻤﺮض ﺿﻤﻮر اﻟﻌﻀﻼت (دوﺷﺎن) واﺳﺘﺨﺪام اﻟﻬﻠﻴﻮم- ﻧﻴﻮن ﻣﻦ اﻟﻄﺮق اﻟﺘﻰ ﻻﺗﺤﺘﺎج اﻟﻰ ﺗﺪﺧﻞ ﺟﺮاﺣﻰ.