The effect of Ginkgo biloba extract on parkinsonism- induced biochemical changes in brain of irradiated rats

A THESIS PRESENTED BY Engy Refaat Abd El-Aziz (M.Sc. - Cairo University) Pharmacist in Drug Radiation Research Department National Centre for Radiation Research and Technology Atomic Energy Authority - Egypt

Submitted for the Degree of Doctor of Philosophy in Pharmaceutical Sciences (Biochemistry) Under the Supervision of Prof. Dr. Amal A. Abd El-Fattah Prof. Dr. Mona A. El-Ghazaly

Prof. of Biochemistry Prof. of Pharmacology Faculty of Pharmacy Chairman of the National Centre for Cairo University Radiation Research and Technology Atomic Energy Authority

Dr. Nermin Abd El-Hamid Sadik Ass. Professor of Biochemistry Faculty of Pharmacy Cairo University

FACULTY OF PHARMACY CAIRO UNIVERSITY 2012

Page 1 of 152 Page 2 of 152 Acknowledgements

I would like to express my gratitude and appreciation to Prof. Dr. Mona Abd El- Lateef El-Ghazaly; Prof. of Pharmacology and Chairman of National Centre for Radiation Research and Technology; for the practical and scientific support, instructive supervision and indispensible efforts that were kindly and generously offered by her; throughout each stage of this study.

I am also very grateful for to Prof. Dr. Amal Ahmed Abd El-Fattah; Prof. of Biochemistry, Faculty of Pharmacy, Cairo University, for suggesting the research point, her kind guidance, great patience and the subjective and precise revision of the thesis in addition to her continuous encouragement for me.

My deep and sincere thanks are extended to Ass. Prof. Dr. Nermin Abd El-Hamid Sadik; Assistant Professor of Biochemistry, Faculty of Pharmacy, Cairo University for her valuable contribution in the experimental work, her subjective criticism and the continuous guidance during the writing and the revision of the thesis, as well as her supportive efforts allover the supervision of this work.

I would like, also; to express my thanks to Dr.Rania Mohsen ; lecturer of pharmacology, Faculty of Pharmacy, Cairo University, for teaching me the model of Parkinsonism and the handling of brain tissue. I am also very grateful for the members of the histology laboratory at the National Centre for Radiation Research and Technology; specially Dr. Seham Abo El-Nour for her assistance in commenting on the electron micrographs.

My gratitude is also extended to my colleagues and friends at Drug Radiation Research Department, National Centre for Radiation Research and Technology, for their continuous and sincere support.

L ast but not least; I would like to thank my family for their support and encouragement which enabled this work to be completed, may God reward them all.

Engy Refaat

Page 3 of 152 ABSTRACT

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. In the present study, neuromodulatory effects of standardized ginkgo biloba extract (EGb761) and low dose whole-body γ-irradiation in a reserpine model of rat Parkinsonism were investigated. Male Wistar rats were pretreated orally with EGb761 (100 mg/kg BW/day for 3 weeks) or low dose whole-body γ-irradiation (0.25Gy once a week for 6 weeks) and their combination (EGb761 was received during the last three weeks of the irradiation period) and then subjected to intraperitoneal injection of reserpine (5mg/kg BW dissolved in 1% acetic acid) 24h after last dose of EGb761or radiation. All rats were sacrificed 24h after reserpine injection. Depletion of striatal dopamine (DA) level, increased oxidative stress indicated via depletion of glutathione (GSH), increased malondialdehyde (MDA) and iron levels; decrease of dopamine metabolites metabolizing enzymes; indicated by decrease of glutathione-S- transferase (GST) and NADPH-quinone oxidoreductase (NQO) activities; mitochondrial dysfunction; indicated by decline of complex I activity and adenosine triphosphate (ATP) level and increased apoptosis; indicated by the decrease of mitochondrial B cell lymphoma-2 protein (Bcl-2) level and as shown by transmission electron microscope (TEM) were observed in brain of reserpine-induced PD model group, along with behavioral study indicated by increased catalepsy score. Moreover, the level of GSH was correlated with the levels of both DA (r = 0.78) and MDA (r = -0.93). The level of Bcl-2 was correlated with the complex I activity (r = 0.94) and ATP level (r = 0.98). Results revealed that either EGb761 or irradiation and their combination ameliorated most of the biochemical and behavioral changes induced by reserpine possibly via replenishment of normal glutathione levels. This study revealed that EGb761, which is a widely used herbal medicine and low dose of whole-body γ-irradiation have protective potential in combating Parkinsonism and can be used in preventing this devastating neurologic disorder.

Keywords: Parkinsonism; reserpine; low dose whole-body γ-irradiation; ginkgo biloba; rats.

Page 4 of 152 List of Contents

No. Item Page no. List of Contents……………………………………………………………….. I List of Tables………………………………………………………………….. III List of Figures………...……………………………………………………….. IV Abbreviations…………………………………………………………………. V 1. Aim of the work………………………………………………………………. 1 2. Introduction…………………………………………………………………... 3 2.1. Brain…………………………………………………………………………... 3 2.2. Parkinson's disease……………………………………………………………. 7 2.2.1. Etiology of Parkinson's disease ………………………………………………. 8 2.2.2. Pathogenesis of Parkinson's disease: Molecular and neurochemical pathways 9 2.2.2.1. Misfolding and aggregation of proteins………………………………………. 9 2.2.2.2. Oxidative stress and mitochondrial dysfunction………………………………. 11 Glutathione, brain and Parkinson's disease ……………………………….…... 14 Role of iron in Parkinson's disease ……………………………………………. 17 Apoptotic cell death………………………………………………………….... 19 2.3. Animal models of Parkinson's disease ………………..……………………… 22 2.3.1. Pharmacological agent-based model (Reserpine model)…………...... 22 2.3.2. Toxin-Based Models…………………………………………………………. 23 2.3.3. Other Parkinsonian agents…………………………………………………….. 25 2.4. Parkinson's disease therapy…………..…………….…………………………. 27 2.4.1. Neurotrophic factor therapy…………………………………………………... 27 2.4.2. Anti-excitotoxin therapy……………………………………………………… 28 2.4.3. Anti-apoptotic therapy……………………………………………...... 29 2.4.4. Antioxidant therapy………………………………………………...... 29 2.4.5. Bioenergetic supplements…………………………………………………….. 29 2.4.6. Cellular transplantation……………………………………………………….. 31 2.4.7. Immunosuppressant therapy………………………………………………….. 32 2.5. Ginkgo biloba extract………………………………………………………… 33 2.6. Ionizing radiation ……………………………………………………………. 35 3. Materials and Methods…………………………………………………….. 38 3.1. Materials……………………………………………………………………... 38 3.1.1. Drugs and Chemicals…………………..…………………………………… 38 3.1.2. Animals and treatment……………………………………………………….. 38 3.1.2.1. Experimental group 1………………………………………………………. 38 3.1.2.2. Experimental group 2………………………………………………………. 39 3.1.2.3. Experimental group 3………………………………………………………. 39 3.1.2.4. Experimental group 4………………………………………………………. 39

Page 5 of 152 3.1.3. Behavioral study……………………………………………………………. 39 3.1.4. Preliminary pilot studies……………………………………………………. 40 3.1.5. Tissue sampling for biochemical studies…………………………………… 40 3.2. Methods……………………………………………………………………….. 42 3.2.1. Determination of dopamine level in brain striata………………….………….. 42 3.2.2. Determination of glutathione level in brain….………………………………... 44 3.2.3. Determination of malondialdehyde level in brain………………………….…. 46 3.2.4. Determination of total iron level in brain…………………………………….. 48 3.2.5. Determination of glutathione-S-transeferase activity in brain………………… 50 3.2.6. Determination of NADPH-quinone oxidoreductase activity in brain…………. 51 3.2.7. Determination of brain mitochondrial complex I activity……………….……. 52 3.2.8. Determination of brain mitochondrial adenosine triphosphate level………… 53 3.2.9. Determination of B cell lymphoma-2 protein level in brain mitochondrial fraction……………………………………………………………………….. 55 3.2.10. Determination of protein content in different brain fractions……………….… 58 3.2.11. Examination of brain tissue by transmission electron microscope ……………. 60 3.3. Statistical analysis …………………………………………………………..… 60 4. Results………………………………………………………………………… 61 4.1. Pilot experiments…………………………………………………………….... 61 4.2. The effect of reserpine on the catalepsy score and the other biochemical parameters in rat brain………………………………………………………. 61 4.3. The effect of pretreatment with EGb761 on the catalepsy score and the other biochemical parameters in rat brain…………………………………….…… 62 4.4. The effect of whole body γ-irradiation on the catalepsy score and the other biochemical parameters in rat brain…………………………………….…… 62 4.5. The effect of the combination of EGb761 and whole body γ-irradiation on the catalepsy score and the other biochemical parameters in rat brain………… 63 4.6. The effect of reserpine and the pretreatment with EGb761, whole body γ- irradiation and their combination on the transmission electron micrograph of striatal neurons in different experimental groups………………………….. 64 5. Discussion…………………….………………………………………………. 79 6. Summary and Conclusion…………………………………………….….…. 92 7. References……………………………………………………………………. 95 Appendix…………………………………………………………………….. i أ .……….……………………………………………………Arabic summary Arabic abstract………………………………………………………..…..….

Page 6 of 152 List of Tables

Table Title Page No. no. 1 Different brain regions and their functions…………………………………... 4 2 Some human studies investigating iron-related genes in PD...... 19 3 Some neurotrophic factors involved in survival and differentiation of developing dopaminergic neurons…………………………………………….. 28 4 Various types of cells of potential benefits in PD treatment………………… 32 5 Different models utilizing LDR as a modulator……………………………… 36 6 The instrumental specifications for iron determination by atomic absorption…. 48 7 Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on catalepsy score and striatal dopamine (DA) level in reserpinized rats. ……………………………………………………… 65 8 Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain levels of glutathione (GSH), malondialdehyde (MDA) and iron in reserpinized rats..……………………. 66 9 Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain activities of glutathione-S-transferase (GST) and NADPH-quinone oxidoreductase (NQO) in reserpinized rats. ……. 67 10 Effect of EGb761, whole body γ-irradiation or their combination on complex I activity as well as levels of adenosine triphosphate (ATP) and B cell lymphoma-2 (Bcl-2) protein in brain mitochondria of reserpinized rats……… 68

Page 7 of 152 List of Figures

Figure Title Page No. no. 1 Different brain regions…………………………………………………………….... 3 2 Schematic representation of the major dopaminergic systems………………….... 6 3 Oxidation of DA……………………………………………………………………. 11 4 Aminochrome metabolism…………………………………………………………. 13 5 A schematic representation of the different pathways involved in GSH metabolism 15 6 The different roles of GSH……………………………………………………….… 17 7 Classification of animal models of PD…………………………………………….. 22 8 Ginkgolides and bilobalides present in ginkgo biloba………………………….... 34 9 A schematic representation of dosage schedule of different studied groups…….. 40 10 Standard curve of DA……………………………………………………….…….. 43 11 Standard curve of GSH……………………………………………….……….…... 45 12 Standard curve of MDA……………………………………………….………...... 47 13 Standard curve of iron………………………………………………………….…. 49 14 Standard curve of ATP……………………………………………..………….….. 54 15 Standard curve of Bcl-2………………………………………………..………..… 57 16 Standard curve of protein……………………………………………………..….. 59 17 Time interval between reserpine administration and sacrifice of rats for A: DA level and B: catalepsy score…………………………………………………….……….. 69 18 Time interval between last irradiation dose and reserpine administration for A: DA level and B: catalepsy score……………………………………………………….. 69 19 Effect of EGb761, whole body γ-irradiation or their combination on the catalepsy score in reserpinized rats.………………………………………………………….. 70 20 Effect of EGb761, whole body γ-irradiation or their combination on the striatal dopamine (DA) level in reserpinized rats. ……………………………………..……. 71 21 Effect of EGb761, whole body γ-irradiation or their combination on the brain levels of glutathione (GSH), malonadialdehyde (MDA) and iron in reserpinized rats…… 72 22 Effect of EGb761, whole body γ-irradiation or their combination on the brain activities of glutathione-S-transeferase (GST) and NADPH-quinone oxidoreductase (NQO) in reserpinized rats…………………….………………………………….… 73 23 Effect of EGb761, whole body γ-irradiation or their combination on complex I activity as well as the levels of adenosine triphosphate (ATP) and B cell lymphoma-2 (Bcl-2) protein in brain mitochondria of reserpinized rats……………. 74 24 Correlation analysis in reserpine-induced Parkinsonism group………………….. 75 25 Correlation analysis in EGb761+reserpine group……………………..…………. 76 26 Correlation analysis in radiation+reserpine group……………………………….. 77 27 Transmission electron micrograph of striatal neurons……………………….……… 78

Page 8 of 152 AIM OF THE WORK

Page 9 of 152 1. Aim of the Work

Par kinson's disease (PD) is an age-related neurodegenerative disease; with almost an equal incidence in both sexes and its prevalence is predicted to increase dramatically in the coming decades. The main clinical features of PD include resting tremor, rigidity and bradykinesia. Pathologically, the disease is characterized by loss of dopaminergic neurons in the substantia nigra (SN) and reduction in striatal dopamine (DA) level with intracellular proteinaceous inclusions called Lewy's bodies (LB). Despite considerable research efforts, the exact causes and mechanisms are not completely understood. Understanding the molecular pathology and finding the cause of dopaminergic cell loss can lead to exploring therapies that could prevent and cure PD.

PD is multifactorial in terms of both etiology and pathogenesis. Several biochemical factors appear to be involved in the pathogenic cascade of events leading to cellular dysfunction and death in PD; including oxidative stress, mitochondrial dysfunction, excitotoxicity, along with accumulation of iron in brain. Mitochondrial dysfunction can result in excessive production of reactive oxygen species (ROS), triggering the apoptotic death of dopaminergic cells reported in PD patients.

Reserpine is a potent, naturally-occurring alkaloid, derived from roots of several members of Rauwolfia genus. Reserpine has the capabability to deplete biogenic amines such as noradrenaline (NA), DA and serotonin; in addition it is a powerful oxidant. The reserpine model was the first model that became available for testing symptomatic anti-PD treatments. The reserpine-induced model in the rat is characterized by akinesia, bradykinesia, hypokinesia, catalepsy and tremors.

Vari ous epidemiological studies indicated that the dietary habits and dietary antioxidant consumption can influence Parkinsonsim. Ginkgo leaf is a widely used herbal medicine. The beneficial effects of Ginkgo biloba substances were known for thousands of years in traditional Chinese medicine. The study of biological activities of EGb761, a standardized extract of ginkgo biloba with a well defined mixture, started more than 20 years ago. EGb761 is an antioxidant, which is reported to possess the capability of scavenging various ROS per se. In addition, EGb761 was shown to exert protective properties against animal models of hypoxia, cerebral ischemia, Parkinsonism and radiation-induced brain injury.

Page 10 of 152 On the other hand, there is a controversy about the effect of whole body exposure to low dose radiation (LDR) on the central nervous system generally, and dopaminergic neurons in particular. Previous studies showed that high doses of ionizing radiation are usually harmful to the nervous system and can lead to peripheral neuropathy, radiation myelopathy, nervous injuries and deoxyribonucleic acid (DNA) damages. However, recent studies suggested that LDR rendered neuroprotection; which had been revealed in different animal models such as inherited glaucoma, optic nerve crush, contusive spinal cord injury and PD models.

The multifactorial etiology of PD suggests that agents with multiple targets such as EGb761 and LDR could have potential benefits for PD; therefore, the present study was undertaken to investigate the pretreatment effects of EGb761 or LDR or their combination on neurological dysfunctions in the reserpine-treated rat model of PD.

Page 11 of 152 INTRODUCTION

Page 12 of 152 2. Introduction 2.1. Brain

The human brain is the center of the human nervous system. Enclosed in the cranium, the human brain has the same general structure as that of other mammals, but is over three times larger than the brain of a typical mammal with an equivalent body size. The adult human brain (Fig. 1) weighs on average about 1.5 kg with a volume of around 1130 cm3 in women and 1260 cm3 in men, although there is substantial inter-individual variations (Cosgrove et al., 2007). Men with the same body height and body surface area as women have on average 100g heavier brains (Ankney, 1992); however, these differences do not correlate in any simple way with the neurons counts or with overall measures of cognitive performance (Gur et al., 1999). At the age of 20, a man has around 176,000 km and a woman about 149,000 km of myelinated axons in their brains (Marner et al., 2003). Table 1 demonstrates different brain areas and their functions.

The brain consumes up to twenty percent of the energy used by the human body, which is the highest share among other body organs. Normally, brain metabolism is completely dependent upon blood glucose as an energy source, since fatty acids do not cross the blood-brain barrier. During times of low glucose (such as starvation), the brain will primarily use ketone bodies as fuel with a smaller requirement for glucose. The brain can also utilize lactate during exercise. However, the brain does store glucose in the form of glycogen to a limited extent, in contrast, for example, to skeletal muscle (Quistorff et al., 2008).

Fig. 1. Different brain regions.

Page 13 of 152 Table 1. Different brain regions and their functions (Palande, 2010) Brain Regions Functions The outermost layer of the cerebral hemisphere, made up of gray matter. It is involved in the functions Cerebral Cortex of learning new information, forming thoughts, making decisions, analyzing sensory data and performing memory functions. Corpus Callosum Connects right and left hemisphere and allows communication between the two hemispheres. Responsible for memory and cognitive functions. Enables the human being to concentrate and attend, it Frontal Lobe also makes him capable of elaboration of thought, judgment and inhibition. Thus involved in personality development and emotional traits. Also helps in voluntary motor activity and motor speech Parietal Lobe Processing of sensory input and sensory discrimination. Helps in body orientation. Concerned with primary visual reception area and primary visual association area which allow for Occipital Lobe visual interpretation. Rules over auditory receptive area and association areas. It takes care of expressed behavior, receptive Temporal Lobe speech and information retrieval. Manages olfactory pathways, amygdala and their different pathways as well as hippocampi and their Limbic System different pathways. Limbic lobes control the functions related to sex, rage, fear; emotions. The system is responsible for integration of recent memory and biological rhythms. These are the subcortical gray matter nuclei which act as processing link between thalamus and motor Basal Ganglia cortex. Their functions include initiation and direction of voluntary movement, balance, postural reflexes and regulation of automatic movement. Sends the incoming sensory nerve impulses to the required appropriate regions of the brain for further processing. Most sensory signals, like auditory signals, visual signals and somatosensory signals pass Thalamus through this relay station before being further interpreted in the brain. Its main function is providing the brain information on what is happening outside the body. Other functions include motor control and control of muscular movements. Integration center of Autonomic Nervous System. It helps regulate body temperature and endocrine functions. It regulates various sensations, such as hunger and thirst. It is responsible for maintaining the Hypothalamus daily sleep and awake cycle. It also controls emotions, autonomic functions and motor functions and maintains homeostasis by exerting control on the pituitary gland. Internal Capsule It contains different motor tracts. Dysfunction leads to paralysis of the opposite side of the body. Reticular Responsible for arousal from sleep, wakefulness and attention. Dysfunction may lead to altered level of Activating consciousness. System Coordinates and controls voluntary movements, maintains balance and equilibrium while walking, Cerebellum swimming, riding, etc., stores memory for reflex motor acts and coordinates simultaneous subconscious actions, like eating while talking or listening etc. Contains auditory and visual reflex centers. It is responsible for the reflex movements of the muscles of Midbrain the head, neck and the eye and provides a passage for different neurons going in and coming out of the cerebrum. It contains the respiratory center. Has control over skin of face, tongue, teeth, muscles of mastication, muscles of eye which rotates eye outward, facial muscles of expression and internal auditory passage. It Pons plays an important role in the level of arousal or consciousness and sleep and is involved in controlling autonomic body functions. It contains the cardiac, respiratory and vasomotor centers and executes the most important function of Medulla the brain; that is, regulating our life processes such as breathing, maintaining a steady heart rate and Oblangeta blood pressure, regurgitation (vomiting), swallowing, urination, defecation and coordination of life saving reflexes.

Page 14 of 152 Nerve cells, or neurons, transmit signals from the environment to the central nervous system (CNS) and from the CNS back to other organs (the periphery). This signal transmission is mediated primarily by small molecules called neurotransmitters (NTs). In general, NTs can be classified as either excitatory or inhibitory. Excitatory NTs increase the activity of the signal receiving (i.e. postsynaptic) neuron, while the inhibitory NTs decrease it. Among the NTs of most interest to researchers are DA, serotonin (5-HT), glutamate, gamma aminobutyric acid (GABA), opioid peptides and adenosine. The neurons that release these substances form the basis of neural circuits that link different areas of the brain in a complex network of pathways and feedback loops. The integrated activity of these circuits regulates mood, activity and the behaviors that may underlie different brain disorders (Gonzales and Jaworski, 1997).

Dopaminergic neurons produce DA from the dietary amino acid tyrosine. The neurons then store the DA in small vesicles in the terminals of their axons. When the dopaminergic neurons are activated, the resulting change in the electrical charges on both sides of the cell membrane (depolarization) induces DA release into the gap separating the neurons (the synaptic cleft) through a process called exocytosis. Then the neurons recapture DA through a specific carrier system located on the cell membrane to terminate the signaling process (Di Chiara, 1997).

Distribution of dopaminergic neurons

Dopaminergic neurons originate in three cell groups located in the brain stem. These cell groups are labeled A8, A9, and A10 and correspond to brain regions called the retrorubral field, the substantia nigra pars compacta (SNpc), and the ventral tegmental area (VTA), respectively. From these cell groups, the axons of dopaminergic neurons extend to discrete regions of the forebrain, forming the following three neuronal systems (Fig. 2): The nigrostriatal system; which comprises dopaminergic neurons that originate in the A9 group and terminate in a region called the dorsal striatum. This region, which includes the caudate nucleus and the putamen (CPU), is involved in learning to execute complex movements triggered by a voluntary command (e.g. driving a car). The degeneration of dopaminergic neurons in the dorsal striatum causes the motor disturbances that occur in PD (Di Chiara, 1997).

A second circuit; the mesolimbic system, originates in the A10 and part of the A9 groups. These neurons terminate in the ventral striatum which plays a role in the learning and performing of certain behaviors in response to motivation (Ungerstedt, 1971). A third group of dopaminergic neurons originates in the A9 and A10 groups and terminates in various regions of the cerebral cortex that are

Page 15 of 152 involved in attention and short-term memory, forming the mesocortical system (Thierry et al., 1973).

Fig. 2. Schematic representation of the major dopaminergic systems (viewed from the top of the head). The nigrostriatal system originates in the A9 cell group and extends to the dorsal striatum, which includes the CPU. The mesolimbic system originates primarily in the A10 cell group and extends to the ventral striatum, which includes the nucleus accumbens (NAc) and the olfactory tubercle (OT). The mesocortical system also originates primarily in the A10 cell group and affects various regions of the cerebral cortex (Di Chiara, 1997).

Page 16 of 152 2.2. Parkinson’s disease

Par kinson’s disease (PD) is the second most common neurodegenerative disease; after Alzehiemer's disease, affecting approximately 1% of the human population aged 65 and above (Schoenberg, 1986; Youdim and Riederer, 1997). The disease is progressive involving neurodegeneration of dopaminergic neurons of the SN which is a part of the midbrain. Clinically, any disease that includes striatal DA deficiency or direct striatal damage may lead to “Parkinsonism”; a syndrome characterized by tremors at rest, rigidity, slowness or absence of voluntary movement, postural instability, and freezing. PD is the most common cause of Parkinsonism, accounting for ~80% of cases. PD tremors occur at rest but decrease with voluntary movement. Rigidity refers to the increased resistance (stiffness) to passive movement of a patient's limbs. Other symptoms include; bradykinesia (slowness of movement), hypokinesia (reduction in movement amplitude) and akinesia (absence of normal unconscious movements such as arm swing in walking). PD patients also manifest a variety of symptoms, including lack of normal facial expression, decreased voice volume (hypophonia), drooling (failure to swallow without thinking about it), decreased speed of handwriting and decreased stride length during walking.

Bradykinesia may significantly impair the quality of life because it takes much longer to perform everyday tasks such as dressing or eating. PD patients may lose normal postural reflexes, leading to falls and, sometimes, confinement to a wheelchair. Freezing, the inability to begin a voluntary movement such as walking (i.e. patients remain “stuck” to the ground as they attempt to begin moving), is a common symptom of Parkinsonism. Abnormalities of cognition also occur frequently; patients may become passive; they may sit quietly unless encouraged to participate in activities. Responses to questions are delayed and cognitive processes are slowed (bradyphrenia). Depression is common and dementia is significantly more frequent in PD, especially in older patients (Dauer and Przedborski, 2003).

O ver time, symptoms worsen, and prior to the introduction of levodopa, the mortality rate among PD patients was three times that of the normal age-matched subjects. While levodopa has dramatically improved the quality of life for PD patients, population-based surveys suggest that these patients continue to display decreased longevity compared to the general population (Hely et al., 1989; Levy et al., 2002). Furthermore, most PD patients suffer from considerable motor disability after 5–10 years of disease, even when expertly treated with available symptomatic medications.

Page 17 of 152 Various Parkinsonian symptoms (particularly resting tremors) were described by Galen (138-201 AD), and an assortment of tremors has even been described in 2500 Before Christ (BC) India (Stern, 1989). It was not until 1817 before the set of symptoms for PD were assembled and described by Dr James Parkinson as “shaking palsy” (Parkinson, 1817). The disease was then renamed PD by Jean- Martin Charcôt (Charcôt, 1886).

2.2.1. Etiology of Parkinson's disease:

PD is multifactorial in terms of pathogenesis, with a complex combination of genetic and non-genetic components. Non-genetic or sporadic form represents the majority of these cases (Ramassamy, 2006). The cause of sporadic PD is unknown. The environmental toxin hypothesis was dominant for much of the 20th century, especially because of the example of post-encephalitic PD and the discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism. The environmental hypothesis suggests that PD-related neurodegeneration results from exposure to a dopaminergic neurotoxin. The finding that people intoxicated with MPTP develop a syndrome nearly identical to PD is an example of how an exogenous toxin can mimic the clinical and pathological features of PD (Langston et al., 1983).

Th e susceptibility of humans to systemic exposure to MPTP lead to its discovery as a selective dopaminergic neurotoxin, where several people had turned up in hospital in northern California in a three week period in 1982 presenting with Parkinsonian symptomatology. The speed in which their symptoms had become remarkable and the fairly young age of the patients suggested against idiopathic PD. Ultimately, it was discovered that these individuals had developed their Parkinsonian movement dysfunction due to intravenous injection of black market street heroin that was contaminated with MPTP which was then identified as a dopaminergic neurotoxin (Langston et al., 1983; Alexi et al., 2000). The herbicide paraquat is structurally similar to 1- methyl-4-phenylpyridinium (MPP+), the active metabolite of MPTP. Like MPP+, the insecticide rotenone is also a mitochondrial poison present in the environment. Human epidemiological studies have reported that residence in a rural environment and related exposure to herbicides and pesticides is associated with an elevated risk of PD (Tanner, 1992).

Another possibility, which does not fit neatly into a genetic or environmental category, is that an endogenous toxin may be responsible for PD neurodegeneration. Distortions of normal metabolism might create toxic substances because of the environmental exposures or the inherited differences in metabolic pathways. One source of endogenous toxins may be the normal

Page 18 of 152 metabolism of DA, which generates harmful ROS (Cohen, 1984). Consistent with the endogenous toxin hypothesis is the report that patients harboring specific polymorphisms in the xenobiotics-detoxifying enzyme cytochrome P450 gene may be at greater risk of developing young-onset PD (Sandy et al., 1996). Furthermore, isoquinoline derivatives, which are toxic to dopaminergic neurons, have been recovered from PD brains (Nagatsu, 1997).

2.2.2. Pathogenesis of Parkinson's disease: Molecular and neurochemical pathways

W hatever insult initially provokes the neurodegeneration process, studies of toxic PD models and the functions of genes implicated in inherited forms of PD suggest two major hypotheses regarding the pathogenesis of the disease. One hypothesis suggests that misfolding and aggregation of proteins are instrumental in the death of SNpc dopaminergic neurons, while the other proposes that the main cause is mitochondrial dysfunction and the associated oxidative stress, including toxic oxidized DA metabolic products. An example of the interaction between different pathways is the finding that oxidative damage to the neuronal protein “α-synuclein” can enhance its ability to misfold and aggregate to form the intracellular inclusion called “Lewy's bodies” (LBs) (Giasson et al., 2000). Another example is the finding that multiple cell death-related molecular pathways are activated during PD neurodegeneration and they ultimately engage to common down stream machinery, such as apoptosis. Clearly, these findings are of great importance in deciding about possible therapeutic strategies for PD (Dauer and Przedborski, 2003).

2.2.2.1. Misfolding and aggregation of proteins

PD is characterized by the presence of the proteinaceous neuronal inclusions LBs in the midbrain (Forno, 1996; Andersen, 2000). Although the composition and location (i.e. intra- or extracellular) of protein aggregates differ from a disease to another, this common feature suggests that protein deposition per se, or some related event, is toxic to neurons. Aggregated or soluble misfolded proteins could be neurotoxic through a variety of mechanisms. Protein aggregates could directly cause damage, either by deforming the cell or interfering with intracellular transmission in neurons. Protein inclusions might also sequester proteins that are important for cell survival (Dauer and Przedborski, 2003). Cytoplasmic protein inclusions may not result simply from precipitated misfolded protein but rather from an active process meant to sequester soluble misfolded proteins from the cellular milieu (Kopito, 2000).

Page 19 of 152 A ccordingly, inclusion formation, while possibly indicative of a cell under attack, may be a defensive measure aimed at removing toxic soluble misfolded proteins (Cummings et al., 2001; Auluck et al., 2002). LBs contain α-synuclein aggregates, ubiquitin (Forno, 1996), parkin (Shimura et al., 1999), the adenosine triphosphatase (ATPase) torsin A (Shashidharan et al., 2000) and other proteasomal pathway proteins (Ii et al., 1997). The Ubiquitin-proteasome system (UPS) is one of the primary pathways for the degradation of cellular, non membrane-bound proteins. Rare familial forms of PD involve genetic mutations in various components of this system. UPS functions to maintain appropriate cellular concentrations of short-lived regulatory/functional proteins and destroy abnormal, misfolded, damaged or toxic proteins. In addition, defects in, or overload of, the UPS might result in accumulation of toxic proteins and cellular dysfunction or death. This UPS overload or dysfunction appears to occur under experimental conditions of increased oxidative stress or mitochondrial complex I inhibition (Betarbet et al., 2005).

α-synuclein is a 140 amino acid protein that consists of a N-terminal region, a hydrophobic central region, and an acidic C-terminal region. The protein appears to possess considerable conformational plasticity and, depending on the environment, can remain unfolded, form alpha-helices or beta-sheets, or exist as a monomer or oligomer (Uversky, 2003); as well as cross link with itself, or with other proteins via advanced glycation end products (Munch et al., 2000) or with neuromelanin (Fasano et al., 2003). The function of α-synuclein, not yet fully clear, appears to involve maintenance and transport of synaptic vesicles and participation in neuronal function through multiple interactions with other proteins and through its involvement in lipid transport and membrane biogenesis (Kahle et al., 2002). Two missense mutations in α-synuclein cause dominantly inherited PD (Polymeropoulos et al., 1997; Kruger et al., 1998). A supporting observation is that a significant deficit in motor performance tests related to nigrostriatal function was developed in mouse with mutated α-synuclein gene (Plaas et al., 2008).

In addition, 6-hydroxydopamine (6-OHDA) toxicity seems to be influenced by α-synuclein, since α-synuclein-deleted mice are more resistant against 6-OHDA than their wild type (Alvarez-Fischer et al., 2008). α-synuclein mutations promote the formation of protofibrils (Conway et al., 2000), which may cause toxicity by permeabilizing synaptic vesicles (Volles et al., 2001; Lashuel et al., 2002), allowing DA to leak into the cytoplasm and participate in reactions that generate oxidative stress. Another possible role of α-synuclein in PD is through its effect on tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis.TH is activated by phosphorylation of the TH regulatory domain; α- synuclein inhibits TH by activating the phosphatase enzyme, which in turn

Page 20 of 152 reduces DA synthesis in vitro (Peng et al., 2005; Alerte et al., 2008). Moreover, it has been proposed that intraneuronal DA levels can be a major modulator of α- synuclein aggregation and inclusion formation (Mazzulli et al., 2006). α- synuclein induced apoptosis in cultured human dopaminergic neurons is dependant on DA and is mediated by ROS (Xu et al., 2002). Parkin, a 465 amino acid protein a component of the UPS that identifies and targets misfolded proteins to the proteasome for degradation (Sherman and Goldberg, 2001). Another study suggests that Parkin is a ligase which acts to ligate α-synuclein to UPS (Shimura et al., 2001).

2.2.2.2. Oxidative stress and mitochondrial dysfunction

Compared to other organs in the body, the brain is more susceptible to oxidative damage due to several factors which include high oxygen utilization (Clark and Sokoloff, 1994), high iron content (Gerlach et al., 1994), presence of excess unsaturated fatty acids which are targets for lipid peroxidation (Halliwell, 1992) and decreased activities of detoxifying enzymes like superoxide dismutase (SOD), catalase and glutathione reductase (GR) (Dringen et al., 2000). Dopaminergic neurons may be a particularly fertile environment for the generation of ROS, as the •- metabolism of DA produces hydrogen peroxide (H2O2) and superoxide radicals (O2 ) (Graham, 1978). Intracellularly, DA is either degraded by monoamine oxidase-A (MAO-A) (Gotz et al., 1994) or by auto- or non-enzymatic oxidation (Fig. 3). DA metabolism by MAO-A leads to the production of dihydroxyphenylacetic acid (DOPAC) and H2O2 with the consumption of O2 and H2O (Maker et al., 1981; Gesi et al., 2001). Intracellular autoxidation of DA generates H2O2 and DA-quinone, a molecule that damages proteins by reacting with cysteine residues (Graham et al., 1978; Sulzer and Zecca, 2000). DA-quinone participates in nucleophilic addition reactions with protein sulfhydryl groups (Stokes et al., 1999), leading to structural modifications of proteins and reduced levels of glutathione (GSH), which is the major cellular redox buffer used to counteract oxidative stress.

H 2O +O 2 H 2O 2+NH3 H O OH OH O M A O -A H O H O O NH2 D opam ine DOPLAD OH O DOPAC

OH NH2

O OH S O NH2 D opam ine quinone oxidation RHN COOR O - , H O 2 2 2 5-S-cysteinyl-dopam ine Aut d e r iv a tiv es

Fig. 3. Oxidation of DA (Hald and Lotharius, 2005). DOPALD, dihydroxyphenylacetaldehyde; DOPAC, dihydroxyphenylacetic acid.

Page 21 of 152 Th e DA-quinone formed instantaneously cyclizes; irreversibly, to form aminochrome (Shen and Dryhurst, 1998). The aminochrome formed can undergo four types of reactions as shown in Fig. 4. The first reaction is the polymerization to form neuromelanin (Fig. 4, reaction 1), a reaction that is dependant on aminochrome concentration. The second reaction undergone by aminochrome is the formation of adducts with α-synuclein (Fig. 4, reaction 2), inducing and stabilizing the formation of neurotoxic protofibrils (Conway et al., 2001), a reaction that is inhibited by the neuroprotective enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase (NQO) or “DT-diaphorase” (Paris et al., 2009).

Am inochrome can also be reduced through one-electron reduction to form leukoaminochrome O-semiquinone radicals (Fig. 4, reaction 3), a reaction that can be one possible source of reactive species (Segura-Aguilar et al., 2001; Smythies, 2002). Leukoaminochrome O-semiquinone radical is a very reactive metabolite (Segura-Aguilar et al., 1998) that autoxidizes in the presence of oxygen or transition metal ions like manganese, copper or iron (Shen and Dryhurst, 1998; Paris et al., 2001), initiating a redox cycling process between aminochrome and leukoaminochrome O-semiquinone (Fig. 4, reaction 4) (Segura-Aguilar and Kostrzewa, 2006). This redox cycling is initiated by flavoenzymes, which catalyze the one-electron reduction of aminochrome using nicotinamide adenine dinucleotide (NADH) or NADPH; resulting in i) the depletion of NADH, which is required for ATP synthesis in the mitochondria; ii) depletion of NADPH, which is required to keep GSH in the reduced state necessary to exert its antioxidant action; iii) depletion of oxygen which is required for the ATP synthesis in mitochondria; •- and iv) formation of O2 , which spontaneously or enzymatically generate H2O2, the precursor of hydroxyl radical (•OH). The leukoaminochrome O-semiquinone radical has been proposed to be an endogenous neurotoxin that induces the neurodegeneration of dopaminergic neurons in PD (Paris et al., 2001; Fuentes et al., 2007).

Page 22 of 152 OH N eu ro m ela n in

DA-quinone OH N H Leukoam inochrom e (1) (5) NADP+ O D T-diaphorase Alpha-Synuclein (2) N A D (P )H O N H Aminochrome

O .- .- 2 O2 NADH/NADPH (4) Flavoenzym es (3) H O 2 2 O2 NAD+/NADP+ P ro tofib rils O

Fe2+ N NADH/NADPH depletion OH H

Leukoam inochrom e O -sem iquinone

Enzym e inactivation .OH O 2 depletion

N eurotoxicity

Fig. 4. Aminochrome metabolism. Aminochrome can participate in four different reactions: (1) polymerization with other aminochrome containing molecules including proteins, lipids, and metals (reaction 1); (2) formation of adducts with alpha-synuclein, inducing and stabilizing the formation of neurotoxic protofibrils (reaction 2); (3) one-electron reduction by flavoenzymes to form the leukoaminochrome O-semiquinone radical (reaction 3), which can autoxidize in the presence of oxygen to generate redox cycling between aminochrome and this radical (reaction 4); and (4) two electron reduction, a reaction catalyzed by NQO, to form leukoaminochrome (reaction 5) (Paris et al., 2009).

Aminochrome can be reduced by two electrons to form leukoaminochrome, a process which is catalyzed by NQO (Fig. 4, reaction 5) (Segura-Aguilar et al., 2001). This process prevents aminochrome from participating in two neurotoxic reactions to form leukoaminochrome O-semiquinone radicals through the one- electron reduction of aminochrome and prevents the formation of neurotoxic protofibrils during the formation of aminochrome adducts with α-synuclein (Segura-Aguilar and Lind 1989; Cardenas et al., 2008). In addition, DA- quinones have been shown to inhibit glutamate and DA transporter function in synaptosomes (Berman et al., 1996; Berman and Hastings, 1997), inhibit TH in cell free systems (Kuhn et al., 1999) and promote H+ leakage from mitochondria resulting in uncoupling of respiration to ATP synthesis (Berman and Hastings, 1999).

Page 23 of 152 Mi tochondria are responsible for generating 90% of the ATP required for all cellular functions, for controlling the cellular redox state and for regulating cytoplasmic calcium levels by acting as the major intracellular sink for this ion. Oxidative damage to the mitochondria might interfere with all of these functions. Mitochondrial dysfunction appears to play a major role in the neurodegeneration associated with the pathology of PD (Albers and Beal, 2000). Mitochondria- related energy failure may disrupt vesicular storage of DA, causing the free cytosolic concentration of DA to rise and allowing harmful DA-mediated reactions to damage cellular macromolecules. The reduced complex I function associated with PD (Schapira, 1994; Sherer et al., 2002) may be dependent on DA, which has been shown to inhibit complex I when injected into the brain ventricles of rats (Ben-Shachar et al., 1995).

A lthough the insecticide rotenone inhibits complex I throughout the brain, it only exerts its toxic effects on dopaminergic neurons; suggesting that dopaminergic neurons have an intrinsic sensitivity to complex I defects due to DA release (Sherer et al., 2002). The possibility that an oxidative phosphorylation defect plays a role in the pathogenesis of PD was supported by the discovery that MPTP blocks the mitochondrial electron transport chain by inhibiting complex I (Nicklas et al., 1987). Interestingly, complex I has been found to be one of the most severely affected mitochondrial enzymes during oxidative stress (Lenaz et al., 1997). In synaptic mitochondria, complex I exerts a major control over oxidative phosphorylation such that a decrease in its activity by 25% drastically affected ATP synthesis and the overall energy metabolism within the cell. On the other hand, inhibition of complex III and IV up to 80% was necessary to show similar effect (Davey et al., 1998).

Nearly 100% of the molecular oxygen is consumed by the mitochondrial respiration, and powerful oxidants are normally produced as byproducts, including •- •- H2O2 and O2 . Inhibition of complex I increases the production of the O2 , which may form toxic •OH or react with nitric oxide to form peroxynitrite. These molecules may cause cellular damage by reacting with nucleic acids, proteins, and lipids. One target of these reactive species may be the electron transport chain itself (Cohen, 2000), leading to mitochondrial damage and further production of ROS. The presence of ROS has been reported to increase the amount of misfolded proteins, and increase the demand on the UPS to remove them. Several biological markers of oxidative damage are elevated in the SNpc of PD brains (Przedborski and Jackson-Lewis, 2000). During oxidative damage of lipids, aldehydes are formed as by-products, the most prevalent of these being 4-hydroxynoneal (4HNE). 4HNE has been proposed to integrate into membranes affecting in vivo membrane fluidity. It has been found that GSH forms conjugate with 4HNE in a

Page 24 of 152 reaction catalyzed by glutathione-S-transeferase (GST) preventing it from incorporating into the membranes (Chen and Yu, 1994; 1996). Additionally, 4HNE may also form adducts with important proteins like Na+/K+ ATPase rendering them inactive. GSH has also been observed to prevent 4HNE from conjugating with proteins (Subramaniam et al., 1997). In the PD brains, the levels of 4HNE adducts have been reported to be elevated, which might be reflective of the loss in GSH in the SN (Yoritaka et al., 1996).

Glutathione, brain and Parkinson's disease

GSH is present in the brain in millimolar concentrations (Dringen et al., 2000). Limited information is available about the exact mechanism of the transport of GSH across the blood brain barrier, its uptake and its release from brain cells and whether these play a role in GSH homeostasis in the brain (Yang et al., 1994; Kannan et al., 1999). The different pathways involved in GSH metabolism are illustrated in Fig. 5.

A part from the antioxidant functions of GSH in the brain, extracellular GSH has been hypothesized to have additional functions as a NT (Janaky et al., 1999) and neurohormone, in the detoxification of glutamate and in leukotriene metabolism (Dringen, 2000). According to evidences accumulated based on coculture experiments, brain astrocytes and neurons appear to interact metabolically with one another in terms of GSH metabolism (Dringen et al., 2000).

Page 25 of 152 Fig. 5. A schematic representation of the different pathways involved in GSH metabolism. GSH is synthesized de novo in the cytoplasm from its constituent amino acids in a two-step reaction. The cellular GSH pool is also contributed to by conversion from GSSG, the oxidized form of GSH. H2O2 produced in the mitochondria is detoxified by the conversion of GSH to GSSG. GSH also participates in the formation of mixed disulfides with thiol-proteins (RSH), which can further become metabolized. GSH that is secreted from the cell is broken down into its constituent amino acids which can be taken up by the cells to be used for de novo GSH synthesis (Bharath et al., 2002). ETC: electron transport chain, SOD: superoxide dismutase.

It has been observed that there is an age-dependent depletion in intracellular GSH of many organisms including humans (Sohal and Weindruch, 1996). In humans, there appears to be a decline in the GSH levels in the cerebrospinal fluid during aging (Cudkowisz et al., 1999). Studies have shown that aged mice have a 30% decrease in levels of GSH compared with younger animals (Chen et al., 1989; Hussain et al., 1995). Since the brain requires extensive ROS detoxification, it is evident that a decrease in GSH content could increase oxidative damage making the brain more susceptible to neurological disorders. GSH plays an important role in the adult brain by removing H2O2 formed during normal cellular metabolism. In general, SN has lower levels of GSH compared to other regions in the brain. Previous experiments (Abbott et al., 1990; Kang et al., 1999) have demonstrated that the relative variations in levels of GSH in different brain regions are cortex > cerebellum > hippocampus > striatum > SN. However, the GSH profiles of all regions are the same through the lifespan, namely, high values during growth dropping to a maturation plateau and then decreasing 30% during aging (Chen et al., 1989). It has been observed that during PD, there is a further reduction in GSH levels within the SNpc (Riederer et al., 1989; Sofic et al., 1992). In fact, GSH depletion is the first indicator of oxidative stress during PD progression suggesting a concomitant increase in ROS. Although GSH is not the only antioxidant depleted during PD, the magnitude of GSH depletion appears to parallel the severity of the disease and occurs prior to other hallmarks of the disease including decreased activity of mitochondrial complex I (Perry and Yong, 1986; Jenner, 1993). Fig. 6 highlights the functions of GSH in counteracting against oxidative stress during PD.

Depletion of brain GSH has been shown to result in decreases in mitochondrial enzyme activities in preweaning rats as well as losses in ATP production in the aging murine brain (Heales et al., 1995; Martinez et al., 1995). The consequences of GSH depletion on mitochondria in dopaminergic neurons of the SN during PD were reported by Jha et al. (2000) who constructed a dopaminergic PC12 cell line model system; wherein the levels of the γ-glutamyl cysteinyl synthase (γ-GCS) enzyme were experimentally decreased resulting in a reduction in GSH synthesis. A decrease in the mitochondrial GSH in these genetically engineered cells resulted in increased oxidative stress and impaired mitochondrial

Page 26 of 152 function as reflected by decreased mitochondrial respiration and ATP synthesis. GSH is known to protect proteins from oxidation by conjugating with oxidized thiol groups to form protein-SS-G mixed disulfides, which can then be re-reduced to protein and GSH by GR or thioredoxin (Ravindranath and Reed, 1990). In dopaminergic cells in vivo, GSH can also bind to quinones formed during oxidation of DA and prevent these compounds from reacting with protein sulfhydryl groups (Fornstedt et al., 1990; Hastings et al., 1996). Moreover, Sriram et al. (1998) have demonstrated that thiol oxidation and loss of mitochondrial complex I activity precede excitatory amino acid mediated neurodegeneration. Both (1)could be prevented by treatment with antioxidant thiol agents. In a previous study, when GSH was administered to PD patients by i.v. injections daily for up to a month, a significant improvement in disease related disability was observed. It can be implied that maintenance of thiol homeostasis is critical for the protection of dopaminergic SN neurons against neurodegeneration (Sechi et al., 1996).

(2) (3)

Fig. 6. The different roles of GSH: a schematic representation of the antioxidant properties of GSH as relevant to SN dopaminergic neuronal cells in PD. Apart from the detoxification of ROS themselves, GSH may protect neurons against the build-up(4) of protein aggregates (1) which form Lewy bodies within the cell; mitochondrial dysfunction (2) due to inhibition of complex I activity; the deleterious effects of the lipid peroxidation by-product 4HNE (3); and protein oxidation (4). Figure cited from Bharath(1et) al. (2002).

Role of iron in Parkinson's disease

(3) Iron concentrations in the(2) brain change over the lifetime: they are highest at birth, decrease during the first two postnatal weeks, then increase again until death

Page 27 of 152 (Aoki et al., 1989; Connor, 1994). Iron is essential for the adequate development and functioning of the brain (Lozoff et al., 2006). It acts as a cofactor in cell proliferation/DNA synthesis, mitochondrial electron transport chain and in myelin and NTs synthesis. Despite the natural increase in brain iron concentrations, reports have regularly described PD brains as having extensive iron deposits, beyond that of non-PD brains of a similar age. A number of postmortem histochemical studies (Gotz et al., 2004) showed increased levels of iron deposits in the SN in Parkinsonian compared with control brains. Imaging studies of living PD patients have confirmed the presence of increased iron deposits in the SN (Martin et al., 2008) and have linked the extent of deposits to the severity of disease (Ye et al., 1996). An experimental study have detected reduction in striatal DA levels in rats with increased brain iron concentrations resulting from injected or infused iron compounds (Wesemann et al., 1994). The iron chelator desferrioxamine has been shown to protect against oxidative stress induced by 6- OHDA, induced DA depletion and prevent impaired motor function in treated animals (Ben-Shachar and Youdim, 1991; Youdim et al., 2004).

Given the observation that males are at an approximately 50% increased risk of developing PD compared to females (Elbaz et al., 2002; Wooten et al., 2004), a hypothesis can be constructed that menstruation is protective for women because it might decrease their body's iron. Interestingly, there is some evidence that smoking, or more particularly exposure to nicotine, might have an effect on iron concentrations in the body. Through traditional chemistry techniques, nicotine has been identified as a strong complexing agent for iron (Linert et al., 1996; Bridge et al., 2004) and appears to decrease the formation of 6-OHDA resulting from 2+ Fe and H2O2 reaction with DA, implying that nicotine affects the Fenton reaction (Linert et al., 1999).

In tracellular iron levels are regulated as a labile iron pool, which provides optimum iron levels for vital biochemical reactions and limits the availability of free iron for generation of ROS. Ferritin is the major iron storage protein in the body, which maintains iron in a nonreactive form in the cell. It is uncertain whether the excess iron observed in the PD brains is in a free form or bound to ferritin (Riederer et al., 1992; Mann et al., 1994). Furthermore, Griffiths et al. (1999) demonstrated that in PD patients, ferritin is heavily loaded with iron implying that even if there is an increase in ferritin levels to counter the excess iron levels, the ferritin molecules are saturated with iron. In the event of a superoxide or catechol-mediated release of iron from loaded ferritin pool, there could be an increase in labile iron pool causing an increase in reactive iron available for generation of ROS (Double et al., 1998). Furthermore, iron promotes autoxidation of DA in SN neurons, releasing additional H2O2 (Ben- Shachar et al., 1995). Iron also catalyses the conversion of excess DA to

Page 28 of 152 neuromelanin, an insoluble black-brown pigment that accumulates in all dopaminergic neurons with age in humans (Sulzer et al., 2000). Neuromelanin in general is neuroprotective and sequesters redox active ions in the cell with a high affinity for Fe3+ ions. However, when bound to excess Fe3+, neuromelanin tends to become a prooxidant and reduces Fe3+ to Fe2+, which then gets released from neuromelanin owing to its weak affinity (Ben-Shachar et al., 1991).

Another possible role for iron in PD pathogenesis is through its effect on UPS. A proposed mechanism for iron in UPS dysfunction was suggested by the observation that the iron chelator desferrioxamine attenuates drug-induced proteasome inhibition and subsequent DA neurons loss and reduces the presence of ubiquitin-positive intracellular inclusions in the SN of mice (Zhang et al., 2005). Furthermore, iron and other metals appear to induce α-synuclein fibril formation (Uversky et al., 2001; Golts et al., 2002) and may contribute to or aggravate the existing LBs aggregation. Furthermore, the α-synuclein messenger ribonucleic acid (mRNA) contains a predicted “iron responsive element” (Friedlich et al., 2007), which implies that the same proteins that influence iron metabolism might influence α-synuclein concentrations. Moreover, different studies have reported the potential association between PD and various genes involved in iron metabolism or homeostasis (Table 2).

Iron storage in the brain is similar to and different from the systemic iron storage in that ferritin is found in the brain and thought to be an important iron storage protein, and also other iron storage mechanisms exist in brain including neuromelanin. Disruptions in the structure of the ferritin complex can result in brain iron accumulation and subsequent increased oxidation and mitochondrial dysfunction as documented for neuroferritinopathy, also known as adult-onset basal ganglia disease or neurodegeneration with brain iron accumulation type-2, which appears to be a result of dysregulation in the ferritin gene (Curtis et al., 2001; Chinnery et al., 2003; Burn and Chinnery, 2006).

Table 2. Some human studies investigating iron-related genes in PD (Rhodes and Ritz, 2008)

Cases/Controls Genes/Markers Reference 216/193 TF: G258S, P570S; TFRC: S82G; HFE: H63D, C282Y; Borie et al., 2002 LTF: N534N 179/2261 HFE: C228Y Buchanan et al., 2002 253/not defined FTL: 460–461 ins Chen et al., 2002 58/24/1417 LRP2: −89 CNT, −35 GNA, L159V Lee et al., 2002a 137/47 HFE: H63D, C282Y Dekker et al., 2003 186/186 FTL: L55L, 460–461 ins, H174H Felletschin et al., 2003 388/505 HFE: H63D, C282Y Aamodt et al., 2007

Page 29 of 152 TF, transferrin; TFRC, transferrin receptor; HFE, hemochromatosis gene; LTF, lactotransferrin or lactoferrin; FTL, ferritin light chain; LRP2, low density lipoprotein- related protein 2.

Apoptotic cell death

Num erous evidences and reports suggest the involvement of apoptosis in the death of neurons following exposure to toxic compounds as well as during development and degenerative disorders (Tatton et al., 2003; Ekshyyan and Aw, 2004). Neurons undergo apoptosis through information received from its environment either internal or external. Internal information depends on cell type, state of differentiation or maturity, developmental history, while external environment like appearance and disappearance of hormones, growth factors, cytokines and cell matrix interactions which affect the cell fate. Various factors get activated on ATP depletion to execute the apoptotic death of dopaminergic neurons in PD. A number of apoptotic cascades have been described, such as mitochondrial and death receptor-mediated, p53-dependent and independent as well as caspase-dependent and independent pathways. The activation of caspases destroys important cellular machinery, preventing the synthesis of new proteins, which ultimately leads to irreversible cell injury (Singh and Dikshit, 2007).

Practica lly, it may be difficult to detect the criteria of apoptosis morphologically because the rate of neuronal loss may be low (McGeer et al., 1988) and apoptotic cells seem to disappear rapidly (Raff et al., 1993). For these reasons, some studies of apoptosis in PD have measured molecular components of apoptosis instead of relying on morphological criteria. For example, investigations of the apoptotic molecule Bax demonstrated an increased number of Bax-positive SNpc dopaminergic neurons in PD (Hartmann et al., 2001a). SNpc dopaminergic neurons with increased expression and subcellular redistribution of the anti-apoptotic protein Bcl-xL and with activated apoptotic effector protease caspase-3 have also been found in greater proportion in PD (Hartmann et al., 2000; 2002). Other molecular markers of apoptosis are altered in PD, including the activation of caspase-8 (Hartmann et al., 2001b) and caspase-9 (Viswanath et al., 2001). Moreover, partial DA loss could also contribute to apoptotic death of dopaminergic neurons through enhancing the activity of the proapoptotic enzyme caspase-3 (Ariano et al., 2005).

Initiation of neuronal apoptosis involves number of factors, which are ROS, reactive nitrogen species (RNS), cytokines levels, deprivation in growth factors and the rise in intracellular calcium and DA. A number of cytokines such as interleukin-β (IL-β), IL-2, IL-4 & IL-6 were significantly increased in PD patients (Mogi et al., 1994; 1995). Moreover, significant increase in the density of cells

Page 30 of 152 expressing tumor necrosis factor (TNF-α) and IL-1β was also observed in SN region of PD patients (Teismann et al., 2003). Increase in cytokines levels could switch on different apoptotic pathways involved in the dopaminergic neuronal death (Singh and Dikshit, 2007). Furthermore, deprivation of different trophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3, 4 and 5- cause initiation of apoptosis in vitro. NGF deprivation led to DNA fragmentation and augmented ROS production in sympathetic neurons apoptosis, while addition of Cu/Zn SOD delayed apoptosis (Greenlund et al., 1995).

Besides the activation of diverse factors; altered calcium homeostasis has also been reported in PD, which involves activation of both ionotropic and metabotropic receptors. This is supported by the fact that α-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA) receptor transmission is functionally overactive in PD and its antagonist showed anti-parkinsonism effects in experimental studies (Klockgether et al., 1991; Konitsiotis et al., 2000). Furthermore, N-methyl-D-aspartate (NMDA) receptor is secondarily involved in glutamatergic synaptic transmission; once the neuron has been depolarized by the activation of AMPA/kainate receptors. On the same line, another study elaborated on AMPA receptor regulation mechanisms, which could be a promising future target for safer neuroprotective drugs (Jayakar and Dikshit, 2004). Activation of these receptors led to an increase in intracellular calcium, leading to excitotoxicity and induction of calcium-dependent gene expression and enzyme activities which could possibly contribute to the degeneration of dopaminergic neurons (Singh and Dikshit, 2007).

Page 31 of 152 2.3. Animal models of Parkinson's disease

Neur ological disorders in humans can be modeled in animals using standardized procedures that recreate specific pathogenic events and their behavioral outcomes. In addition to providing an indispensable tool for basic research, animal models of human disorders allow to investigate the therapeutic strategies as a prerequisite to their testing in patients (Cenci et al., 2002). Since the common pharmacological therapies are limited to the symptomatic treatment of PD without arresting the course of the disease, it is extremely important to develop experimental animal models that replicate several aspects of this human disease. Availability of suitable animal models could further allow analyzing the mechanisms that underlie the etiology of this disease and may facilitate the development of novel neuroprotective agents or therapeutic strategies.

Ce rtain criteria must be present in PD animal models. As they should reproduce the main characteristics of the human disease, such as: selective lesion of dopaminergic neurons and depletion of DA from the striatum. In addition, from the behavioral point of view, a Parkinsonian syndrome should be observed, ideally with akinesia, rigidity and resting tremors (Halbach, 2005). General classification of PD models is illustrated in Fig. 7.

Animal models of PD

Pharmacological agent-based model Toxin-based models Other Parkinsonian agents

MPTP Methamphetamine model

Reserpine model 6-Hydroxydopamine

Intranigral injection of iron (II) Rotenone

Paraquat Miscellaneous agents

Fig. 7. Classification of animal models of PD.

2.3.1. Pharmacological agent-based model (Reserpine model)

Page 32 of 152 Reserpine is a potent, naturally-occurring alkaloid, derived from roots of several members of Rauwolfia genus (Doyle et al., 1955). Reserpine has the capabability to deplete biogenic amines such as NA, DA and 5-HT; in addition, it is a powerful oxidant (Metzger et al., 2002). Clinically, reserpine is an important antihypertensive drug, but its use is limited by its side-effects including organ damaging oxidative stress (Al-Bloushi et al., 2009).

The reserpine model was the first model that became available for testing symptomatic anti-PD treatments. The partial removal of the reserpine syndrome in the rat (akinesia, bradykinesia, hypokinesia, catalepsy and tremors) by levodopa led to the development of the levodopa therapy, which has remained the cornerstone of anti-PD treatment up to the present. The mechanism of action of reserpine is thought to be through a reversible functional reduction of dopaminergic neurotransmission, following a diminished capacity of the storage vesicles for storing DA (Gerlach and Riederer, 1996).

2.3.2. Toxin-Based Models

Am ong the neurotoxins used to induce dopaminergic neurodegeneration are: MPTP, 6-OHDA, and more recently paraquat and rotenone have received the most attention. Presumably, all of these toxins provoke the formation of ROS. Rotenone and MPTP are similar in their ability to potently inhibit complex I, but they differ in their ease of use in animals.

In 1982, young drug users developed a rapidly progressive parkinsonian syndrome traced to i.v. use of a street preparation of 1-methyl-4-phenyl-4- propionoxypiperidine (MPPP), an analog of the narcotic meperidine (Demerol) (Langston et al., 1983). MPTP was the responsible neurotoxic contaminant, inadvertently produced during the illicit synthesis of MPPP in a basement laboratory. In humans and monkeys, MPTP produces an irreversible and severe Parkinsonian syndrome characterized by all of the features of PD, including tremor, rigidity, slowness of movement, postural instability and freezing. In MPTP-intoxicated humans and nonhuman primates, the beneficial response to levodopa and development of long-term motor complications to medical therapy are virtually identical to that seen in PD patients. Also similar to PD, the susceptibility to MPTP increases with age in both monkeys and mice (Irwin et al., 1993; Ovadia et al., 1995).

Af ter systemic administration, MPTP, which is highly lipophilic, crosses the blood-brain barrier within minutes (Markey et al., 1984). Once in the brain, the pro-toxin MPTP is oxidized to 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+) by MAO-B. It is then converted to MPP+ (probably by spontaneous

Page 33 of 152 oxidation), the active toxic molecule, and released by an unknown mechanism into the extracellular space. Since MPP+ is a polar molecule, it depends on the plasma membrane carriers to enter cells.

In side the dopaminergic neurons, MPP+ can follow one of three routes: concentration into mitochondria through an active process; interaction with cytosolic enzymes and sequestration into synaptic vesicles via the vesicular monoamine transporters (VMAT). Within the mitochondria, MPP+ blocks complex I, which interrupts the transfer of electrons from complex I to ubiquinone (Q). This perturbation enhances the production of ROS and decreases the synthesis of ATP. Neurological effects following systemic application of MPTP have been found in a variety of animal families, including monkeys, mice, dogs, cats, sheep and even goldfish (Zigmond and Stricker, 1989; Tipton and Singer, 1993). However, the marked differences in sensitivity to the neurotoxic action of MPTP are notable. Even large doses of MPTP elicit only slight neurotoxic effects in rats and guinea pigs. Therefore; in order to produce DA losses similar to those seen in monkeys in even the most sensitive strain of mice, the C57/Black mouse, a 50-fold dose of MPTP is required (Gerlach and Riederer, 1996).

6-OH DA, the first animal model of PD associated with SNpc dopaminergic neuronal death, was introduced more than 40 years ago (Ungerstedt, 1968). Interstingly, there is a range of sensitivity to 6-OHDA among the ventral midbrain dopaminergic neuronal groups; greatest loss is observed in the SNpc, while other neurons are almost completely resistant (Jonsson, 1980). Inside neurons, 6- OHDA accumulates in the cytosol, generating ROS and inactivating biological macromolecules by generating quinones that attack nucleophilic groups (Cohen and Werner, 1994).

Becau se 6-OHDA cannot cross the blood-brain barrier, it must be administered by local injection into the SN, median forebrain bundle (MFB), or the striatum to target the nigrostriatal dopaminergic pathway (Javoy et al., 1976; Jonsson, 1983). After 6-OHDA injections into SN or the MFB, dopaminergic neurons start degeneration within 24 hr and die without apoptotic morphology (Jeon et al., 1995). When injected into the striatum, however, 6-OHDA produces degeneration of nigrostriatal neurons, which lasts for 1–3 weeks (Sauer and Oertel 1994; Przedborski et al., 1995). For striatal lesions, 6-OHDA is injected unilaterally, with the contralateral side serving as control (Ungerstedt, 1971). These injections produce an asymmetric circling behavior in the animals, the magnitude of which depends on the degree of the nigrostriatal lesion (Hefti et al., 1980; Przedborski et al. 1995). The unilateral lesion can be quantitatively assayed; thus, a notable advantage of this model is the ability to assess the efficacy of new drug therapies (Jiang et al., 1993). However, it is not clear

Page 34 of 152 whether the mechanism by which 6-OHDA kills dopaminergic neurons shares key molecular features with PD.

Rotenone is the most potent member of the rotenoids, a family of natural cytotoxic compounds extracted from tropical plants; it is widely used as an insecticide. Rotenone is highly lipophilic and readily gains access to all organs (Talpade et al., 2000). It binds (at the same site as MPP+) and act by inhibiting mitochondrial complex I activity. The administration of low-dose i.v. rotenone to rats was reported to produce selective degeneration of nigrostriatal dopaminergic neurons accompanied by α-synuclein-positive LB-like inclusions (Betarbet et al., 2000). Rotenone-intoxicated animals developed abnormal postures and slowness of movement. Nevertheless, this model was the first to link an environmental toxin of possible relevance to PD to the pathologic hallmark of α-synuclein aggregation, similar results were also observed in cell culture studies (Uversky et al., 2001; Lee et al., 2002b).

The herbicide paraquat (N,N′-dimethyl-4-4′-bipiridinium) also induces a toxic model of PD. Interestingly, paraquat shows structural similarity to MPP+. Exposure to paraquat may confer an increased risk for PD (Liou et al., 1997). However, paraquat does not easily penetrate the blood brain barrier (Shimizu et al., 2001), and its CNS distribution does not parallel any known enzymatic or neuroanatomic distribution (Widdowson et al., 1996a; b). The toxicity of •- paraquat appears to be mediated by the formation of O2 (Day et al., 1999). Systemic administration of paraquat to mice leads to SNpc dopaminergic neuron degeneration accompanied by α-synuclein containing inclusions, as well as increases in α-synuclein immunostaining in frontal cortex (Manning-Bog et al., 2002; McCormack et al., 2002). It remains to be seen whether the dopaminergic toxicity is selective or whether other cell types are similarly affected. Paraquat ability to induce dopaminergic neuronal loss and α-synuclein-positive inclusions in a reliable fashion is valuable for studies of the role of α-synuclein in neurodegeneration.

2.3.3. Other Parkinsonian agents

The amphetamines are psychostimulatory drugs with an addictive potential. Their activity is primarily associated with their DA-releasing mechanism (McMillen, 1983). However, in very high doses these indirectly acting psychostimulants do also have a neurotoxic activity in rodents (rats, mice and guinea pigs) and non-human primates (Wagner et al., 1980). Single dose or multiple applications of methamphetamine to rats or mice was reported to cause a reduction of the DA, DOPAC and homovanillic acid (HVA) concentrations in the striatum. This reduction seems to be restricted to the nigrostriatal dopaminergic

Page 35 of 152 system as DA concentrations are unchanged in the extra-striatal brain regions, such as the frontal cortex, hippocampus and hypothalamus (Ricaurte et al., 1980; Ohmori et al., 1993). Beside the lowered concentrations of DA and its metabolites, diminished TH activity is also found. These biochemical changes are accompanied by the degeneration of dopaminergic nerve endings (Ricaurte et al., 1982) and a transient diminished number of DA reuptake sites (Ikawa et al., 1994).

Th e unilateral injection of 50µg of iron (III) into the SN of the rat produces strongly altered motor behaviour in the animals. Even three weeks after application, the altered motor behaviour is expressed by a reduction in spontaneous locomotor activity, a lower frequency of getting up onto the hind- legs, a transient appearance of "freezing" phenomena and spontaneous rotations. The unilateral nigral injection of iron (III) evokes a considerable reduction of the DA concentration in the ipsilateral striatum, as well as the DA metabolites DOPAC and HVA (Ben-Shachar and Youdim, 1991). It is assumed that iron (III) is taken up into the neurons by transferring receptors (Arendash et al., 1993). It is probably reduced in the cytosol to iron (II), which shows a cytotoxic action in primary cultures of neurons derived from the mesencephalon of rat embryos (Michel et al., 1992). Another possible mechanism is that excess iron • (II) could catalyze the formation of OH from H2O2, which is an endogenous product of enzymatic DA metabolism. This proposed mechanism for the neurotoxic effect is indirectly confirmed by the demonstration of increased amounts of thiobarbituric acid (TBA)-reactive compounds following intranigral injection of iron (III) (Arendash et al., 1993; Wesemann et al., 1993).

In addition, a variety of Parkinsonian agents have been tested in animals, with the purpose of studying a possible cause or risk factor for the development of PD (Tolwani et al., 1999). For example, the microorganism nocardia asteroids affects nigral neuron survival (Kohbata and Beaman, 1991) and its investigation is helping to understand the roles of infection and inflammation in nigral cell degeneration (Emborg, 2004). Another example is the isoquinoline derivatives (endogenous metabolites), which act as weak inhibitors of the mitochondrial complex I and have been shown to be neurotoxic for dopaminergic neurons (Nagatsu and Yoshida, 1988).

Page 36 of 152 2.4. Parkinson's disease therapy

Despite advances in modern therapy, patients with PD continue to experience an unacceptable disability. Currently, medical therapy continues to be levodopa mixed with a peripheral decarboxylase inhibitor, carbidopa (Savitt et al., 2006). Although in the early stages of the disease, the symptoms are relatively well controlled with levodopa and other dopaminergic agents, but they lack significant disease-modifying effect. Thus, the majority of patients experience levodopa- related motor complications and the disease progresses with the development of features such as freezing, falling, autonomic dysfunction and dementia that do not adequately respond to dopamine replacement therapies (Schapira and Olanow, 2004; Savitt et al., 2006). These disorders have not been cured because the neurons of CNS cannot regenerate on their own after cell death or damage (Rachakonda et al., 2004). Thus, the main challenge is to develop a neuroprotective therapy that can be administered early in the course of the disease to slow, stop or reverse the disease progression (Schapira and Olanow, 2004; Abdin and Hamouda, 2008).

A large number of techniques have been tried in the laboratory, with several of them having advanced to clinical trials. Some experimental studies attempt to interfere with neurotoxic mechanisms while others try to bypass these mechanisms and promote trophic mechanisms, while still other approaches involve replacing cells via transplantation. Tested compounds include neurotrophic factors, antioxidants, anti-excitotoxins, bioenergetic supplements, immunosuppressants and antiapoptotic therapy. The most promising approaches for PD are neurotrophic factors and cellular transplantation, with several newer approaches such as bioenergetic supplements and antioxidants holding considerable promise (Alexi et al., 2000).

2.4.1. Neurotrophic factor therapy

Neurotrophic factors have the potential for not only protecting against cellular degeneration, but also to enhace growth and biochemical or metabolic functions of the cell. Most neurotrophic factors that are localized to the nigrostriatal system support the survival and differentiation of developing dopaminergic neurons in culture (Beck, 1994; Hefti, 1997), as shown in Table 3. Although some studies have failed to find neurotrophic factor treatment effective in reversing dopaminergic and Parkinsonian functional deficits in animal models of PD, the vast majority have found positive, and in some cases, very dramatic protective effects. Neurotrophic factors do have the shortcoming that they are large molecules that will not bypass the blood-brain barrier. This makes them difficult

Page 37 of 152 to deliver to the brain in the clinical setting. However, the various CNS delivery methods that are evolving are overcoming this difficulty (Alexi et al., 2000).

Table 3. Some neurotrophic factors involved in survival and differentiation of developing dopaminergic neurons

Factor Reference Basic fibroblast growth factor (bFGF) (Knüsel et al., 1990) Brain derived neurotrophic factor (BDNF) (Knüsel et al., 1991) Epidermal growth factor (EGF) (Alexi and Hefti, 1993) Transforming growth factor-α (TGFα) (Widmer et al., 1993) Insulin-like growth factors (IGF-I and -II) (Alexi and Hefti, 1993) Glial cell line-derived neurotrophic factor (GDNF) (Lin et al., 1993) Neurotrophin-4/5 (NT4/5) (Hynes et al., 1994) Transforming growth factor-β2 and β3 (TGF-β2 & TGF-β3) (Poulsen et al., 1994)

2.4.2. Anti-excitotoxin therapy

A ntagonizing the excitotoxicity has been considered to have a therapeutic potential for the treatment of PD. Glutamate neurotransmission plays an integral role in basal ganglia functioning especially in the striatum (where the balance of glutamate and DA is critical) and the SN which receives glutamatergic input from the cortex (Rodriguez et al., 1998). Nigral dopaminergic neurons possess glutamate receptors (Kosinski et al., 1998) and they degenerate when exposed to excitotoxins (Miranda et al., 1999). Neuroprotection from MPTP toxicity to dopaminergic neurons is afforded by treatment with excitotoxin antagonists, especially those for the NMDA receptor or its modulatory glycine site (Ossowska, 1994). A large number of studies have found that various excitotoxin antagonists especially the NMDA antagonists MK801 and 3-((2)-2- carboxypiperazin-4-yl)-propyl-1-phosphoric acid (CPP), and the anticonvulsants riluzole (2-amino-6-tri-fluoromethoxy-benzothiazole) and remacemide which interfere with glutamate neurotransmission, can improve Parkinsonian behavioral parameters such as akinesia and rigidity in experimental animals lesioned with MPTP or 6-OHDA (Klockgether and Turski, 1990; Barnéoud et al., 1996). Moreover, Parkinsonian patients have shown symptomatic improvements after thalamotomy surgery; which removes the excess excitatory drive from the subthalamic nucleus. Removing the subthalamic nucleus helps restore neurotransmission flow within the basal ganglia circuitry to normal (Blandini and Greenamyre, 1998) and may be `neuroprotective by removing the source of excess glutamate to the SN (Rodriguez et al., 1998). Antagonising the excitatory neurotransmission in the SN may provide symptomatic improvements but does not appear to be truly neuroprotective.

Page 38 of 152 2.4.3. Anti-apoptotic therapy

V ar ious studies conducted on post mortem PD nigral cells suggest that apoptosis could play a role in the cell death that occurs in this disease (Burke and Kholodilov, 1998). In addition, transgenic mice over-expressing the anti- apoptotic gene B cell lymphoma-2 (Bcl-2) are resistant to MPTP toxicity (Offen et al., 1998; Yang et al., 1998). Dopaminergic terminals were found to be damaged, showing that although neurons were exposed to potentially cytotoxic concentrations of MPTP, the lack of p53 (a tumour suppressor which has a pro- apoptotic function) allowed neurons to survive the insult. In addition, p53 knock- out mice showed resistance to methamphetamine lesioning of striatal dopaminergic terminals (Hirata and Cadet, 1997). Inhibitors of caspase-3 proteases have been found to attenuate MPP+ lesioning of dopaminergic neurons in vitro (Dodel et al., 1998). In fact, the anti-apoptotic strategies are considered an approach and they are technically a more complex strategy due to the variant genetic mechanisms that occur during apoptosis and to the general role of apoptosis-related signals in normal physiological functioning. Nevertheless, anti- apoptotic therapy is a promising approach that requires further investigation (Esposito and Cuzzocrea, 2010).

2.4.4. Antioxidant therapy

A s there is a large body of evidence suggesting that neurodegeneration occurring in PD may involve oxidative stress (Cassarino and Bennett, 1999), antioxidant therapy is expected to be a good candidate for PD therapy. The antioxidant deprenyl (selegiline) has been found to protect against MPTP neurotoxicity and akinesia (Tatton and Greenwood, 1991). As an inhibitor of MAO, deprenyl was reported to decrease the accumulation of H2O2 and thus likely decrease the level of oxidative stress and subsequent cell death (Knoll, 1998). In addition, 7-nitroindazole (7-NI); an inhibitor of nitric oxide synthase (NOS) (which generates the reactive species peroxynitrite) was found to be effective in preventing the loss of striatal DA (Schulz et al., 1995) and nigral cell degeneration in MPTP Parkinsonian mice (Przedborski et al., 1996), in MPP+ treated rats (Matthews et al., 1997) and baboons (Hanatraye et al., 1996).

2.4.5. Bioenergetic supplements

Based on the considerable evidences suggesting that the mitochondrial dysfunction and the oxidative damage may play a role in the pathogenesis of PD; several agents are being studied for their beneficial effects in animal models of PD

Page 39 of 152 that can modulate cellular energy metabolism and/or exert antioxidative effects (Beal, 2003).

Creatin e is a guanidine compound found in meat-containing products and produced endogenously by the liver, kidneys and pancreas (Tarnopolsky and Beal, 2001). Creatine is taken up into brain as well as cardiac and skeletal muscles by sodium-dependant transporter (Sora et al., 1994). The creatine/phosphocreatine (PCr) system functions as a spatial energy buffer between the cytosol and mitochondria, using a unique mitochondrial creatine kinase (CK) isoform (Brdiczka and Wallimann, 1994). Both creatine and PCr can attenuate peroxynitrite-mediated mitochondrial CK inactivation and the consequent dimerization and opening of the mitochondrial permeability transition pore (whose opening promotes apoptosis) (O'Gorman et al., 1997). Another potential neuroprotective effect of creatine administration is by increasing glutamate uptake into synaptic vesicles, which has been shown to be energy dependant and which can be fueled by PCr (Xu et al., 1996). Creatine was found to produce a dose-dependant protection against dopamine loss, as well as an attenuation of neuron loss in the SN of mice treated with MPTP (Matthews et al., 1999; Beal, 2003).

Coenzyme Q10 (CoQ10) is an important cofactor of the electron transport chain where it accepts electrons from complexes I and II (Beyer, 1992; Dallner and Sindelar, 2000). CoQ10 has been shown to exert neuroprotective effects in the CNS in several in vivo models. It was found that oral administration of CoQ10 significantly attenuated ATP depletion and produced dose-dependant neuroprotective effects against striatal lesions produced by the mitochondrial toxin malonate (Beal et al., 1994). Furthermore, in an experimental model of Parkinsonism induced by rotenone in rats, administration of coenzyme Q10 provided a significant increase in striatal complex I activity, ATP levels and Bcl-2 expression (Abdin and Hamouda, 2008)

Nicotinamide is a precursor of NADH, which is a substrate for complex I of the electron transport chain. It is also an inhibitor of polyADP-ribose polymerase (PARP), an enzyme that is activated by DNA damage and that, in turn, depletes both NADH and ATP. Several studies have shown that nicotinamide, like other PARP inhibitors, protects against MPTP neurotoxicity (Cosi and Marien, 1998).

Carnitine and acetyl-L-carnitine are agents that facilitate the entry and exit of fatty acids from mitochondria. Carnitine facilitates the entry of long chain fatty acids into mitochondria for subsequent β-oxidation. Acetyl-L-carnitine has better brain penetration and may be useful as an agent for elevating brain carnitine levels (Beal, 2003). Carnitine delays mitochondrial depolarization in response to a

Page 40 of 152 variety of stressors including oxidative damage (Di-Lisa et al., 1985). Acetyl-L- carnitine increases the cellular respiration and mitochondrial membrane potential in hepatocytes of rats (Hagen et al., 1998). Carnitine and acetyl-L-carnitine were reported to attenuate neuronal damage produced by rotenone and MPTP in vitro (Virmani et al., 1995).

Lipoic acid is a disulfide compound that is found naturally in mitochondria as a coenzyme for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and also has antioxidant effects. It possesses a proven neuroprotective effect in rodent models of both focal and global cerebral ischemia (Wolz and Kreiglstien, 1996). Supplementation with α-lipoic acid in old rats improved the brain mitochondrial function and decreased the oxidative damage (Liu et al., 2002).

2.4.6. Cellular transplantation

For many years, fetal tissue has been used for treatment of human disorders, including fetal pancreatic transplants to treat diabetes mellitus and fetal thymic transplants to treat lymphogenic immunological deficiency. The defining basic research that opened investigations on fetal tissue and brain transplantation was undertaken by Olson and Seiger (1972). They showed that fetal tissue grafted in the anterior chamber of the eye has the capacity to integrate with the host target neurons and that these graft-host connections were functional. The aim of stem cell therapy for PD is to reconstruct nigro-striatal neuronal pathways using endogenous neural stem/precursor cells or grafted dopaminergic neurons. As an alternative, transplantation of stem cell-derived dopaminergic neurons into the striatum has been attempted, with the aim of stimulating local synapse formation and/or release of DA and cytokines from grafted cells (Takahashi, 2007). A large variety of cell replacement strategies are under investigation in animal models of PD, which began with the success of transplanted fetal neurons in reconstructing the lesioned nigrostriatal pathway and ameliorating behavioral impairments (Perlow et al., 1979). Cell transplant therapy for PD has been in use for over 2 decades as an experimental treatment. Different cell types have been proposed as better therapeutic alternatives (Vidaltamayo et al., 2010). Various types of cells have been tested for their potential usefulness in PD (Table 4). Fetal ventral mesencephalic cells transplanted into the striatum two weeks after 6-OHDA lesioning in rats have been found to survive out to a full two years with many TH neurons remaining and forming functional synaptic connections with host striatum, improving DA content and successfully eliminating methamphetamine- induced rotations (Nishino et al., 1990). Precursor cells isolated from the fetal rat ventral mesencephalon and engineered with an extra copy of the TH gene were able to ameliorate apomorphine-induced rotations (Anton et al., 1994).

Page 41 of 152 Table 4. Various types of cells of potential benefits in PD treatment (Raymon et al., 1997; Rosenthal, 1998)

Cell origin or type Characteristics Cells from the embryonic ventral mesencephalon (which contains the primordial SN) Secretion of dopamine Neuronal stem or progenitor cells and/or neurotrophic Dopaminergic cell lines factors Engineered non-neuronal cells (fibroblasts or astrocytes) Adrenal medullary cells Testis-derived Sertoli cells Rich in trophic factors Carotid body epithelial glomus cells Synthesize dopamine

2.4.7. Immunosuppressant therapy

Immunos uppressant therapy was originally used in combination with cellular transplantation to minimize host rejection of grafted tissue. Immunosuppressant drugs bind to receptor binding proteins called immunophilins forming a complex which inhibits the calmodulin-stimulated phosphatase calcineurin. Calcineurin normally activates interleukin-2 which stimulates T-cell proliferation, thus, immunosuppressants inhibit the immune reaction. Calcineurin also activates NOS which could lead to peroxynitrite-induced oxidative stress (Dawson et al., 1993). In addition, immunosuppressants have been found to have neurotrophic actions (Snyder et al., 1998). Interestingly, analogues of immunophilin ligands which do not have immunosuppressive activity and do not inhibit calcineurin, have also been found to have neurotrophic actions, although this is controversial (Costantini et al., 1998).

Page 42 of 152 2.5. Ginkgo biloba extract

Ginkgo leaf is a widely used herbal medicine. The beneficial effects of Ginkgo biloba substances were known for 5000 years in traditional Chinese medicine. The study of biological activities of EGb761, a standardized extract of ginkgo biloba with a well defined mixture started more than 20 years ago. EGb761 contains 24% of flavonoids and 6% of terpenes giving this extract its unique polyvalent pharmacological action. The flavonoid fraction is composed of 3 flavonols: quercetin, kaempferol and isorhamnetin, which are combined with sugar forming glucosides.

The terpene lactones are represented by the Ginkgolides A, B, C, J and M and bilobalide (Fig. 8). The Ginkgolides are platelet-activating factor antagonists (Braquet, 1986; Smith et al., 1996) which is able to reduce platelet activation and aggregation, and therefore having the potential to improve blood circulation. In addition, ginkgolide K was reported to exert a neuroprotective effect in a rat model of cerebral ischemia reperfusion (Ma et al., 2012). Bilobalide, a sesquiterpene trilactone, can reduce damage from cerebral ischemia (Chandrasekaran et al., 2003). Several studies have highlighted the potential of EGb761 and its constituents as antioxidants and free radical scavengers such as • •– OH, O2 and peroxyl radicals (Marcocci et al., 1994; Maitra et al., 1995). EGb761 can also prevent lipid oxidation (Ramassamy et al., 1999). On synaptosomal preparations from striatum of mice, EGb761 has been shown to prevent the alteration of the neuronal DA uptake system and the modifications of the membrane fluidity induced by a pro-oxidant system ascorbic acid/Fe2+ (Ramassamy et al., 1992a; 1993). The flavonoids fraction of EGb761 was implicated in these protections. This efficacy was also observed in vivo, where the dopaminergic neurons were reported to be protected against the neurotoxin MPTP when mice received EGb761 two weeks before the MPTP administration (Ramassamy et al., 1990).

Du ring the past few decades, there has been an exponential growth in the number of reports describing the therapeutic benefits of the Ginkgo biloba extract EGb761 on symptoms associated with cognitive disorders. In 1998, a meta- analysis study reviewed over 50 clinical studies using EGb761 for treatment of dementia and cognitive functions associated with Alzheimer's disease (Oken et al., 1998; DeFeudis and Drieu, 2000; DeKosky and Fuberg, 2008). However, therapeutic use of EGb761 is approved for the treatment of dementia in Germany. This potential was observed in a study on 1465 women over 75 years in France (Andrieu et al., 2003). Moreover, a double-blind trial including 400 patients aged

Page 43 of 152 50 years or above with Alzheimer's disease or vascular dementia (VaD), randomized to receive EGb761 or placebo for 22 weeks (Napryeyenko et al., 2007); in their study EGb761 improved dementia scores while subjects receiving the placebo experienced a deterioration on scores.

Fig. 8. Ginkgolides and bilobalides present in ginkgo biloba (Ramassamy, 2006).

In the presence of controversial results for its beneficial effects in Alzheimer's disease, EGb761 has been the focus of two phase III clinical trials, the GEM study (Ginkgo Evaluation of Memory Study) in the United States and the Guide Age study in France both with more than 3,000 individuals/study older than 70 years old. On the other hand, the role of EGb761 in the protection against the amyloid β peptide-induced toxicity has received much attention. Thus EGb761 is able to protect and to rescue primary hippocampal neurons and PC12 cells against the toxicity of the amyloid β peptide (Bastianetto et al., 2000; Yao et al., 2001). Accordingly, EGb761 is also able to attenuate the basal as well as the induced levels of H2O2-related ROS in neuroblastoma cell lines. This protection was also observed in transgenic Caenorhabditis elegans constitutively expressing human amyloid β peptide. Among individual EGb761 components tested, kaempferol and quercetin provided maximum attenuation in both models (Smith and Luo, 2003).

Page 44 of 152 2.6. Ionizing radiation

Ionizing radiation (IR) is an invaluable diagnostic and therapeutic tool that is widely used in medicine. Previously, the majority of studies focused mainly on effects of high doses of radiation, such as those received during radiation treatment of primary and metastatic brain tumors, head and neck cancers as well as leukemia/lymphoma (Monje et al., 2002; Ohizumi et al., 2002). High-dose radiation exposure was proven to cause severe functional and morphological changes in brain tissue (Tofilon and Fike, 2000; Limoli et al., 2004), declines in the hippocampal proliferation and neurogenesis, debilitating cognitive declines, as well as learning and memory deficits (Monje and Palmer, 2003).

Ho wever, studies investigating the effects of IR on the CNS suggested that LDR rendered neuroprotection (Kipnis et al., 2004; Anderson et al., 2005). Fortunately, in everyday life, the brain is frequently exposed to LDR due to diagnostic, therapeutic, occupational, and environmental sources. The neuroprotection offered by LDR had been found in different animal models such as inherited glaucoma, optic nerve crush and contusive spinal cord injury (Kipnis et al., 2004; Liang et al., 2006).

Radi ation hormesis (also called radiation homeostasis) is the hypothesis that low doses of IR (within the region and just above natural background levels) are beneficial, activating the repair mechanisms that protect against diseases, that are not activated in absence of IR (Feinendegen, 2005). Radiation hormesis is highly controversial. Edward Calabrese found that hormesis usually occurred at doses about five times lower than the toxic threshold. Environmental Protection Agency (EPA) set the acceptable exposure limit which was 20 times lower than the toxic threshold (Calabrese and Baldwin, 2003). The dose for radiation hormesis research was usually very low: from 0.025 Gray (Gy) to 0.2 Gy (Ren et al., 2006; Zhang et al., 2006), which was determined on the toxic threshold of lymphocyte. Earlier studies have reported cognitive impairments and deficits in neural functions following brain irradiation in the dose of 5–10Gy in rats and mice. So in the study on neuroprotection, the whole body irradiation dose of radiation hormesis should be lower than 2Gy. It is a very low dose for nervous system, but a high dose for immune system. So it was supposed that the neuroprotection induced by low dose γ-irradiation was probably related to the immune system injury rather than radiation hormesis (Zhang et al., 2009).

In (2006), Liang et al. reported a potential neuroprotection in PD models (C57BL/6 mice administered with MPTP) when the mice were pretreated with

Page 45 of 152 3.5Gy total body irradiation (TBI). But the neuroprotection disappeared when the dose rose to 5.5Gy. Furthermore, it was reported that exposure of mice to 8 Gy of X-rays; about 30% of the mice survived 30 days after exposure. However, pre- irradiation of rats with 0.005 Gy several hours before the 8 Gy exposure increased the survival rate to 70% (Yonezawa et al., 1996). Adaptive or hormetic effects of LDR have been extensively observed in previous studies with in vitro cultured cells and in vivo animal models are shown in Table 5.

Table 5. Different models utilizing LDR as a modulator

Model Reference LDR decreased the frequency of chromatid aberrations (Cai and Liu, 1990) following a challenging radiation dose in human and rabbit lymphocytes in vitro and in bone marrow and germ cells in vivo. LDR induced an adaptive response to dominant lethal mutations (Cai et al., 1993) and chromosome aberrations in mice spermatocytes in vivo. LDR induced an adaptive response to high dose radiation- (Cai and Wang, 1995) induced DNA and chromosomal damage in spleenocytes, bone marrow cells and spermatocytes of the mice offspring in vivo. LDR induced an adaptive response to high dose radiation- (Kim et al., 1997) induced cell death and DNA fragmentation in mouse lymphoma cells in vitro. LDR elevated the antioxidant potency in a model of MPTP- (Kojima et al., 1999) induced brain damage, depletion of endogenous antioxidants and free radical scavenging enzymes in mice. LDR induced an adaptive response to the inhibition of SOD-like (Yamaoka et al. 1999) substances and antioxidant activity induced by X-irradiation of mice. LDR induced an adaptive response to the testicular damage (Zhang et al., 2000) induced by subsequent high-radiation dose in hybrid strain mice. Elevation of the antioxidant potency by LDR in a model of (Yamaoka et al., 2002) Fe3+-induced lipid peroxidation associated with decreased activities of the antioxidant enzymes in mice brain. LDR induced an adaptive response to high dose radiation- (Liu et al., 2006) induced cytogenetic damage in mouse male germ cells in vivo. LDR induced an adaptive response to high dose radiation- (Lu et al., 2009) induced gene mutation in human cell line. Low dose x-rays attenuated the diabetes-induced testicular (Zhao et al., 2010) dysfunction in rats. LDR induced an adaptive response to the mitochondrial (Lu et al., 2011) dysfunction caused by a higher dosage of radiation.

Page 46 of 152 It was supposed that two mechanisms might take part in the neuroprotection: increasing of GSH activity induced by low dose irradiation (Kojima et al., 1999) and proliferation of adaptive immunity induced by radiation hormesis (Kipnis et al., 2004). Radiation exposure of mammalian cells has been shown to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), to induce Ca2+ oscillations, and to activate the mitogen activated protein kinase (MAPK) pathway. The latter has been suggested to have a cytoprotective effect and promote survival of affected cells (Silasi et al., 2004).

The use of a whole body irradiation before standard radiation therapy has been suggested by Jin et al. (2007). This technique has the disadvantage of exposing the entire body to unnecessary radiation. It also may trigger a protective adaptive response in the cancerous tissue thereby reducing the kill probability from the standard treatment. However, more study is needed to evaluate the full effects and potential of this protocol. It is important to study and discern whether low-level exposure always constitutes a significant risk to patients, or if, in fact, under certain prescribed conditions, some exposure can be protective. In particular, the research of Blankenbecler (2010) was a step in showing that the protective effect can be used to reduce the damage to healthy cells from the high exposure used in standard therapy.

Page 47 of 152 MATERIALS AND METHODS

Page 48 of 152 3. Materials and Methods 3.1. Materials 3.1.1. Drugs and chemicals Reserpine (methyl reserpate 3, 4, 5-trimethoxybenzoic acid) and EGb761 were kindly donated as gifts from Novartis (Cairo, Egypt) and Al-Amriya (Alexanderia, Egypt) pharmaceutical companies, respectively. The standardized extract of ginkgo biloba contains approximately 24% flavone glycosides (primarily composed of quercetin, kaempferol, and isorhamnetin) and 6% terpene lactones (2.8-3.4% ginkgolides A, B, and C, and 2.6-3.2% bilobalide). Ginkgolide B accounts for about 0.8% of the total extract, and bilobalide accounts for about 3% of the extract. Other constituents include proanthocyanidins, glucose, rhamnose, organic acids, D- glucaric acid and ginkgolic acid (at most 5 ppm) (Ramassamy, 2006).

Coenzym e-Q1, ATP, DA hydrochloride, dichlorophenolindophenol, bovine serum albumin (BSA), 1,1',3,3' tetraethoxypropane, GSH, 5,5'-dithiobis- (2- nitrobenzoic) acid (DTNB) and all the high performance liquid chromatography (HPLC)-grade chemicals and solvents used in ATP assay (potassium dihydrogen phosphate (KH2PO4), acetonitrile and methanol) were purchased from Sigma- Aldrich, Inc. (St. Louis, MO, USA). Folin Ciocalteus reagent was purchased from Fluka Chemie GmbH (Buchs, Switzerland). All other chemicals were of pure analytical grade. 3.1.2. Animals and treatment Male Wistar rats obtained from the Animal House of National Research Centre, Cairo, Egypt, weighing 180–200 g were maintained on a 12-h dark–light cycle and provided free access to pellet diet and water ad libitum. Animal care was supervised and approved by the Ethics Committee of faculty of Pharmacy, Cairo University, Cairo, Egypt. After one week of acclimatization, rats were randomly divided into four experimental groups as follows: 3.1.2.1. Experimental group I The animals in this group were used to evaluate Parkinsonism induction by reserpine. Animals were divided into three sub-groups, 8 rats each: Group 1: normal; Group 2: received single intraperitoneal injection of reserpine in a dose of 5mg/kg body weight (BW) dissolved in 1% acetic acid (Kaur and Starr, 1997; DiMarzo et al., 2000); Group 3: received the vehicle of reserpine; single intraperitoneal injection of 1% acetic acid solution in distilled water in a dose of 2ml/Kg BW.

Page 49 of 152 3.1.2.2. Experimental group II The animals in this group were used to study the pretreatment effect of EGb761 against reserpine-induced PD. Animals were divided into three sub-groups, 8 rats each: Group 4: rats were pretreated with EGb 761 in 2% gum acacia, given orally in a dose of 100 mg/kg BW/day for 3 weeks (Ahmad et al., 2005) and then PD was induced as in group 2; Group 5: rats were pretreated orally with the vehicle of EGb761; 2% gum acacia in distilled water, in a dose of 2ml/kg/day for 3 weeks and then PD was induced as in group 2; Group 6: rats received EGb761 in 2% gum acacia as in group 4. Reserpine was injected 24h after the last dose of EGb761. 3.1.2.3. Experimental group III The animals in this group were used to study the pretreatment effect of whole body LDR against reserpine-induced PD. Animals were divided into two sub- groups, 8 rats each: Group 7: rats were pretreated by exposure to whole body γ- radiation at a dose level of 0.25Gy once a week for 6 weeks (Slyshenkov et al., 1999; Shukitt-Hale et al., 2007) and then PD was induced as in group 2; Group 8: rats were exposed to whole body γ-radiation only as in Group 7. Whole body γ- irradiation was performed at the National Centre for Radiation Research and Technology, Cairo, Egypt, using Gamma cell-40; Caesium-137 irradiation unit manufactured by the Atomic Energy of Canada Limited (AECL). Radiation dose levels were delivered at a rate of 0.05 Gy/sec. Reserpine was injected 24h after the last dose of γ-irradiation. 3.1.2.4. Experimental group IV The animals in this group (8 rats) were used to study the combined pretreatment effect of low dose whole body γ-radiation and EGb761 against reserpine-induced PD. Group 9: received EGb761 as in group 4 during the last three weeks of the irradiation period as in groups 7 and 8 and then PD was induced as in group 2. Reserpine was injected 24h after the last doses of EGb 761 and γ-irradiation. A schematic representation of dosage schedule of different studied groups is illustrated in Fig. 9. 3.1.3. Behavioral study Th e development of Parkinsonism was detected by the occurrence of tremors, bradykinesia and rigidity in rats that were further quantified by a catalepsy test known as “grid test”; where the rat was hung by its paws on a vertical grid (25.5 cm wide and 44 cm high with a space of 1 cm between each two wires), and the time for the rats to move its paws or any sort of first movement was recorded (Alam and Schmidt, 2004).

Page 50 of 152 6 weeks 0 3 weeks Normal ● 0 Day 21 Day 42 24h Reserpine 24h 0 ● Acetic acid ● 24h24h EGb761+ 0 Daily EGb761 administration 24h 24h● reserpine 0 Gum acacia+ Daily Gum acacia administration EGb761reserpine ● 0 Daily EGb761 administration 48h ● 0 24h 24h Radiation + Irradiation once/week rRadiationeserpine ● 24h48h24h● Radiation + 0 IrradiationIrradiation once/week once/week EGb761+ Daily EGb761 administration reserpine ● Fig. 9. A schematic representation of dosage schedule of different studied groups.

: Reserpine injection; ● : Sacrifice 3.1.4. Preliminary pilot studies A pilot study was carried out to determine an appropriate interval between reserpine injection and animal sacrifice. Rats received reserpine and the behavioral effects (tremors, bradykinesia and rigidity) and striatal DA levels were assessed at 18, 20, 24 and 30h intervals. The optimum behavioral effects and the decrement in striatal DA level were observed after 24h. Anothe r pilot experiment was carried out to determine the optimum time interval between the last irradiation dose and reserpine administration. Rats received reserpine at intervals of 18, 24, 48h and 1week after the last irradiation dose, and the behavioral effects and striatal DA levels were assessed at these time intervals. The optimum protection offered by radiation was observed when reserpine was injected at 24h after the last irradiation dose 3.1.5. Tissue sampling for biochemical studies At the end of the experimental period; in the preliminary pilot studies and all the experimental groups, rats were sacrificed by decapitation and their skulls were split on ice. The whole brain was separated. Striata of the two hemispheres were isolated, weighed and homogenized in 9 volume ice cold acidified n-butanol (85µl conc HCl/100ml butanol) using Glas-Col® homogenizer (Terre Haute, Indiana, USA). The homogenate was centrifuged at 450xg for 5 min at 4ºC. 1.25ml of the resulting supernatant was mixed with 0.8ml 0.2N acetic acid and 2.5ml heptane,

Page 51 of 152 vortexed and centrifuged at 450xg for 5 min at 4ºC. The upper organic layer was discarded and the aqueous layer was stored at -70ºC for the assay of striatal DA level. The remaining brain tissue was weighed and homogenized in 9 volume of the collecting buffer (pH 7.8): 10 mM Tris-HCl, 1 mM ethylene glycol tetracetic acid (EGTA) and 0.32 M sucrose. Two aliquots of homogenate were mixed with ice cold 6% w/v metaphosphoric acid (1:2 ratio) and 2.3% w/v KCl (1:1 ratio), centrifuged at 1,000xg for 15 min at 4ºC, and the resulting supernatant was used for the assay of brain levels of GSH and malondialdehyde (MDA), respectively. Another aliquot of the homogenate was used for the determination of total iron level. Another aliquot of the brain homogenate was centrifuged at 11,000xg for 15 min at 4ºC using Hettich Mikro 22R (Germany) centrifuge. The resulting supernatant was used for the assay of GST and NQO activities. A last aliquot of brain homogenate was used for separation of brain mitochondrial fraction. The homogenate was centrifuged at 700xg for 10 min at 4ºC; the supernatant was centrifuged for 20 min at 10,000xg to obtain mitochondria pellets that were washed once with the collecting buffer to remove microsomal and cellular contamination. Finally the mitochondria were resuspended in 9 volumes of the collecting buffer to prepare 10% mitochondrial fraction (Turpeenoja et al., 1988) used for the assay of complex I (NADH:ubiquinone oxidoreductase) activity and levels of ATP and Bcl-2.

Page 52 of 152 3.2. Methods 3.2.1. Determination of dopamine level in brain striata Principle DA level was determined according to the method of Guo et al. (2009). It depends on the reduction of Fe (III) to Fe (II) by DA. Fe (II) reacts with potassium ferricyanide to form a soluble Prussian blue (K Fe (III) [Fe (II) (CN) 6]) which is measured colorimetrically at 735nm. Reagents 1- 0.015 mM potassium ferricyanide: 1.2347g in 250ml bidistilled water. 2-0.015 mM ferric chloride: 1.0137g in 250ml bidistilled water. 3- Standard stock DA solution: 0.1g DA hydrochloride/ 100ml of 0.2N acetic acid. This solution was used to prepare different dilutions of 6, 4.8, 4, 1.6, 0.2 and 0.05 μg/ml. Procedure 1- 1ml of brain homogenate supernatant or standard solution was mixed with 1ml of potassium ferricyanide and 1ml ferric chloride. 2- This mixture was diluted to 25ml using bidistilled water and left to stand for 35 min at room temperature. 3- The absorption of this solution was then measured at 735nm using UV-Vis spectrophotometer; Double beam PC scanning spectrophotometer UVD-2950 (LABOMED, INC., USA), against blank which was prepared by using bidistilled water instead of the sample. The assay was calibrated using different concentrations of standard DA (Fig. 10). Calculations DA level was expressed as ng/mg protein. DA level = Absorbance of sample x concentration of standard Absorbance of standard x mg protein in the fraction used

Page 53 of 152 Fig. 10. Standard curve of DA.

Page 54 of 152 3.2.2. Determination of glutathione level in brain Principle Br ain level of GSH was determined according to the method of Beutler et al., (1963). It depends on the reaction of the free SH-group of the GSH molecule with DTNB yielding a yellow colored product (2 nitro-5- thiobenzoic acid) that can be measured colorimetrically at 412 nm. Reagents

1- Phosphate solution (0.3M disodium hydrogen phosphate): 4.26 g Na2HPO4 in100ml bidistilled water. 2- DTNB reagent: 40 mg DTNB in 100ml 1% sodium citrate. 3- GSH standard solution: 15mg GSH/100ml 1% metaphosphoric acid. Different concentrations were prepared by serial dilution of the stock solution to get standard solutions of 0.92, 1.86, 3.7, 7.4 and 15 mg GSH per 100 ml.

Procedure 1- 0.5 ml of the brain homogenate supernatant or standard solution was mixed with 2 ml of phosphate solution, followed by the addition of 0.25 ml of DTNB reagent.

2- The absorbance of this mixture was measured at 412 nm within 5 min of the addition of DTNB reagent against blank which was prepared using 0.5ml of 1% metaphosphoric acid instead of the sample. The assay was calibrated using different concentrations of standard GSH solutions (Fig.11). Calculation GSH level was expressed as mg/g wet tissue. GSH level = Absorbance of sample x concentration of standard Absorbance of standard x gram tissue in the fraction used

Page 55 of 152 Fig. 11. Standard curve of GSH.

Page 56 of 152 3.2.3. Determination of malondialdehyde level in brain Principle Br ain MDA level was determined according to the method of Uchiyama and Mihara (1978). It depends on the reaction of MDA with TBA in an acidic medium to give a colored TBA-complex which can be measured colorimetrically at 535 and 520 nm after extraction with n-Butanol. Reagents 1- TBA: 0.67 % W/V. 2-Trichloroacetic acid (TCA): 0.5% W/V. 3-n-Butanol 4-Standard malondialdehyde (1, 1', 3, 3' tetraethoxypropane): 15µl of standard MDA was diluted to 10 ml using bidistilled water. One ml of this solution was further diluted to 100 ml with bidistilled water to prepare the working standard solution; which was used to prepare serial dilutions of 5, 10, 15, 25 and 30 nmol per 0.5 ml. Procedure 1- 0.5ml of the brain homogenate supernatant was mixed with 3 ml of TCA and 1ml of TBA solution, the mixture was placed in a boiling water bath for 45min. 2- After cooling, 4ml n-butanol were added and mixed using vortex mixer. The mixture was centrifuged for 10 min at 1,000xg to separate the butanol layer. Absorbance of the n-butanol layer was then measured at 535 and 520 nm against a blank which was prepared by using 0.5 ml of bidistilled water instead of the sample and the difference in absorbance between the two determinations (ΔA) was calculated. The assay was calibrated using different concentrations of standard MDA (Fig. 12). Calculation: MDA level was expressed as nmole/mg protein. MDA level = ΔA of test x concentration of standard (nmol/ ml) ΔA of standard x mg protein in the fraction used

Page 57 of 152 Fig. 12. Standard curve of MDA.

Page 58 of 152 3.2.4. Determination of total iron level in brain Principle Total brain iron level was determined using the atomic absorption spectrophotometry according to the method of Kingston and Jassie (1988), after sample digestion (Parker et al., 1967; Subramania, 1995). Procedure 1-The homogenate was digested in a mixture of conc. nitric acid and hydrogen peroxide in 5:1 ratio using microwave sample digester (Lab Station, MLS-1200 MEGA, Italy). 2-The samples were atomized under the instrumental condition shown in Table 6. 3-The total iron level in digested samples was estimated by using SOLAR system Unicam, 939 Atomic Absorption Spectrometer (England), equipped with deuterium background corrections. All solutions were prepared with ultra pure water with specific resistance of 18 Ω cm-1, using ultra pure water station (ELGA, England) using reverse osmosis technique and mixed bed ion exchanger for removal of residual salts that may interfere with iron determination. 4- The assay was calibrated using different concentrations of standard iron solutions (Fig. 13).

Table 6. The instrumental specifications for iron determination by atomic absorption

Instrumental Condition Value (for iron determination) Wave length (nm) 248.3 Band pass (nm) 0.2 Lamb current (mA) 7-11 Integration period 4 Sec Air flow rate (L/m) 5 Acetylene flow rate (L/m) 0.8-1.1 Sensitivity: Flame (mg/L) 0.06 Furnace (pg) 1.5

Page 59 of 152 Calculation Iron level was expressed as µg/mg protein. Iron level = Absorbance of sample x concentration of standard Absorbance of standard x mg protein in the fraction used

Fig. 13. Standard curve of iron.

Page 60 of 152 3.2.5. Determination of glutathione-S-transeferase activity (EC 2.5.1.18) in brain Principle Th e determination of GST activity was carried out according to the method of Habig et al. (1974); using UV kinetic kit provided by Bio-diagnostic® (Cairo, Egypt). The assay depends on the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH, which is accompanied by an increase in absorbance of the reaction product. The rate of this increase is directly proportional to GST activity in sample. GSH + CDNB GST adduct product [S-(2,4 diphenyl)-GSH] Reagents 1- R1: phosphate buffer pH 7.4 2- R2: GSH. 3- R3: CDNB reagent 4- R4: trichloroacetic acid Procedure 1- R2 was reconstituted in 5ml distilled water. 2- 50µl of the post mitochondrial fraction was mixed with 1ml R1 and 0.1ml R2, and the mixture was incubated at 37ºC for 5 min. 3- 0.1ml R3 was added to the reaction mixture and was incubated at 37ºC for 5 min. Then 0.1ml R4 was added. 4- The increase in absorbance at 340 nm was monitored at 0, 1, 2 and 3 min (ΔA/min was calculated).

Calculation GST activity was expressed as U/mg protein. The enzyme unit of GST is defined as the amount of enzyme that catalyzes the formation of 1 nmol of CDNB conjugate per minute. Enzyme activity = ΔA/min x 2.812 mg protein in the fraction used

Page 61 of 152 3.2.6. Determination of NADPH-quinone oxidoreductase activity (EC 1.6.99.2) in brain Principle NQO activity was determined according to the method of Schlager et al. (1993). It depends on monitoring the decrease in absorbance produced by the reduction of dichlorophenolindophenol (DCPIP) by NQO at 600nm. Reagents 1- 0.07% BSA. 2- 0.2% Tween 20. 3- 25mM Tris pH 7.8: 0.3028g in 100ml bidistilled water. 4- 0.2mM NADPH: 0.0149g in 100ml bidistilled water. 5- 0.04mM DCPIP: 0.0012g in 100ml bidistilled water. Procedure 1- To a mixture of 51.4ml tris and 34ml tween 20; the following reagents were added in order: 11.9ml BSA, 0.2ml DCPIP and 2.5ml NADPH to prepare the reaction mixture. 2-The reaction was initiated by the addition of 50μl of post mitochondrial fraction to 1ml of the reaction mixture; the decrease in absorbance per min was monitored at 600nm for 3 min. Calculation Using the molar extinction coefficient of NADPH of 6.22µmol-1cm-1, the activity of the enzyme was calculated. One unit of NQO was defined as the amount of enzyme oxidizing 1nmol of NADPH per min under specified conditions. NQO activity was expressed as U/mg protein. Enzyme activity = ΔA/min x total volume of the assay (1.05ml) 6.22 x mg protein in the mitochondrial fraction used

Page 62 of 152 3.2.7. Determination of brain mitochondrial complex I activity (NADH:ubiquinone oxidoreductase; EC 1.6.99.3) Principle Determination of mitochondrial complex I activity was carried out based on the method of Whitfield et al. (1981). It depends on the reaction catalyzed by mitochondrial complex I by following the decrease in the absorbance due the oxidation of NADH at 340 nm coupled with the reduction of ubiquinone-1 (Coenzyme-Q1) to ubiquinol-1 with the use of extinction coefficient =6.81 l/mmol/cm. Reagents 1- 0.1mM ethylene diamine tetra acetic acid (EDTA): 0.00292g in 100ml bidistilled water. 2- 20mM potassium dihydrogen phosphate (KH2PO4) pH 8.0: 0.272g in 100ml bidistilled water. 3-0.04mM Co-Q1: 1mg in 100mL bidistilled water. 4- 0.1mM NADH: 0.0067g in 100ml bidistilled water. 5- 2mM sodium azide (NaN3): 13mg in100ml bidistilled water. 6- BSA: 0.15g in100ml bidistilled water. Procedure 1- Reaction mixture was prepared by mixing 67.5ml BSA, 0.5ml Co-Q1, 5.5ml NaN3, 1.5ml EDTA and 122ml KH2PO4. 2- 50μL of the mitochondrial fraction was mixed with 0.9ml of the reaction mixture and left at room temperature for 3 min for equilibrium. 3- 0.1ml NADH was added to initiate the reaction. The decrease in absorbance per min was monitored at 340nm for 3 successive min. Calculation Using the combined extinction coefficient for Co-Q1 and NADH of 6.81µmol- 1cm-1, the activity of the enzyme was calculated. One unit of Mitochondrial complex I was defined as the amount of enzyme oxidizing 1nmol of NADH per min under specified conditions. Mitochondrial complex I activity was expressed as U/mg protein. Enzyme activity = ΔA/min x total volume of the assay (1.05ml) 6.81 x mg protein in the mitochondrial fraction used

Page 63 of 152 3.2.8. Determination of brain mitochondrial adenosine triphosphate level Brain mitochondrial ATP level was determined using HPLC according to the method of Botker et al. (1994). Reagents

1-4.8M perchloric acid (HClO4): 52.32ml perchloric acid in100ml bidistilled water. 2-1mM EDTA: 0.0585g EDTA in 20ml bidistilled water. 3-2M potassium bicarbonate (KHCO3): 10.012g EDTA in 50ml bidistilled water. 4- ATP standard solution (90μM): 5mg ATP/100ml deionized HPLC water. Serial dilutions were prepared immediately prior to use, containing 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 and 0.1μM. 5- Mobile phase (pH 6.3): 0.2M potassium dihydrogen phosphate KH2PO4: acetonitrile: methanol (9.6:0.3:0.1); all constituents of the mobile phase were of HPLC grade. Procedure A- Separation of adenosine triphosphate from mitochondrial fraction: 1-2ml of mitochondrial fraction were deproteinized with 0.6ml ice cold perchloric acid and left for 5min on ice. Then, 4ml EDTA was added with repeated mixing on ice for 10 min and centrifuged at 40xg for 15 min at 4ºC.

2- 2ml of the resulting supernatant was neutralized with 0.66ml KHCO3 and left for few min on ice until CO2 evolves and centrifuged at 40xg for 10min at (to separate KClO4 precipitate). The obtained clear supernatant was used directly for the HPLC assay or stored at -70◦C. B- Operation of HPLC system:

TM 1- C18 column (Supelcosil ) was eluted at a flow rate of 0.5 ml/min with the mobile phase. The system was set to operate at 23-25◦C with and an operating pressure of 11.5mPa. 20μL of each sample or standard was injected on the HPLC system through a 100μL PEEK injection loop (Scientific Products & Equipment, Canada), using a Rheodyne syringe loading injector.

2- The wavelength was set at 254 nm for detection and quantification by Spectra- Physics variable wavelength ultraviolet detector (Spectra 100, Spectra-Physics Inc., USA). The assay was calibrated using different concentrations of standard ATP (Fig. 14). Recovery of ATP was determined by measuring the retention time in relation to that of the standard solutions.

Page 64 of 152 3-Detector output was recorded by an integrator (Hewlett-Packard Integrator USA), and digitalized using the Peak Simple® software. . Calculation:

The concentration of ATP was calculated using a standard curve generated by determining ratio between three known amounts of the standard and represented as μmol/mg protein.

ATP level = Peak area of sample x μmoles standard in the assay volume (20μL) Peak area of standard x mg protein in the mitochondrial fraction used

Fig. 14. Standard curve of ATP

Page 65 of 152 3.2.9. Determination of B cell lymphoma-2 protein level in brain mitochondrial fraction Principle Bcl -2 protein level was measured in mitochondrial fraction with a monoclonal antibody-based enzyme linked immunosorbant assay ELISA kit (Biovendor® Laboratory and Medicine, Inc., Cezch republic). It depends on the binding of Bcl-2 protein in the samples and standards to the antibody coated (adsorbed) on the plate. A biotin-conjugated antibody is added and binds to protein captured by the first antibody. Streptavidin-horse raddish peroxidase (HRP) is added and binds to the biotin-conjugated antibody. The substrate solution is added to the wells to form the colored products. The reaction is then terminated by addition of acid and absorbance is measured.

Reagents 1- Antibody coated microtiter strips with monoclonal antibody to Bcl-2. 2- Biotin-conjugate: anti- Bcl-2 monoclonal antibody. 3- Streptavidin-HRP. 4- Standard Bcl-2 lyophilized (64 ng/ml upon reconstitution). 5- Sample diluent. 6- Assay buffer concentrate 20x: phosphate buffered saline (PBS) with 1% Tween 20 and 10% BSA. 7- Wash buffer concentrate 20x: PBS with 1% Tween 20. 8- Lysis buffer concentrate 10x. 9- Tetramethyl-benzidine (TMB) substrate solution. 10- Stop Solution: 1M phosphoric acid. Procedure 1 - Preparation of standard solution a- Seven tubes were labeled, one for each standard point: S1, S2, S3, S4, S5, S6 and S7. b- 1:2 serial dilutions for the standard curve were prepared as follows: c- 225 µl of sample diluent was pipetted into each tube. d- 225 µl of reconstituted standard was pipetted into the first tube, labeled S1 (32ng/ml). e- 225 µl of S1 was pipetted into the second tube, labeled S2, and mixed thoroughly before the next transfer. f- Serial dilutions were repeated 5 more times thus creating the points of the standard curve. The sample diluent serves as blank.

Transfer 225 µl Discard 225 µl

Page 66 of 152 Reconstituted Bcl-2 2- The microwell strips were washed twice with approximately 400 µl wash buffer per well with thorough aspiration of microwell contents between washes. Wash Buffer was allowed to sit in the wells for about 10-15 sec before aspiration. After the last wash step, wells were emptied and microwell strips were tapped on absorbent pad or paper towel to remove excess wash buffer. Microwell strips were placed upside down on a wet absorbent paper for 15 min. 3- 100 µl of sample diluent was added in duplicate to the blank wells. 4- 80 µl of sample diluent was added to the sample wells. 5- 20 µl of each sample was added in duplicate to the sample wells. 6- 50 µl of Biotin-conjugate was added to all wells. 7- The microplate was covered with an adhesive film and incubated at room temperature (18-25°C) for 2 h, on a microplate shaker set at 100 revolutions per min (rpm). 8- The adhesive film was removed and wells were emptied. The microplate was washed 3 times according to point B. of the test protocol. 9-100 µl of diluted streptavidin-HRP was added to all wells, including the blank wells. 10- Microplate was covered with an adhesive film and incubated at room temperature (18°- 25°C) for 1h, on a microplate shaker set at 100 rpm. 11- The adhesive film was removed and wells were emptied. The microplate was washed 3 times according to point B. of the test protocol. 12-100 µl of TMB substrate solution was pipetted to all wells. 13- Microplate was covered with an adhesive film and incubated at room temperature (18°-25°C) for about 10 min, avoiding direct exposure to intense light. 14- The enzyme reaction was stopped by quickly pipetting 100 µl of stop solution into each well. 15- Absorbance of each microwell was read using a spectrophotometeric microplate reader (Sunostik® SPR-960B, U.K.) at 450 nm. The plate reader was blanked according to the manufacturer's instructions by using the blank wells. The absorbance of both the samples and the standards was recorded. The standard curve is shown in Fig. 15.

Calculation

Level of Bcl-2 (ng/mg protein) was obtained from the plotted standard curve.

Page 67 of 152 Fig. 15. Standard curve of Bcl-2.

Page 68 of 152 3.2.10. Determination of protein content in different brain fractions Principle The protein content of different brain fractions used in assays was determined using the method of Lowry et al. (1951). The final color was produced from the reduction of phosphomolybdic-phosphotungestic acid reagent (Folin Ciocalteus reagent) by the copper-treated protein in alkaline medium at 500nm. Reagents 1- Solution A: 2% sodium carbonate (Na2CO3) in 0.1N (NaOH). 2- Solution B: 0.5% copper sulphate (CuSO4.5H2O) in 1% sodium potassium tratarate (Na-K tartarate). 3- Solution C: freshly prepared by mixing 50ml solution A with 1ml solution B. 4- Solution D: Folin Ciocalteus reagent. 5- Protein standard solution: 100mg BSA/100ml bidistilled water. This stock solution was then diluted to prepare standard solutions of 100, 80, 60, 40 and 20 μg/0.1ml. Procedure 1- A known volume of each of three brain fractions (20µl-50µl) was diluted up to 0.5ml with bidistilled water. 2-2.5ml of solution C was added and allowed to stand for exactly 10 min at room temperature. 0.25ml of solution D was added, mixed thoroughly and allowed to stand for 30 min at room temperature. 3- The blue color produced was measured at 500 nm against a blank which was prepared using bidistilled water instead of the sample. The assay was calibrated using different concentrations of standard BSA solutions (Fig. 16). Calculation: Concentration of protein in the fraction used = Absorbance of sample x mg protein of the standard used in the assay Absorbance of standard

Page 69 of 152 Fig. 16. Standard curve of protein.

Page 70 of 152 3.2.11. Examination of brain tissue by transmission electron microscope (TEM) Imm e diately after animal sacrifice, straiata were fixed in 3% glutaraldehyde/phosphate buffer pH 7.2 dehydrated in degraded series of ethanol. Each specimen was embedded in a mould (capsule/plate) filled with epon resin and kept in an oven at 70ºC for 3 days. After complete polymerization, samples were ready for ultra-microtomy. Semi-thin sections (1.0µm) were cut using an LKB ultra-microtome (Leica Microsystems, Wetzlar, Germany) and stained with toluidine blue for light microscope survey. Then, ultra-thin sections were double- stained with uranyl acetate/lead citrate and were examined under a Jeol 1200EXII electron microscope (JEOL Ltd., Tokyo, Japan), operated at 80kV.

3.3. Statistical analysis Values are represented as means ± standard error of the mean (SEM). Data were subjected to one-way analysis of variance (ANOVA) followed by Tukey–Kramer test to analyze differences among the groups. Statistical analysis was carried out using Graph Pad prism5 (Graph Pad Software Inc, CA, USA). The correlation between the parameters was determined using the Pearson's correlation coefficient. A P-value < 0.05 was considered significant. Microsoft Excel program was used for figure representation.

Page 71 of 152 RESULTS

Page 72 of 152 4. Results 4.1. Pilot experiments The 24h was selected as an interval time to either scarify rats after reserpine administration to induce Parkinsonism (Fig. 17A and B) or to administer reserpine after last pre-irradiation dose (Fig. 18A and B). The 24 h was found optimum, based on results observed in striatal DA level and catalepsy score.

4.2. The effect of reserpine on the catalepsy score and the other biochemical parameters in rat brain Results of the present study showed that reserpine treated rats (group II) exhibited a significant increase in catalepsy score up to 40-fold and a significant reduction in striatal level of DA that amounted to 17% in comparison to the normal group (Table 7, Fig.19, 20). Moreover, striatal DA level was found to be negatively correlated with catalepsy score (Fig. 24A; r = -0.87, P = 0.005). As shown in Table 8 and Fig. 21, this model of reserpine-induced Parkinsonism caused increased state of oxidative stress in brain demonstrated as significant decrease of GSH level that was accompanied by increases in MDA and iron levels. The decrease in GSH level amounted to 57%, while the levels of MDA and iron reached 240% and 135% as compared to the normal group value, respectively. As shown in rats treated with reserpine alone, the level of GSH was positively correlated with the levels of both DA (Fig. 24B; r = 0.78, P = 0.023) and negatively correlated MDA (Fig. 24C; r = -0.93, P = 0.008). In addition, significant decreases of brain activities of GST and NQO were also revealed in reserpine-group. Their activities amounted to 22% and 20% in comparison to the normal group value, respectively (Table 9, Fig. 22). The data demonstrated in Table 10 and Fig. 23 show significant decreases in mitochondrial complex I activity and the levels of ATP and Bcl-2 when compared to the normal group, indicating mitochondrial dysfunction-induced apoptosis in reserpine-induced Parkinsonism. The changes in the three parameters amounted to 93%, 91% and 96% as compared to the normal group, respectively. In reserpinized rats, the level of Bcl-2 was positively correlated with the complex I activity (Fig. 24D; r = 0.94, P = 0.0005) and ATP level (Fig. 24E; r = 0.98, P = 0.0001). Moreover, the activity of complex I and ATP level were also positively correlated (Fig. 24F; r = 0.94, P < 0.0006).

Page 73 of 152 4.3. The effect of pretreatment with EGb761 on the catalepsy score and the other biochemical parameters in rat brain Partial restoration of catalepsy score and striatal DA level (Table 7 and Fig. 19, 20) were recorded in the reserpinized rats pretreated with EGb761 (group IV) in comparison to the reserpinized group; where DA level amounted to 5-fold of the value recorded in the reserpinized group. In addition, catalepsy sore was diminished to 4-fold, while DA level reached 90% of normal value. The striatal DA level was found to be negatively correlated with catalepsy score in this group (Fig. 25A; r = - 0.93, P = 0.0006). Furthermore, the results shown in Table 8 and Fig. 21 indicated that the effects of reserpine on brain levels of GSH, MDA and iron were significantly masked by the pretreatment of rats with EGb761, where a rise in GSH level up to 115% of the normal value accompanied by a significant decrement in brain levels of MDA and iron to 114% and 63% of the values recorded in normal rats, respectively. Moreover, the GSH level reached about 2.5-fold, while the levels of MDA and iron were diminished to less than 1/2 the reserpinized group value. In addition, significant increase in both brain GST activity (Table 9, Fig. 22) and mitochondrial Bcl-2 level (Table 10, Fig. 23), were demonstrated in EGb761+resperine group; GST activity and Bcl-2 level amounted to 52% and 280% of the normal value, respectively. Moreover, the GST activity and Bcl-2 level reached 2.4-fold and 87-fold in comparison to the reserpine group, respectively. In this group the level of GSH was positively correlated with the levels of both DA (Fig. 25B; r = 0.94, P = 0.0005) and MDA (Fig. 25C; r = -0.88, P = 0.0035). Moreover, the level of Bcl-2 was positively correlated with the complex I activity (Fig. 25D; r = 0.72, P = 0.039) and ATP level (Fig. 25E; r = 0.98, P < 0.0001). On the same line, rats which received EGb761 prior to induction of Parkinsonism by reserpine showed a rise in the mitochondrial complex I activity and ATP level up to 96% and 84% of the normal value, respectively, while their values amounted to about 15-fold and 10-fold of the reserpinized group values, respectively (Table 10, Fig. 23). In addition, the activity of complex I and ATP level were positively correlated (Fig. 25F; r = 0.84, P = 0.008). On the other hand, no change in brain NQO activity was revealed in group IV in comparison to the reserpine group.

4.4. The effect of whole body γ-irradiation on the catalepsy score and the other biochemical parameters in rat brain The results of the current study showed a significant attenuation of the reserpine effects on catalepsy score and striatal DA level (Table 7 and Fig. 19, 20) in the group of irradiated reserpinized rats (group VII). In this group, the values of these

Page 74 of 152 two parameters amounted to 24% and about 4-fold of the reserpine treated group, respectively. In addition, the catalepsy score amounted to 10-fold, while the DA level reached 83% of the normal value. Moreover, striatal DA level was found to be negatively correlated with catalepsy score in this group (Fig. 26A; r = -0.97, P < 0.0001). Furthermore, radiation + reserpine group (VII) demonstrated a rise in brain GSH level which amounted to 2.4-fold with a decrement in level of iron and no significant change in MDA level in comparison to the reserpinized group value (Table 8, Fig. 21). On the other hand, the level of GSH was normalized while those of iron and MDA reached 47% and 195%; respectively, of the normal value. Moreover, in the irradiated+reserpine group, the level of GSH was positively correlated with the level of DA (Fig. 26B; r = 0.76, P = 0.027). Interestingly, the reserpine-induced inhibition of mitochondrial complex I activity and the depletion of ATP and Bcl-2 levels were attenuated by whole body γ- irradiation of rats before reserpine administration. Their values amounted to 43%, 51% and 123% of the values recorded in normal rats, respectively. Moreover, their values reached 6.8-fold, 6.25-fold and 38-fold of those of the reserpinized group, respectively (Table 10, Fig. 23). Moreover, in the pre-irradiated-reserpine group, level of Bcl-2 was positively correlated with ATP level (Fig. 26C; r = 0.79, P = 0.019). On the other hand, no significant change in brain GST activity was observed while NQO activity was amounted to 3.2-fold in the radiation + reserpine group when compared to the reserpinized group. In contrast, this group showed a rise in NQO activity which reached 66% of the normal value (Table 9, Fig. 22). In addition, significant rises in striatal DA level (Table 7, Fig. 20); brain GSH level (Table 8, Fig. 21) and NQO activity (Table 9, Fig. 22) were observed in the irradiated group (group VIII) in comparison to the normal group. The values of these three parameters amounted to 137%, 146% and 150% of the normal group, respectively.

4.5. The effect of the combination of EGb761 and whole body γ-irradiation on the catalepsy score and the other biochemical parameters in rat brain In the group (IX) pretreated with radiation and EGb761, reserpine treated rats demonstrated a decreased catalepsy score and an increased DA level as compared to the values detected in reserpinized group; where the DA level amounted to 9-fold as compared to the reserpinized group, while the values of catalepsy score and DA level amounted to 120% and 160% of the normal value, respectively (Table 7, Fig.19, 20). Furthermore, this group showed increases of brain GST activity (Table 9, Fig. 22) and mitochondrial Bcl-2 level (Table 10, Fig. 23) and a decrease in iron

Page 75 of 152 level (Table 8, Fig. 21); the values of these three parameters reached 55%, 225% and 50% of the normal value, respectively. Additionally, the values of these parameters reached 2.5-fold, 70-fold and 0.36 of the reserpinized group values. In addition, group (IX) showed normalization of brain levels of GSH and MDA (Table 8, Fig. 21). The values of GSH level, NQO activity, mitochondrial complex I activity and ATP level (Tables 8-10, Fig. 21-23) amounted to about 3-, 5-, 11- and 10-fold of the reserpinized group values, respectively. 4.6. The effect of reserpine and the pretreatment with EGb761, whole body γ- irradiation and their combination on the transmission electron micrograph of striatal neurons in different experimental groups

Examination of brain tissue by TEM (×20,000) revealed normal ultra structure of striatal neurons of normal rats (Fig. 27A), which showed spheroid nucleus (N) with unstained dispersed chromatin, abundant cytoplasm and normal nucleolus (Nu) and multiple intact mitochondria (M). Reserpine group (Fig. 27B) and gum acacia + reserpine group (Fig. 27E) showed ultra-structure abnormalities indicated by active lysosomes (L) with the nucleus (N) shows migration of chromatin to the nuclear membrane (NM) which is characteristic of apoptotic nuclear morphology. Improvement was revealed in reserpine groups pretreated with EGb761 (Fig. 27D), radiation (Fig. 27G) and their combination (Fig. 27 I) in some ultrastructures (as indicated by arrows) by the normal intact, slightly swollen (I) and less dense mitochondria and almost normal (less destructed) mylein sheath. Normal ultra structural were revealed in the groups treated with acetic acid (Fig. 27C), EGb761(Fig. 27F) and radiation (Fig. 27H).

Page 76 of 152 Table 7. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on catalepsy score and striatal dopamine (DA) level in reserpinized rats.

Catalepsy score Striatal DA Group (sec.) (ng/mg protein)

Normal (group I) 1.21 ± 0.14 116.7 ± 5.51

a a Reserpine/acetic acid (group II) 54.00 ± 3.71 20.76 ± 2.18

b b Acetic acid (group III) 0.90 ± 0.09 133.3 ± 3.03

b b EGb761/gum acacia+ reserpine (group IV) 5.75 ± 0.67 105.8 ± 16.97

ac ac Gum acacia + reserpine (group V) 62.63 ± 5.10 45.61 ± 5.18

EGb761/gum acacia (group VI) 1.56 ± 0.21 127.2 ± 9.11

ab b Radiation+ reserpine/acetic acid (group VII) 13.00 ± 1.85 97.15 ± 8.93

a Radiation (group VIII) 1.90 ± 0.37 160.5 ± 6.72

b abc Radiation+EGb761+reserpine (group IX) 1.53 ± 0.30 187.4 ± 11.28

Values are represented as means ± S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 77 of 152 Table 8. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain levels of glutathione (GSH), malondialdehyde (MDA) and iron in reserpinized rats.

Parameters GSH MDA Total iron (mg/g wet tissue) (nmol/mg protein) (µg/mg protein) Groups

Normal (group I) 1.99 ± 0.15 1.48 ± 0.17 176.0 ± 10.12

a a a Reserpine/acetic acid (group II) 0.85 ± 0.03 3.57 ± 0.22 241.6 ± 22.92

b b b Acetic acid (group III) 2.05 ± 0.10 2.20 ± 0.23 171.7 ± 7.13

b b ab EGb761/gum acacia+ reserpine (group IV) 2.29 ± 0.18 1.69 ± 0.12 112.4 ± 4.25

ac ac ac Gum acacia + reserpine (group V) 0.98 ± 0.15 2.94 ± 0.28 256.7 ± 18.86

b EGb761/gum acacia (group VI) 1.95 ± 0.11 2.04 ± 0.20 158.5 ± 7.35

b ac ab Radiation+ reserpine/acetic acid (group VII) 2.05 ± 0.16 2.91 ± 0.20 84.43±2.32

a Radiation (group VIII) 2.91 ± 0.14 1.37 ± 0.19 204.6±6.99

b b ab Radiation+EGb761+reserpine (group IX) 2.49 ± 0.09 2.34 ± 0.28 88.98 ± 6.45

Values are represented as means ± S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 78 of 152 Table 9. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain activities of glutathione-S-transferase (GST) and NADPH-quinone oxidoreductase (NQO) in reserpinized rats.

Parameters GST NQO (U /mg protein) (U/mg protein) Groups

Normal (group I) 105.6 ± 11.96 88.82 ± 9.14

a a Reserpine/acetic acid (group II) 23.26 ± 3.85 18.02 ± 0.97

b b Acetic acid (group III) 84.24 ± 9.29 92.56 ± 7.50

ab a EGb761/gum acacia+ reserpine (group IV) 55.92 ± 6.94 29.50 ± 2.23

ac ac Gum acacia + reserpine (group V) 16.95 ± 1.67 10.40 ± 1.01

EGb761/gum acacia (group VI) 98.57 ± 6.89 88.86 ± 5.88

a b Radiation+ reserpine/acetic acid (group VII) 40.34 ± 2.09 58.45 ± 9.57

a Radiation (group VIII) 104.9 ± 7.46 132.60 ± 6.46

ab bc Radiation+EGb761+reserpine (group IX) 58.99 ± 3.71 92.99 ± 18.47

Values are represented as means ± S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 79 of 152 Parameters Complex I ATP Bcl-2 (U/mg protein) (nmol/mg protein) (ng/mg protein) Groups

Table 10. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on complex I activity as well as the levels of adenosine triphosphate (ATP) and B cell lymphoma-2 (Bcl-2) protein in brain mitochondria of reserpinized rats.

Page 80 of 152 Normal (group I) 232.9 ± 29.21 243.5 ± 26.78 33.15 ± 2.08

a a a Reserpine/acetic acid (group II) 14.67 ± 1.20 19.82 ± 1.82 1.075 ± 0.13

b b b Acetic acid (group III) 186.9 ± 14.61 271.1 ± 12.21 32.48 ± 2.69

b b ab EGb761/gum acacia+ reserpine (group IV) 225.5 ± 23.69 205.6 ± 20.78 93.24 ± 13.12

ac ac ac Gum acacia + reserpine (group V) 21.76 ± 4.09 31.73 ± 3.83 1.238 ± 0.10 EGb761/gum acacia (group VI) 182.1 ± 14.48 233.0 ± 24.16 39.26 ± 4.59

abc ab bc Radiation+ reserpine/acetic acid (group VII) 99.64 ± 3.05 124.00 ± 9.99 40.92 ± 3.94

Radiation (group VIII) 174.6 ± 29.35 224.4 ± 25.10 31.43 ± 2.98

b b ab Radiation+EGb761+reserpine (group IX) 166.5 ± 17.13 187.7 ± 20.39 74.68 ± 6.87

Values are represented as means ± S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 81 of 152 A B

Fig. 17. Time interval between reserpine administration and scarifice of rats for A: DA level and B: catalepsy score. 24h interval (indicated by arrow) was selected. Values are represented as mean ± % S.E.M (n = 8 observations) a : Significant difference from 0h group at p < 0.05 b : Significant difference from 24h group at p < 0.05

A B

Fig. 18. Time interval between last irradiation dose and reserpine administration for A: DA level and B: catalepsy score. 24h (indicated by arrow) was selected. Values are represented as mean ± % S.E.M (n = 8 observations) a : Significant difference from 0h group at p < 0.05 b : Significant difference from 24h group at p < 0.05 c : Significant difference from 48h group at p < 0.05

Page 82 of 152 Fig. 19. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on catalepsy score in reserpinized rats.

Values are represented as % of normal group ± % S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 83 of 152 Fig. 20. Effect of standardized ginkgo biloba extract (EGb761), whole body γ-irradiation or their combination on striatal dopamine (DA) level in reserpinized rats. Values are represented as % of normal group ± % S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 84 of 152 Fig. 21. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain levels of glutathione (GSH), malondialdehyde (MDA) and iron in reserpinized rats.

Values are represented as % of normal group ± % S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 85 of 152 Fig. 22. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on brain activities of glutathione-S-transferase (GST) and NADPH-quinone oxidoreductase (NQO) in reserpinized rats.

Values are represented as % of normal group ± % S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 86 of 152 Fig. 23. Effect of standardized ginkgo biloba extract (EGb761), whole body γ- irradiation or their combination on complex I activity as well as the levels of adenosine triphosphate (ATP) and B cell lymphoma-2 (Bcl-2) in brain mitochondria of reserpinized rats.

Values are represented as % of normal group ± % S.E.M (n = 8 observations) a : Significant difference from normal group at p < 0.05 b : Significant difference from reserpine/acetic acid group at p < 0.05 c : Significant difference from EGb761/gum acacia + reserpine group at p < 0.05

Page 87 of 152 Fig. 24. Correlation analysis in reserpine-induced Parkinsonism group of striatal DA level with catalepsy score (A), GSH level with DA (B) and MDA (C);Bcl-2 level with complex I activity (D) and ATP level (E) and complex I activity with ATP level (F) using Pearson's correlation coefficient.

75 Page 88 of 152 F Fig. 25. Correlation analysis in EGb761 + reserpine group of striatal DA level with catalepsy score (A), GSH level with DA (B) and MDA (C); Bcl-2 level with complex I activity (D) and ATP level (E) and complex I activity with ATP level (F) using Pearson's correlation coefficient.

Page 89 of 152 76

Fig. 26. Correlation analysis in radiation + reserpine group of striatal DA level with catalepsy score (A), GSH level with DA (B) and ATP level with Bcl-2 level (C) using Pearson's correlation coefficPageient. 90 of 152 Fig.27. Transmission electron micrograph of striatal neurons (x20, 000) of A: normal, B: reserpine, C: acetic acid, D: EGb761 + reserpine, E: gum acacia + reserpine, F: EGb761, G: radiation+ reserpine, H: radiation and I: radiation+ EGb761 + reserpine. Nucleus (N), mitochondria (M), nucleolus (Nu), mylein sheath (MS), microvascular endothelial cell (MEC), nuclear membrane (NM), concentric cuticle structure (CS), lysosomes (L). Normal ultra-structure (A, C, F and H) and ultra-structure abnormalities (B and E), which is characteristic of apoptosis, were shown. Improvement (D, G and I) was revealed in some ultrastructures by the normal intact, slightly swollen (I) and less dense mitochondria (D) and almost normal (less destructed) mylein sheath (G) (as indicated by arrows).

78 Page 91 of 152 DISCUSSION

5. Discussion

Page 92 of 152 Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Its prevalence is predicted to increase in the coming decades due to the aging of the population. Evidences provided by previous studies suggest the involvement of oxidative damage (Przedborski and Jackson-Lewis, 2000) and mitochondrial dysfunction (Albers and Beal, 2000) in the pathogenesis of PD. Thus, agents that can modulate cellular energy metabolism and/or exert antioxidative effects could be of beneficial effects in modulating the course of PD. Despite the discovery of levodopa revolutionized the treatment of PD, most PD patients suffer considerable motor disability after 5–10 years of disease and dementia that do not adequately respond to DA replacement therapies (Schapira and Olanow, 2004; Savitt et al., 2006). Thus, finding a neuroprotective therapy that can be administered early in the course of the disease to slow, stop, or reverse disease progression is the main challenge (Schapira and Olanow, 2004). Ginkgo leaf is a widely used herbal medicine. Its standardized extract (EGb761) was reported to protect neurons against damages induced by a variety of injuries in different experimental paradigms (Ramassamy et al., 1990; Yao et al., 2001; Ahmad et al., 2005). However, in everyday life, the brain is frequently exposed to LDR due to diagnostic, therapeutic, occupational, and environmental sources. The neuroprotection offered by LDR had been found in different animal models such as inherited glaucoma, optic nerve crush and contusive spinal cord injury (Kipnis et al., 2004; Liang et al., 2006). Therefore, the present study was undertaken to investigate the pretreatment effects of EGb761 and LDR on neurological dysfunction in the reserpine-treated rat model of PD. The present study showed that reserpine produced a model of Parkinsonism resembling the basic findings in human, where bradykinesia and rigidity were manifested as an increase in catalepsy score with evident decline in striatal DA level as compared to the normal rats. Striatal DA level was found to be negatively correlated with catalepsy score (Fig. 24A, r = - 0.87). Reserpine is an oxidant and a monoamine depletory, which prevents the storage of DA in neuronal synaptic vesicles. Reserpine interferes with the VMAT, causing an increase in cytosolic DA which results in its oxidative metabolism by MAO (Abílio et al., 2003; Naidu et al., 2003; Bilska et al., 2007). The brain is more susceptible to oxidative stress when compared to

Page 93 of 152 other organs or systems, due to its high oxygen utilization, high content of unsaturated membrane lipids, excitatory amino acids, low levels of antioxidant defenses and autoxidizable NTs (Halliwell and Gutteridge, 1999). Sussman et al. (1997) showed that reserpine administration causes a decrease in striatal DA levels and an increase in the ratios of dihydroxyphenylacetic acid to DA (DOPAC/DA) and the ratio of homovanillic acid to DA (HVA/DA) in rats. In addition DA depletion may be mediated through inhibitory effect of reserpine on the plasma membrane DAT activity of mice neurons in vitro (Egana et al., 2009). Since DA uptake through DAT is used to replenish the synaptic vesicle stores for subsequent release of DA into the synaptic cleft; inhibition of DAT by reserpine results into DA depletion from its synaptic stores. As shown in the present study, reserpine produced a state of oxidative stress revealed as decreased GSH level with concomitant increase in MDA level in brain of reserpine-induced Parkinsonism rats as compared to normal rats. This finding is in accordance with the results of Bilska et al. (2007). This enhanced state of oxidative stress can be attributed to the accelerated DA metabolism that can lead to the formation of reactive metabolites and hydrogen peroxide (H2O2) in dopaminergic neurons (Abílio et al., 2003; Naidu et al., 2003; Bilska et al., 2007). This was further assessed in the current study with the positive correlation of GSH level with the level of DA (Fig. 24B, r = 0.78) and its negative correlation with the level of MDA (Fig. 24C, r = -0.93) observed in the reserpine–treated group. Furthermore, autoxidation of DA produce aminochrome that can be subjected to one- electron reduction to form the leukoaminochrome O-semiquinone radical, which is thought to be one of the major sources of endogenous reactive species involved in the degenerative processes (Fuentes et al., 2007). During PD, a further reduction in GSH levels within the SNpc has been observed (Riederer et al., 1989). GSH can bind to quinones formed during oxidation of DA (Fornstedt et al., 1990). Significant reduction in enzyme activities of GST and NQO, both involved in phase II of detoxification, has been revealed in reserpine-treated rats in the present study in comparison to their activities in normal rats. GST catalyzes the reactions of O-quinones that are formed during autoxidation of catecholamines with GSH yielding S-conjugates (Segura-Aguilar et al., 1997). Therefore, reduction in GST activity; also reported by Ahmad et al.

Page 94 of 152 (2012), can be attributed to brain GSH depletion. In addition to GST, also NQO (via two-electron reduction of the redox-labile O-quinones) is implicated in their detoxification (van Muiswinkel et al., 2000). Glutathione S-transferase catalyze the conjugation of glutathione peptides to 4HNE forming the adduct molecule GS-HNE which is a potent inhibitor of the activity of GST (Sharma et al., 2004). Addition of reserpine to a cell line derived from rat SN was reported to cause an inhibition of NQO activity (Fuentes et al., 2007). Additionally, 4HNE, a lipid peroxidation by-product may also form adducts with enzymes proteins rendering them inactive (Subramaniam et al., 1997). Mitochondrial dysfunction appears to play a major role in the neurodegeneration associated with the pathology of PD (Fiskum et al., 2003). Mitochondria was found to play essential roles in regulation of key steps in both apoptotic and necrotic cell death by affecting energy metabolism, participating in intracellular Ca2+ homeostasis, regulating the activity of caspases, and releasing ROS (Kowaltowski et al., 2004). Mitochondria are responsible for generating 90% of the ATP required for all cellular functions. This reserpine model confirmed the implication of mitochondrial dysfunction in the pathogenesis of Parkinsonism as there was a decrease in striatal complex I activity and ATP level; those are considered as potential biomarkers for diagnosis of PD (Savitt et al., 2006). This observation is in accordance with that of Osubor and Nwanze (1994); where reserpine injection caused a decrease in the respiratory chain activity in frontal cortex and striatum of rats one hour after administration. Depletion of brain GSH has been reported previously to result in decreases in mitochondrial enzyme activities as well as losses in ATP production in the brain (Heales et al., 1995). The consequences of mitochondrial GSH depletion in dopaminergic neurons of SN during PD were revealed in a dopaminergic PC12 cell line model system; wherein, the level of the γ-GCS enzyme was decreased resulting in reduction in GSH synthesis. The decrement in the mitochondrial GSH in these genetically engineered cells resulted in increased oxidative stress and impaired mitochondrial function as reflected by decreased pyruvate-mediated mitochondrial respiration and ATP synthesis (Jha et al., 2000). In addition, reduction in the activity of mitochondrial complex I in the SN have been reported as a major biochemical feature in the pathogenesis of PD (Haas et al., 1995). Interestingly, complex I has been found to be one of the most severely affected mitochondrial enzymes during oxidative stress (Lenaz et al., 1997).

Page 95 of 152 In synaptic mitochondria, complex I exert a major control over oxidative phosphorylation such that a decrease in its activity by 25% drastically affected ATP synthesis and the overall energy metabolism within the cell, whereas inhibition of complex III and IV up to 80% was necessary to show similar effect (Davey et al., 1998). One of the possible reasons for the susceptibility of complex I to oxidative damage is thiol oxidation and the presence of accessible oxidation sensitive iron-sulfur centers within this enzyme complex (Keyer and Imlay, 1996). Therefore, the observed reduction of brain GSH level, which is the chief molecular player in maintaining the SH groups of protein in reduced state can be associated with reduction of the activity of complex I.

The depletion in brain mitochondrial ATP level evoked by reserpine administration herein is compatible with the results reported by Kirpekar and Lewis (1959); where reserpine caused depletion of ATP in isolated rat brain. Uncoupling agents such as reserpine can disturb metabolism by depressing the formation of high energy phosphate bonds without depressing the oxygen consumption by mitochondria (Brody, 1955). Moreover, reserpine was reported to enhance ATP hydrolysis in intact rat liver mitochondria (Weinbach et al., 1983).

Accumulating experimental evidence suggest that the rise of the free cytosolic concentration of DA due to disruption of its vesicular storage could be attributed to mitochondria-related energy failure and inhibition of the mitochondrial respiratory system (Brenner-Lavie et al., 2008; 2009). Elevated rat brain DA concentrations following chronic administration of levodopa was reported to result in a reduction of mitochondrial complex I activity and ATP level in the striatum (Przedborski et al., 1993; Chan et al., 1994). Moreover, in a neuronal cell line; DA induced a reduction in cellular ATP levels without affecting cell viability (Ben-Shachar et al., 2004). Furthermore, in disrupted mitochondria from both rat brain and human platelets, DA reversibly inhibited complex I activity but not that of complexes IV and V (Ben-Shachar et al., 1995; Khan et al., 2005). The existence of a relationship between DA level and complex I activity was reported previously. Two mechanisms have been suggested for DA interference with mitochondrial respiration: the first involves DA enzymatic catabolism or autoxidation to highly ROS which in turn affect the normal mitochondrial functions leading to cell death in PD. The second proposed mechanism is through a direct reversible inhibition of complex I activity, which can disrupt

Page 96 of 152 mitochondrial activity leading to abnormal neuronal transmission, rather than cell death (Ben-Shachar et al., 2004). Thus, depletion of DA stores by reserpine in the present study might have caused a transient rise in DA levels before its oxidation into other metabolites; with a subsequent further inhibition in mitochondrial complex I activity.

Besides the decreased level of GSH and the impaired mitochondrial complex I activity, a third component supporting the role of oxidative stress in PD is the increased brain iron level in reserpine-treated rats in the present work in comparison to normal rats. Sofic et al. (1991) have demonstrated that total iron levels in the SN of PD patients are higher than age matched controls. Similar findings utilizing various methods have been reported previously (Dexter et al., 1989; Griffiths et al., 1999); where Griffiths et al. (1999) demonstrated that in PD patients, ferritin is heavily loaded with iron implying that even if there is an increase in ferritin levels to counter excess iron levels, the ferritin molecules are saturated with iron. Iron also catalyses the conversion of excess DA to neuromelanin, an insoluble black-brown pigment that accumulates in all dopaminergic neurons with age in humans (Sulzer et al., 2000). Neuromelanin in general is neuroprotective and sequesters redox active ions in the cell with a high affinity for Fe3+ ions. However, when it is bound to excess Fe3+, neuromelanin tends to become a prooxidant and reduces Fe3+ to Fe2+, which then gets released from neuromelanin owing to its weak affinity (Ben-Shachar et al., 1991), thus increases the neuronal iron pool and also the fraction of iron capable of reacting with H2O2.

Moreover, increased brain iron level in reserpine-injected rats may be due to the increased intestinal iron absorption reported by Ganchev et al. (1998). An increase in reactive iron available for generation of ROS may result from the superoxide or catechol-mediated release from loaded ferritin pool (Double et al., 1998). Hence, it is suggested that oxidative stress produced during PD is likely the consequence of H2O2 production due to a combination of DA oxidation, GSH depletion and Fe2+ generated by neuromelanin or released from ferritin, thus allowing the Fenton reaction to proceed at a considerable rate resulting in neuronal death.

The mechanism by which inhibition of complex I lead to degeneration of dopaminergic neurons, involves activation of mitochondria-dependent apoptotic molecular pathways (Waldmeier and Tatton, 2004; Ekstrand et

Page 97 of 152 al., 2007). In addition, oxidative stress promotes the expression and/or intracellular distribution of proapoptotic proteins to the mitochondrial outer membrane (Savitt et al., 2006). The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role of Bcl- 2 proteins in regulating either mitochondrial physiology or mitochondria- dependent caspase activation. In the current study, the antiapoptotic protein Bcl-2 level was found to be extremely reduced in the mitochondria of rats with reserpine-induced Parkinsonism. This was further confirmed in rats treated with reserpine alone, herein, where the level of Bcl-2 was positively correlated with both the mitochondrial activity of complex I (Fig. 24D, r = 0.94) and ATP level (Fig. 24E, r = 0.98). Moreover, the activity of complex I and ATP level were positively correlated (Fig. 24F, r = 0.94).

Apoptosis was also confirmed by transmission electron microscope (TEM) examination in the present work. Ultra structure abnormalities indicated by active lysosomes with the nucleus showing migration of chromatin to the nuclear membrane which is characteristic of apoptotic nuclear morphology were revealed in reserpinized rats in the present study. Apoptosis is controlled in part by the Bcl-2 family of regulatory proteins (Bcl-2, Bcl-x, Bax, and others). Bcl-2 can prevent or delay apoptosis (Reed, 1997). In a previous study, Cantarella et al. (2009) reported increased expression of the proapoptotic protein Bax and decreased expression of the antiapoptotic protein Bcl-2 in gastric mucosa of rats injected with reserpine. Interestingly, in the current study, the reduction in Bcl-2 level observed in the reserpine treated group might be at least in part, due to the oxidative stress caused by reserpine; as H2O2 was reported to reduce Bcl-2 gene expression in T-cell line (Kane et al., 1993; Gottlieb et al., 2000).

Moreover, Abdin and Hamouda (2008) reported that although levodopa administration caused symptomatic improvement in the form of reduction of catalepsy score with restoration of striatal DA levels, but it did not show any significant effects on either striatal complex I activity, ATP or Bcl-2 levels, pointing to the lack of its disease-modifying role. Therefore, understanding and finding the cause of dopaminergic cell loss will lead to exploring therapies that prevent and cure the disease. Results of the present study demonstrate the ameliorating effect of pretreatment with EGb761 on reserpine-induced Parkinsonism group where it caused partial restoration of the striatal DA level and catalepsy score as compared to the reserpinized group. This finding is supported by the negative

Page 98 of 152 correlation revealed between striatal DA level and catalepsy score in group IV (Fig. 25A; r = -0.93). Ginkgo biloba is a potent inhibitor of MAO, which would prevent the degradation of DA and increase its availability (Sloley et al., 2000). Pretreatment with EGb761 has been reported to restore or increase striatal DA level in 6-OHDA and MPTP models of Parkinsonism, respectively (Wu and Zhu, 1999; Ahmad et al., 2005).

Besides the inhibition of MAO, this effect of EGb761 could be also attributed to its free radical scavenging property (Ahlemeyer and Krieglstein, 2003); as such a decrease of DA uptake has been considered as a consequence of the deleterious action of free radicals on uptake mechanisms through the peroxidation of membrane components. These findings are in agreement with other previous findings, in which in vivo administration of EGb761 protected the level of DA in the nigrostriatal system (Ahmad et al., 2005). In line with this, EGb761 was reported to prevent not only the decrease of DA uptake but also its autoxidation in mice cerebral cortex synaptosomes in a concentration-dependent manner (Ramassamy et al., 1992b).

This free radical scavenging and antioxidant activity of EGb761 is confirmed in the current work via restoration of GSH and MDA levels along with significant increase of GST activity in brain as compared with the values recorded in normal rats. These findings are in agreement with other previous findings, in which EGb761 or some of its constituents increased GSH level, activity of GST and reduced MDA level in brain, enhanced brain antioxidant enzymatic status in Parkinsonism-model induced in mice (Ahmad et al., 2005) and showed strong iron chelating property in in vivo and in vitro assays (Perez et al., 2008; Reznichenko et al., 2010). Another supporting observation was reported in the study of Ma et al. (2012); where the administration of ginkgolide K to rats prior to the induction of cerebral ischemia markedly reversed the level of MDA in both serum and the ischemic cerebral section. These findings are also supported by the positive correlation of the level of GSH with the levels of DA (Fig. 25B; r = 0.94) and its negative correlation with MDA (Fig. 25C; r = -0.88) revealed in the present work.

A significant reduction in brain iron level was recorded in reserpinized rats pretreated with EGb761 in the present study as compared to the reserpine- treated group. This action could be attributed to the polyphenolic content of the ginkgo extarct, including flavonoids, which has been reported to possess

Page 99 of 152 metal chelating properties (Sgaragli et al., 1993; Bars et al., 1994). Furthermore, a number of polyphenols (such as those found in ginkgo biloba) was previously reported to protect DA neurons from neurotoxins such as 6- OHDA in in vitro assays (Mercer et al., 2005). Furthermore, EGb761 was able to attenuate the degeneration of DA neurons and symptoms caused by the neurotoxins MPTP and 6-OHDA in both in vitro and in vivo conditions (Chen et al., 2007). Thus, it could be concluded that metal chelation may be a possible mechanism responsible for this effect.

Concerning the effect of EGb761 on the brain activity of NQO enzyme; it failed to restore the enzyme activity when administered prior to induction of Parkinsonism by reserpine. In contrast, oral treatment with EGb761 and bilobalide caused a dose-dependent elevation in NQO activity in mouse liver (Sasaki et al., 2002). This contradiction might be attributed to the different animal species and/or the organ under investigation used in their study.

The results of the present study demonstrated the effect of enhanced brain antioxidant status; due to pretreatment with EGb761 in amelioration of the mitochondrial dysfunction induced apoptosis where it caused restoration of complex I activity, ATP level and an increase in level of the antiapoptotic protein Bcl-2 as compared to reserpine-injected group. Moreover, in the current study, the level of Bcl-2 was positively correlated with the complex I activity (Fig. 25D; r = 0.72) and ATP level (Fig. 25E; r = 0.98) and the activity of complex I and ATP level were found to be positively correlated (Fig. 25F; r = 0.84). EGb761 was reported previously to alleviate mitochondrial dysfunctions in vitro and in vivo resulting in restoration of the activities of complexes I, IV and V in mice (Abdel-Kader et al., 2007); increase the mitochondrial NADH dehydrogenase mRNA level (Tendi et al., 2002) and cellular ATP levels (Abdel-Kader et al., 2007) in PC12 cells.

These data appears to be in harmony with that of Janssens et al. (1999); where the activity of complex I together with ATP level was elevated in liver mitochondria isolated from rats treated with bilobalide. The non-flavone fraction bilobalide and to a lesser extent terpenoid; ginkgolide B were found to be responsible for increasing ATP level. Bilobalide easily crosses the blood brain barrier and reaches extracellular concentrations in the brain that allow efficient interaction with target molecules such as NTs receptors (Lang et al., 2010). The exact mechanism whereby bilobalide operates on complexes I and III seems to be questioned. However, several hypotheses can

Page 100 of 152 -• be suggested; bilobalide could act as an antioxidant by scavenging O2 generated by electron leakage (Nohl et al., 1993) and could be by itself an electron transporter, thus increasing electron transfer from complex I to complex III (Janssens et al., 1999).

The increase of the reduced mitochondrial Bcl-2 level in reserpine+ EGb761 group is comparable with the previous studies of Koh (2009) and Jiang et al. (2009). It is speculated that EGb761 may be a potential neuroprotective agent against apoptosis through the differential expressions of the Bax and Bcl-2 in brain (Mak et al., 2006). Bcl-2 increase has been previously shown to act at multiple steps of mitochondrial mediated apoptosis including: preventing the release of apoptogenic factors, such as cytochrome c and apoptosis-inducing factor from mitochondria (Susin et al., 1996), increasing the maximal mitochondrial Ca+2 uptake capacity (Murphy et al., 1996) and preventing oxidative stress (Hockenbery et al., 1993; Voehringer, 1999). There are also some indications that Bcl-2 may function as an antioxidant and in this way it exerts the anti-apoptotic activity (Hochman et al., 1998; Gottlieb et al., 2000). Furthermore, there was evidence that Bcl-2 can promote regeneration of retinal axons in vitro, independent of its antiapoptotic effects, suggesting its neuroprotective effects, as well as restorative effects, in promoting regrowth of dopaminergic axons in PD (Chen et al., 1997). The anti-apoptotic effect of EGb761 was also confirmed by TEM examination in the present study, which is also in harmony with other previous studies (Smith et al., 2002).

Pretreatment with 1.5Gy whole body γ-radiation restored the reduced level of DA and decreased catalepsy score caused by reserpine. This finding is further supported by the negative correlation revealed between striatal DA level and catalepsy score in group VII (Fig. 26A; r = -0.97). Similarly, Liang et al. (2006) demonstrated that low dose whole-body γ-irradiation of mice rendered neuroprotection against MPTP-mediated damage of striatal dopaminergic nerve fibers. This neuroprotective effect of low dose-whole body irradiation may be attributed to inhibiting MAO activity in cerebral cortex, cerebellum and hippocampus as reported previously by Catravas and McHale (1974). Hence, this inhibition of MAO activity by radiation might cause reduced DA metabolism and consequent rise in its striatal level observed in the present work. This mechanism is further supported by the observed increase in striatal DA level in rats exposed to whole body γ- radiation without reserpine injection in the current work.

Page 101 of 152 Interestingly, the results of the present study showed that pretreatment with low dose-whole body γ-irradiation restored GSH level and reduced the level of brain iron level as compared to reserpine group. This finding is in harmony with the study of Kojima et al. (1999), which attributed the increment of GSH level to the elevation of thioredoxin (TRX) activity in 0.5 Gy-pretreated MPTP model. TRX is not only a potential endogenous antioxidant, but also contributes to the biosynthesis of GSH by promoting cystine transport into cells and is also a key protein for the control of cellular redox status (Okamoto et al., 1992). Moreover, in our study, the level of GSH was positively correlated with level of DA (Fig. 26B; r = 0.76) in rats pre-irradiated, prior to reserpine administration.

Liang et al. (2006) supposed that two mechanisms might be responsible for the neuroprotection induced by low dose irradiation: increasing of GSH level (Kojima et al., 1999) and activation of immune function (Nogami et al., 1993) and of enzymatic DNA repair induced by radiation hormesis (Kipnis et al., 2004). Increased brain GSH level by radiation was revealed in the group of rats pretreated with irradiation only in the present study. Kipnis et al. (2004) reported that low dose γ-irradiation was accompanied by an increased incidence of activated T cells, leading to accumulation of self- reactive T cells in injured CNS and neuroprotection through the release of various cytokines. In addition, Safwat (2000) reported that immune enhancement, rather than direct radiation cell killing, is one of the suggested mechanisms by which low-dose total body irradiation (TBI) can exert its effect; where the data from animal experiments have shown that low-dose TBI could enhance the immune response through augmenting the proliferative reactive response of the T cells to mitogenic stimulation; altering cytokine release, particularly the activation of interferon-γ (IF-γ) and IL-2 production; increasing the expression of IL-2 receptors on the T-cell surface; facilitating signal transduction in T lymphocytes; increasing splenic catecholamine content and lowering the serum corticosterone level and eliminating a particularly radiosensitive subset of the suppressor T-cells.

In the present work, irradiation of rats prior to reserpine administration resulted in a significant attenuation of the rise in brain iron level evoked by reserpine administration. A contradictory observation was reported in the study of Robello et al. (2009). The authors reported a significant increase in the total iron level of the developing fetal rat brain embryos when exposed to

Page 102 of 152 local brain γ-irradiation at a dose level of 1Gy; one to four hours post- irradiation, as compared to its level in non-irradiated brain. This contradiction might be a result of the difference between tissue responses to local and whole body irradiation and/or the higher radiation sensitivity of the developing brain than that of the adult brain. The more immature antioxidant defenses and the higher abundance of labile Fe found in the developing CNS, together with the high proportion of dividing neuroblasts, might be the reasons for the high radiosensitivity of developing brain (Guelman et al., 2004).

In addition, restoration of reduced NQO activity was evoked by pre- irradiation of reserpinized rats in the current work when compared to the reserpine-injected group. The finding of the current work is further confirmed by the significant rise in NQO activity observed in brains of rats exposed to whole body γ-radiation only. This observation is in harmony with a previously reported one; where in vitro irradiation of human lung cancer cell line at a dose level of 4Gy caused a long-lasting increment of NQO protein expression and activity (Choi et al., 2007). Furthermore, IR at different dose levels (2.5, 4 and 4.5Gy) caused a significant elevation of NQO expression and activity in human and murine tumor cells (Park et al., 2005; Suzuki et al., 2006).

Significant increases in brain mitochondrial complex I activity and ATP level were observed in reserpinized rats pretreated with radiation as compared to the reserpine group. These apparent increases are comparable with other studies using different irradiation dose levels (Gong et al., 1998; Sattler et al., 2010). In the former study; the level of mitochondrial NADH dehydrogenase mRNA was increased one hour after exposure of human glioblastoma cell line to low dose IR (0.05 Gy X-rays), with elevated expression persisting for at least 24h.

In the study of Sattler et al. (2010); where tumour xenografts derived from human head and neck squamous cell carcinoma were irradiated with 30 fractions within 6 weeks showed increased ATP level in tumor cells. Another study revealed that whole body acute γ-irradiation of developing and adult rats with low or high doses was able to inhibit nucleotide hydrolysis. Low dose of radiation inhibited ADP and AMP hydrolysis while high dose inhibited only ATP hydrolysis in purified synaptic plasma membrane (Stanojević et al., 2009).

Page 103 of 152 On the other hand, a significant inhibition of liver mitochondrial oxidative phosphorylation was observed at 72h following 8Gy-whole-body X- irradiation of rats (Alexander et al., 1972). Generally, many of the conflicting reports on the effects of whole body irradiation on mitochondrial function in various tissues appear to be arguing due to two major factors; the amount of IR actually reaching the organ and the length of time after irradiation at which the effect is measured (Hall et al., 1963).

In the current study, irradiated rats showed significant reduction in apoptosis as revealed by the increase in mitochondrial Bcl-2 level and TEM observations. The significant increase of mitochondrial Bcl-2 level in the pre- irradiated-reserpine group may be attributed to amelioration of GSH depletion and mitochondrial dysfunction in brain. This is further supported by the positive correlation between the levels of Bcl-2 and ATP in the present study (Fig. 26C; r = 0.79). This may be attributed to the increase in Bcl-2 gene expression in the anterior segments of porcine eyes following irradiation as previously reported; where irradiation exerted a profound preservative anti-apoptotic effect on these cells (Akeo et al., 2006).

Low dose irradiation at 0.01- 0.5 Gy dose levels were reported to decrease the apoptosis in the mice spleenocytes (Bogdándi et al., 2010). On the other hand, the same study showed that exposure to 2 Gy increased apoptosis in these cells. Furthermore, localized low dose γ-irradiation (4 Gy) induced apoptosis in human follicular lymphoma cells (Ganem et al., 2010). Another recent study reported that low dose irradiation decreased the number of apoptotic cells in the glomeruli of nephrectomized rats, while high dose showed a dramatic increase in apoptotic cells in the glomeruli at week 2 (Aunapuu et al., 2010).

In addition, P. falciparum infection-induced apoptosis in cultured human peripheral blood mononuclear cells (PBMCs) was inhibited by 0.07 Gy low dose irradiation of cells (Singh et al., 2009). At the molecular level, the authors of this study proved that low dose irradiation reduced the expression of pro-apoptotic proteins and induced survival proteins in PBMCs. A further supporting observation was pointed out by the study of Zhao et al. (2010) that showed that significant attenuation of diabetes-induced testicular cell death and mitochondrial dysfunction and increased expressions of the pro- apoptotic Bax mRNA and protein were obtained by repetitive exposures to low dose radiation.

Page 104 of 152 Apart from the higher level of DA which exceeds the value detected in the normal group, the combined pretreatment with low dose whole body γ- irradiation and EGb761 produced comparable effects to either treatment alone.

It could be concluded that replenishment of normal GSH level within the brain may hold an important key to protective management of PD. EGb761 and low dose 1.5Gy whole body γ-irradiation produced neuroprotective effects against reserpine-induced Parkinsonism on several levels of the proposed mechanisms, including improvement of oxidant status, mitochondrial function and intervention with neuronal apoptosis. A further research to investigate other apoptosis-targeted compounds will open a new era in the protective management as well as the treatment of Parkinsonism.

Page 105 of 152 SUMMARY AND CONCLUSION

Page 106 of 152 6. Summary and Conclusion Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, primarily affecting people of ages over 65 years. PD is a slowly progressive neurodegenerative disorder clinically manifested by resting tremors, rigidity, bradykinesia and postural instability. It is widely accepted that the main behavioral disturbances in PD are the consequence of a substantial loss of dopaminergic neurons within the SNpc and a depletion of DA in the striatum, with intracellular inclusions called LB. Reserpine is an alkaloid, derived from roots of several members of Rauwolfia genus. Reserpine has been used clinically for treatment of hypertension and as a tranquilizer, but its use has been limited because of its side effects. Reserpine is an oxidant and a monoamine depletory, which prevents the storage of DA. The association of an elevation in DA metabolism in reserpine-induced animal model with neurochemical, behavioral and neuropathological features of PD makes it suitable to study neuroprotective strategies Besi des being a free radical scavenger, Ginkgo biloba has been reported to enhance activities of various enzymatic antioxidants in sriatum, SN and hippocampus, the major sites damaged in PD. The standardized extract of Ginkgo biloba (EGb761) was reported to exhibit protective properties against neuronal dysfunction in several animal models of brain injury. Generally, irradiation is known to evoke harmful effects on nervous system and can lead to peripheral neuropathy and DNA damages. However, recent studies suggested that low dose whole-body γ-irradiation rendered neuroprotection in several animal models. Th erefore, the present study was undertaken to investigate the pretreatment effects of EGb761, low dose whole-body γ-irradiation or their combination on neurological dysfunctions in the reserpine-treated rat model of PD. In order to perform such investigations, male Wistar rats were pretreated orally with EGb761 (100 mg/kg BW/day for 3 weeks) or low dose whole-body γ- irradiation (0.25 Gy once a week for 6 weeks) or their combination (EGb761 was received during the last three weeks of the irradiation period) and then subjected to intraperitoneal injection of reserpine (5mg/kg BW dissolved in 1% acetic acid) 24h after last dose of EGb761or radiation. All rats were sacrificed 24h after reserpine injection. Depletion of striatal DA level, increased oxidative stress indicated via depletion of GSH, increased MDA and iron levels; decrease of DA metabolites

Page 107 of 152 metabolizing enzymes; indicated by decrease of GST and NQO activities; mitochondrial dysfunction; indicated by decline of complex I activity and ATP level and increased apoptosis; indicated by the decrease of mitochondrial Bcl-2 level and by TEM were observed in brain of reserpine-induced PD model group, along with behavioral study indicated by increased catalepsy score. Moreover, the level of GSH was positively correlated with the level of DA (r = 0.78) and negatively correlated to MDA (r = -0.93). The level of Bcl-2 was positively correlated with both complex I activity (r = 0.94) and ATP level (r = 0.98). Restoration of catalepsy score and striatal DA level was observed in the groups pretreated with either EGb761 (group IV) or 1.5Gy whole body γ-irradiation (group VII) in comparison to the normal group (group I). In addition, striatal DA level was found to be negatively correlated with catalepsy score in group IV (r = - 0.93) and group VII (r = -0.97). Normalization of brain levels of GSH and MDA; mitochondrial complex I activity and level of ATP in comparison to the normal group as well as significant increase of brain GST activity, mitochondrial Bcl-2 level and decrease of iron level in comparison to the reserpine group were demonstrated in EGb761 + reserpine group. In this group the level of GSH was positively correlated with the level of DA (r = 0.94) and negatively correlated with MDA (r = -0.88). Moreover, the level of Bcl-2 was positively correlated with both complex I activity (r = 0.72) and ATP level (r = 0.98). Furthermore, the activity of complex I and ATP level were positively correlated (r = 0.84). On the other hand, no change in brain NQO activity was also revealed in group IV. In addition, no change was revealed in the group administered EGb761 dissolved in gum acacia. The group administered gum acacia+reserpine (group V) did not differ from the reserpine group confirming the inertness of gum acacia. Pre-irradiated-reserpine group (VII) demonstrated restoration of GSH level and NQO activity in brain and mitochondrial Bcl-2 level in comparison to the normal group along with significant decrease of brain iron level in comparison to the reserpine group. Moreover, in the pre-irradiated-reserpine group, the level of GSH was positively correlated with the level of DA (r = 0.76) and the level of Bcl-2 was positively correlated with the ATP level (r = 0.79). Significant increases in mitochondrial complex I activity and ATP level, and no significant change in brain MDA level and GST activity were observed in the radiation +reserpine group in comparison to the reserpine group. Significant increase in striatal DA level; brain GSH level and NQO activity was observed in the group pretreated with irradiation only (group VIII) in comparison to the normal group. In the group (IX) pretreated with radiation and EGb761, rats demonstrated increased DA level which was higher than the values detected in normal group as well as increases of brain GST activity and mitochondrial Bcl-2 level, decrease in

Page 108 of 152 iron level and normalization of brain levels of GSH and MDA, NQO activity, mitochondrial activity of complex I, level of ATP and catalepsy score. The results of the present study revealed that EGb761 or low dose 1.5Gy whole body γ-irradiation ameliorated the state of oxidative stress, mitochondrial dysfunction and apoptosis observed in reserpine injected rats. It could be concluded that replenishment of normal GSH level within the brain may hold an important key to the protective management of PD. EGb761 and low dose 1.5Gy whole body γ-irradiation produced neuroprotective effects against reserpine-induced Parkinsonism on several levels of the proposed mechanisms, including improvement of oxidant status, mitochondrial function and intervention with neuronal apoptosis. A further research to investigate other apoptosis-targeted compounds will open a new era in the protective management as well as treatment of Parkinsonism.

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Page 147 of 152 ARABIC SUMMARY

Page 148 of 152 ﺍﻟﻤﻠﺨﺹ ﺍﻟﻌﺭﺒﻲ

ﯾﻌﺘﺒﺮ ﻣﺮض اﻟﺸﻠﻞ اﻟﺮﻋﺎش ﺛﺎﻧﻲ أﻛﺜﺮ أﻣﺮاض اﻟﻀﻤﻮر اﻟﻌﺼﺒﻲ ًإﻧﺘﺸﺎرا ﺑﻌﺪ ﻣﺮض اﻟﺰھﺎﯾﻤﺮ، و ھﻮ ﯾﺼﯿﺐ اﻷﺷﺨﺎص اﻟﺬﯾﻦ ﺗﺘﻌﺪى أﻋﻤﺎرھﻢ اﻟﺨﺎﻣﺴﺔ و اﻟﺨﻤﺴﯿﻦ ًﻋﺎﻣﺎ. و ﺗﺘﻠﺨﺺ أﻋﺮاض ھﺬا اﻟﻤﺮض ﻓﻲ اﻟﺘﯿﺒﺲ، رﺟﻔﺔ اﻟﻌﻀﻼت ، ﺑﻂء اﻟﺤﺮﻛﺔ و ﻋﺪم إﺳﺘﻘﺮار اﻟﻘﺎﻣﺔ، و ﻣﻦ اﻟﻤﺘﻌﺎرف ﻋﻠﯿﮫ أن اﻟﺴﺒﺐ اﻟﺮﺋﯿﺴﻲ ﻟﺤﺪوث ھﺬه اﻷﻋﺮاض ھﻮ ﻓﻘﺪان اﻟﺨﻼﯾﺎ اﻟﻌﺼﺒﯿﺔ ﻓﻲ ﻣﻨﻄﻘﺔ "ﺳﺎﺑﺴﺘﺎﻧﺸﯿﺎ ﻧﯿﺠﺮا" و ﻧﻀﻮب ﻣﺎدة اﻟﺪوﺑﺎﻣﯿﻦ ﻓﻲ ﻣﻨﻄﻘﺔ ﺍﻟﺠﺴﻡ ﺍﻟﻤﺨﻁﻁ (ﺍﻹﺴﺘﺭﺍﻴﺎﺘﻡ) ﺒﺎﻟﻤﺦ، ﻣﻊ وﺟﻮد ﺗﺮﺳﺒﺎت داﺧﻞ اﻟﺨﻼﯾﺎ ﺗﻌﺮف ﺑﺈﺳﻢ أﺟﺴﺎم ﻟﻮي. و ﯾﻌﺘﺒﺮ اﻟﺮﯾﺰرﺑﯿﻦ ھﻮ أﺣﺪ اﻟﻘﻠﻮﯾﺪات اﻟﻤﺴﺘﺨﺮﺟﺔ ﻣﻦ ﺟﺬور ﺑﻌﺾ ﻧﺒﺎﺗﺎت اﻟﺮاوﻟﻔﯿﺎ. و ﯾﺴﺘﺨﺪم اﻟﺮﯾﺰرﺑﯿﻦ ًإﻛﻠﯿﻨﯿﻜﯿﺎ ﻓﻲ ﻋﻼج إرﺗﻔﺎع ﺿﻐﻂ اﻟﺪم ﻛﻤﺎ ﯾﺴﺘﺨﺪم ﻛﻤﮭﺪئ ﻟﻸﻋﺼﺎب و ﻟﻜﻦ أﻋﺮاﺿﮫ اﻟﺠﺎﻧﺒﯿﺔ ﺗﺤﺪ ﻣﻦ إﺳﺘﺨﺪاﻣﮫ ﻋﻠﻰ ﻧﻄﺎق واﺳﻊ. ﯾﻌﻤﻞ اﻟﺮﯾﺰرﺑﯿﻦ ﻛﻤﺎدة ﻣﺆﻛﺴﺪة ﻛﻤﺎ ﯾﺆدي إﻟﻰ ﻧﻀﻮب اﻷﻣﯿﻨﺎت اﻷﺣﺎدﯾﺔ و اﻟﺬي ﯾﻤﻨﻊ ﺗﺨﺰﯾﻦ اﻟﺪوﺑﺎﻣﯿﻦ ﺑﺎﻟﺨﻼﯾﺎ. و ﯾﺘﻤﯿﺰ ﻧﻤﻮذج إﺣﺪاث اﻟﺸﻠﻞ اﻟﺮﻋﺎش ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ ﺑﻤﻼﺋﻤﺘﮫ ﻟﺪراﺳﺔ اﻟﺸﻠﻞ اﻟﺮﻋﺎش و ﻃﺮق ﺗﺤﺴﯿﻦ اﻷﻋﺮاض اﻟﻌﺼﺒﯿﺔ و اﻟﻜﯿﻤﯿﺎﺋﯿﺔ اﻟﺤﯿﻮﯾﺔ اﻟﻤﺼﺎﺣﺒﺔ ﻟﮫ. ﯾﺴﺘﺨﺪم ﻧﺒﺎت اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ ﻣﻨﺬ اﻟﻘﺪم ﻓﻲ اﻟﻄﺐ اﻟﺸﻌﺒﻲ اﻟﺼﯿﻨﻲ ﻟﻌﻼج اﻟﻌﺪﯾﺪ ﻣﻦ اﻷﻣﺮاض. و ﯾﻌﻤﻞ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﻛﻤﻀﺎد ﻟﻸﻛﺴﺪة، ﻛﻤﺎ ﯾﻌﻤﻞ ﻋﻠﻰ ﺗﺤﻔﯿﺰ ﻧﺸﺎط اﻟﻌﺪﯾﺪ ﻣﻦ اﻹﻧﺰﯾﻤﺎت اﻟﻤﻀﺎدة ﻟﻸﻛﺴﺪة ﻓﻲ أﻛﺜﺮ ﻣﻨﺎﻃﻖ اﻟﻤﺦ ﺗﺄﺛ ًﺮا ﻓﻲ ﺣﺎﻟﺔ اﻟﺸﻠﻞ اﻟﺮﻋﺎش. ﻛﻤﺎ أﻇﮭﺮت اﻟﺪراﺳﺎت اﻟﺤﺪﯾﺜﺔ ﺗﺄﺛﯿﺮ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﻋﻠﻰ اﻟﻮﻗﺎﯾﺔ ﻣﻦ اﻹﻋﺘﻼل اﻟﻌﺼﺒﻲ ﻓﻲ اﻟﻜﺜﯿﺮ ﻣﻦ اﻟﻨﻤﺎذج اﻟﻤﺮﺿﯿﺔ اﻟﻤﺤﺪﺛﺔ ﻓﻲ ﺣﯿﻮاﻧﺎت اﻟﺘﺠﺎرب. ﯾﻌﺘﺒﺮ اﻟﺘﻌﺮض ﻟﻺﺷﻌﺎع، ﻋﻠﻰ وﺟﮫ اﻟﻌﻤﻮم، ﻣﺆﺛﺮ ﺳﻠﺒﻲ ﻋﻠﻰ اﻟﺠﮭﺎز اﻟﻌﺼﺒﻲ و اﻟﺬي ﯾﻤﻜﻦ أن ﯾﺆدي إﻟﻰ إﻋﺘﻼل ﻓﻲ اﻷﻋﺼﺎب اﻟﻄﺮﻓﯿﺔ و ﺗﺪﻣﯿﺮ اﻟﺤﻤﺾ اﻟﻨﻮوي. و ﻟﻜﻦ اﻟﺪراﺳﺎت اﻟﺤﺪﯾﺜﺔ أﻇﮭﺮت ﻗﺪرة اﻟﺠﺮﻋﺎت اﻟﻤﻨﺨﻔﻀﺔ ﻣﻦ اﻹﺷﻌﺎع اﻟﻤﺆﯾﻦ (ﺟﺎﻣﺎ) ﻋﻠﻰ وﻗﺎﯾﺔ اﻷﻋﺼﺎب ﻓﻲ أﻛﺜﺮ ﻣﻦ ﻧﻤﻮذج ﻣﺮﺿﻲ ﻓﻲ ﺣﯿﻮاﻧﺎت اﻟﺘﺠﺎرب. و ﻣﻦ ﺛﻢ، ﻓﻘﺪ ﺗﻢ إﺟﺮاء اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ ﻟﻠﺘﺤﻘﻖ ﻣﻦ ﻣﺪى اﻟﺘﺄﺛﯿﺮ اﻟﻮاﻗﻲ ﻟﻜﻞ ﻣﻦ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ و اﻟﺠﺮﻋﺔ اﻟﻤﻨﺨﻔﻀﺔ اﻟﻤﻘﺴﻤﺔ ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ اﻹﻋﺘﻼل اﻟﻌﺼﺒﻲ ﻓﻲ ﻧﻤﻮذج اﻟﺸﻠﻞ اﻟﺮﻋﺎش اﻟﻤﺤﺪث ﻋﻦ ﻃﺮﯾﻖ اﻟﺮﯾﺰرﺑﯿﻦ ﻓﻲ اﻟﺠﺮذان. و ﻟﺘﺤﻘﯿﻖ اﻟﮭﺪف ﻣﻦ ھﺬه اﻟﺪراﺳﺔ، ﺗﻤﺖ ﻣﻌﺎﻟﺠﺔ ذﻛﻮر اﻟﺠﺮذان ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ- ٧٦١ (١٠٠ﻣﺠﻢ/ﻛﺠﻢ ﻣﻦ وزن اﻟﺠﺴﻢ ﯾﻮﻣﯿًﺎ ﻟﻤﺪة ٣ أﺳﺎﺑﯿﻊ، ﻣﺬاب ﻓﻲ ﺻﻤﻎ اﻷﻛﺎﺷﯿﺎ) أو ﺗﺸﻌﯿﻌﮭﻢ ﻋﻨﺪ ﻣﺴﺘﻮى ﺟﺮﻋﺔ ٠٫٢٥ ﺟﺮاي ﻛﻞ أﺳﺒﻮع ﻟﻤﺪة ٦ أﺳﺎﺑﯿﻊ، أو اﻟﺠﻤﻊ ﺑﯿﻦ اﻟﺘﺸﻌﯿﻊ و اﻟﻌﻼج ﺑﺎﻟﻤﺴﺘﺨﻠﺺ (ﻓﻲ اﻷﺳﺎﺑﯿﻊ اﻟﺜﻼﺛﺔ اﻷﺧﯿﺮة ﻣﻦ اﻟﺘﺸﻌﯿﻊ). و ﺑﻌﺪ ﻣﺮور٢٤ ﺳﺎﻋﺔ ﻋﻠﻰ آﺧﺮ ﺟﺮﻋﺔ ﺗﺸﻌﯿﻊ أو ﻋﻼج ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﺗﻢ ﺣﻘﻦ اﻟﺠﺮذان ﺑﺠﺮﻋﺔ واﺣﺪة ﻣﻦ

Page 149 of 152 اﻟﺮﯾﺰرﺑﯿﻦ ﻓﻲ اﻟﻐﺸﺎء اﻟﺒﺮﯾﺘﻮﻧﻲ (٥ ﻣﺠﻢ/ﻛﺠﻢ ﻣﻦ وزن اﻟﺠﺴﻢ ﻣﺬاب ﻓﻲ ١٪ ﺣﻤﺾ اﻟﺨﻠﯿﻚ) و ﺗﻢ ذﺑﺢ اﻟﺠﺮذان ﺑﻌﺪ ﻣﺮور٢٤ ﺳﺎﻋﺔ ﻋﻠﻰ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ. أدى اﻟﺮﯾﺰرﺑﯿﻦ إﻟﻰ ﻧﻀﻮب اﻟﺪوﺑﺎﻣﯿﻦ ﻓﻲ ﻣﻨﻄﻘﺔ اﻟﺠﺴﻢ اﻟﻤﺨﻄﻂ (اﻟﺴﺘﺮاﯾﺎﺗﻢ) ﺑﺎﻟﻤﺦ، ﻛﻤﺎ أدى إﻟﻰ ﺗﻮﺗﺮ ﺗﺄﻛﺴﺪي ﺑﺎﻟﻤﺦ ﻣﺘ ًﻤﺜﻼ ﻓﻲ إﻧﺨﻔﺎض ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن و إرﺗﻔﺎع ﻣﺴﺘﻮى ﻛﻞ ﻣﻦ اﻟﻤﺎﻟﻮﻧﺪاﯾﺎﻟﺪھﺎﯾﺪ و اﻟﺤﺪﯾﺪ ﻓﻲ اﻟﻤﺦ. و ﻗﺪ أدى إﻟﻰ ﺗﺜﺒﯿﻂ ﻧﺸﺎط اﻹﻧﺰﯾﻤﺎت اﻟﺘﻲ ﺗﻌﻤﻞ ﻋﻠﻰ اﻟﺘﺨﻠﺺ ﻣﻦ ﻧﻮاﺗﺞ أﯾﺾ اﻟﺪوﺑﺎﻣﯿﻦ وھﻲ اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ﺗﺮاﻧﺴﻔﯿﺮاز و اﻟﻜﯿﻨﻮن أوﻛﺴﯿﺪورﯾﺪاﻛﺘﯿﺰ، ﻛﺬﻟﻚ أدى اﻟﺮﯾﺰرﺑﯿﻦ إﻟﻰ إﺧﺘﻼل ﻋﻤﻞ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ و اﻟﺬي ﻇﮭﺮ واﺿﺤﺎً ﻣﻦ ﺧﻼل ﺗﺜﺒﯿﻂ ﻛﻞ ﻣﻦ ﻧﺸﺎط اﻟﻤﺮﻛﺐ ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت و ﻣﺴﺘﻮى ﻛﻞ ﻣﻦ اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت و ﺑﺮوﺗﯿﻦ ﺑﻲ- ﺳﻲ إل - ٢ اﻟﻤﻀﺎد ﻟﻤﻮت اﻟﺨﻼﯾﺎ و ﻛﺬﻟﻚ ﻧﺘﺎﺋﺞ اﻟﻔﺤﺺ ﺑﺎﻟﻤﯿﻜﺮوﺳﻜﻮب اﻹﻟﯿﻜﺘﺮوﻧﻲ. ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ذﻟﻚ، أﻇﮭﺮت اﻟﺠﺮذان زﯾﺎدة ﻓﻲ ﻋﺪد ﻧﻘﺎط اﻟﺘﺨﺸﺐ اﻟﻌﺼﺒﻲ ﺑﻌﺪ ﺣﻘﻨﮭﺎ ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ. و ﻗﺪ إرﺗﺒﻂ إ ًﺣﺼﺎﺋﯿﺎ ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ﺑﺎﻟﻤﺦ ًإﯾﺠﺎﺑﯿﺎ ﺑﻤﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ r) (0.78 = و ًﺳﻠﺒﯿﺎ اﻟﻤﺎﻟﻮﻧﺪاﯾﺎﻟﺪھﺎﯾﺪ (r = -0.93). ﻛﻤﺎ إرﺗﺒﻂ ًإﯾﺠﺎﺑﯿﺎ ﻣﺴﺘﻮى ﺑﻲ- ﺳﻲ إل - ٢ ﺑﻜﻞ ﻣﻦ ﻧﺸﺎط اﻟﻤﺮﻛﺐ ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت (r = 0.94) و ﻣﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت (r = 0.98) إ ًﺣﺼﺎﺋﯿﺎ. و ﻗﺪ أوﺿﺤﺖ ﻧﺘﺎﺋﺞ اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ أن ًﻛﻼ ﻣﻦ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ أو اﻟﺘﺸﻌﯿﻊ اﻟﻜﻠﻲ ﻟﻠﺠﺴﻢ ﺑﺄﺷﻌﺔ ﺟﺎﻣﺎ (١٫٥ ﺟﺮاي) ﻗﺪ أدى إﻟﻰ إﺳﺘﻌﺎدة ﻣﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ و ﻋﺪد ﻧﻘﺎط اﻟﺘﺨﺸﺐ اﻟﻌﺼﺒﻲ إﻟﻰ اﻟﻤﺴﺘﻮﯾﺎت اﻟﻄﺒﯿﻌﯿﺔ. ﺑﺎﻹﺿﺎﻓﺔ ﻟﺬﻟﻚ ﻓﻘﺪ وﺟﺪ أن ﻣﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ ﻣﺮﺗﺒﻂ إ ًﺣﺼﺎﺋﯿﺎ ﺑﺸﻜﻞ ﺳﻠﺒﻲ ﺑﻌﺪد ﻧﻘﺎط اﻟﺘﺨﺸﺐ اﻟﻌﺼﺒﻲ ﻓﻲ ﻛﻞ ﻣﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ (r = -0.93) و ﺗﻠﻚ اﻟﻤﻌﺮﺿﺔ ﻟﻺﺷﻌﺎع (r = -0.97) ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ. ﻛﻤﺎ أﻇﮭﺮت اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ إﺳﺘﻌﺎدة اﻟﻌﺪﯾﺪ ﻣﻦ اﻟﺪﻻﻻت اﻟﻜﯿﻤﯿﺎﺋﯿﺔ اﻟﺤﯿﻮﯾﺔ (ﻣﺴﺘﻮى ﻛﻞ ﻣﻦ اﻟﺠﻠﻮﺗﺎﺛﯿﻮن و اﻟﻤﺎﻟﻮن داي اﻟﺪھﺎﯾﺪ ﻓﻲ اﻟﻤﺦ و ﻧﺸﺎط اﻟﻤﺮﻛﺐ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت و ﻣﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت ﻓﻲ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ) إﻟﻰ ﻣﺴﺘﻮﯾﺎﺗﮭﺎ اﻟﻄﺒﯿﻌﯿﺔ ﻋﻨﺪ ﻣﻘﺎرﻧﺘﮭﺎ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻄﺒﯿﻌﯿﺔ. وﻗﺪ ﺻﺎﺣﺐ ذﻟﻚ زﯾﺎدة ﻣﻠﺤﻮﻇﺔ ﻓﻲ ﻧﺸﺎط اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ﺗﺮاﻧﺴﻔﯿﺮاز و ﻣﺴﺘﻮى ﻛﻞ ﻣﻦ ﺑﻲ- ﺳﻲ إل-٢ ﻓﻲ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ و اﻟﺤﺪﯾﺪ ﻓﻲ اﻟﻤﺦ، ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﺤﻘﻮﻧﺔ ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ. و ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ إرﺗﺒﻂ ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ًإﺣﺼﺎﺋﯿﺎ ﺑﺸﻜﻞ إﯾﺠﺎﺑﻲ ﺑﻤﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ (r = 0.94) و ﺑﺸﻜﻞ ﺳﻠﺒﻲ ﺑﻤﺴﺘﻮى اﻟﻤﺎﻟﻮن داي اﻟﺪھﺎﯾﺪ (r = -0.88)، ﻛﻤﺎ إرﺗﺒﻂ ﻣﺴﺘﻮى اﻟﺒﻲ - ﺳﻲ إل-٢ ًإﯾﺠﺎﺑﯿﺎ ﺑﻜﻞ ﻣﻦ ﻧﺸﺎط اﻟﻤﺮﻛﺐ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت (r = 0.72) و ﻣﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت (r = 0.98). ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ذﻟﻚ، ﻓﻘﺪ إرﺗﺒﻂ ﻧﺸﺎط اﻟﻤﺮﻛﺐ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت ًإﯾﺠﺎﺑﯿﺎ ﺑﻤﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت (r = 0.84). و ﻣﻦ ﻧﺎﺣﯿﺔ أﺧﺮى، ﻟﻢ ﯾﺤﺪث ﺗﻐﯿﺮ ﻓﻲ ﻧﺸﺎط اﻟﻜﯿﻨﻮن أوﻛﺴﯿﺪورﯾﺪاﻛﺘﯿﺰ ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ ﻋﻨﺪ اﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﺤﻘﻮﻧﺔ ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ. ﻛﻤﺎ ﻟﻢ ﺗﻈﮭﺮ أي ﺗﻐﯿﺮات ﻓﻲ أي ﻣﻦ اﻟﺪﻻﻻت اﻟﻤﻘﺎﺳﺔ ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﻤﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ - ٧٦١ ﺑﺪون ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ و ﻛﺬﻟﻚ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﺼﻤﻎ اﻷﻛﺎﺷﯿﺎ.

Page 150 of 152 أﻣﺎ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺮﺿﺔ ﻟﻺﺷﻌﺎع ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ ﻓﻘﺪ أﻇﮭﺮت ﻋﻮدة ﻛﻞ ﻣﻦ ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن، ﻧﺸﺎط إﻧﺰﯾﻢ اﻟﻜﯿﻨﻮن أوﻛﺴﯿﺪورﯾﺪاﻛﺘﯿﺰ و ﻣﺴﺘﻮى اﻟﺒﻲ- ﺳﻲ إل-٢ ﻓﻲ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ إﻟﻰ اﻟﻤﺴﺘﻮﯾﺎت اﻟﻄﺒﯿﻌﯿﺔ. ﻛﻤﺎ أﻇﮭﺮت ﺗﻠﻚ اﻟﻤﺠﻤﻮﻋﺔ إﻧﺨ ًﻔﺎﺿﺎ ﻣﻠ ًﺤﻮﻇﺎ ﻓﻲ ﻣﺴﺘﻮى اﻟﺤﺪﯾﺪ ﻓﻲ اﻟﻤﺦ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﺤﻘﻮﻧﺔ ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ. و ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺎﻟﺠﺔ ﺑﺎﻟﺘﺸﻌﯿﻊ ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ إرﺗﺒﻂ ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ًإﺣﺼﺎﺋﯿﺎ ﺑﺸﻜﻞ إﯾﺠﺎﺑﻲ ﺑﻤﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ (r = 0.76)، ﻛﻤﺎ إرﺗﺒﻂ ﻣﺴﺘﻮى اﻟﺒﻲ- ﺳﻲ إل-٢ ﺑﻤﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت ًإﯾﺠﺎﺑﯿﺎ (r = 0.79). و ﻗﺪ أﻇﮭﺮت ھﺬه اﻟﻤﺠﻤﻮﻋﺔ، أﯾﻀﺎً، إرﺗﻔﺎﻋﺎً ﻣﻠﺤﻮﻇﺎً ﻓﻲ ﻧﺸﺎط اﻟﻤﺮﻛﺐ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت و ﻣﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت، ﺑﯿﻨﻤﺎ ﻟﻢ ﺗﻈﮭﺮ أي ﺗﻐﯿﺮ ﻓﻲ ﻣﺴﺘﻮى اﻟﻤﺎﻟﻮﻧﺪاﯾﺎﻟﺪھﺎﯾﺪ و ﻧﺸﺎط اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ﺗﺮاﻧﺴﻔﯿﺮاز ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﺤﻘﻮﻧﺔ ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ. ﻓﻲ ﺣﯿﻦ أن اﻟﺠﺮذان اﻟﻤﻌﺎﻟﺠﺔ ﺑﺎﻹﺷﻌﺎع ﺑﺪون ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ ﻗﺪ أﻇﮭﺮت زﯾﺎدة ﻓﻲ ﻣﺴﺘﻮى ﻛﻞ ﻣﻦ اﻟﺪوﺑﺎﻣﯿﻦ و اﻟﺠﻠﻮﺗﺎﺛﯿﻮن و ﻓﻲ ﻧﺸﺎط اﻟﻜﯿﻨﻮن أوﻛﺴﯿﺪورﯾﺪاﻛﺘﯿﺰ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺔ اﻟﻄﺒﯿﻌﯿﺔ. أﻣﺎ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻤﻌﺮﺿﺔ ﻟﻺﺷﻌﺎع و ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ - ٧٦١ ﻣﻌﺎً ﻗﺒﻞ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ ﻓﻘﺪ أﻇﮭﺮت زﯾﺎدة ﻣﺴﺘﻮى اﻟﺪوﺑﺎﻣﯿﻦ إﻟﻰ ﻣﺴﺘﻮى أﻋﻠﻰ ﻣﻦ ذﻟﻚ اﻟﻤﺴﺠﻞ ﻓﻲ ﺟﺮذان اﻟﻤﺠﻤﻮﻋﺔ اﻟﻄﺒﯿﻌﯿﺔ. وﻗﺪ ﻛﺎﻧﺖ ﺑﺎﻗﻲ اﻟﻨﺘﺎﺋﺞ اﻟﻤﺴﺠﻠﺔ ﻓﻲ ﺗﻠﻚ اﻟﻤﺠﻤﻮﻋﺔ ﻛﺎﻟﺘﺎﻟﻲ: زﯾﺎدة ﻧﺸﺎط اﻟﺠﻠﻮﺗﺎﺛﯿﻮن ﺗﺮاﻧﺴﻔﯿﺮاز و ﻣﺴﺘﻮى اﻟﺒﻲ- ﺳﻲ إل-٢، إﻧﺨﻔﺎض ﻣﺴﺘﻮى اﻟﺤﺪﯾﺪ ﻣﻊ ﻋﻮدة ﻛﻞ ﻣﻦ ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن و اﻟﻤﺎﻟﻮﻧﺪاﯾﺎﻟﺪھﺎﯾﺪ و اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت و ﻧﺸﺎط ﻛﻞ ﻣﻦ اﻟﻜﯿﻨﻮن أوﻛﺴﯿﺪورﯾﺪاﻛﺘﯿﺰ و اﻟﻤﺮﻛﺐ١ ﻓﻲ ﺳﻠﺴﻠﺔ ﻧﻘﻞ اﻹﻟﯿﻜﺘﺮوﻧﺎت و ﻣﺴﺘﻮى اﻷدﯾﻨﻮزﯾﻦ ﺛﻼﺛﻲ اﻟﻔﻮﺳﻔﺎت ﯾﺎﻹﺿﺎﻓﺔ إﻟﻰ ﻋﺪد ﻧﻘﺎط اﻟﺘﺨﺸﺐ اﻟﻌﺼﺒﻲ ﻟﻤﺴﺘﻮﯾﺎﺗﮭﻢ اﻟﻄﺒﯿﻌﯿﺔ. وﻗﺪ أوﺿﺤﺖ ﻧﺘﺎﺋﺞ ھﺬه اﻟﺪراﺳﺔ ﻗﺪرة ﻛﻼً ﻣﻦ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ و اﻟﺠﺮﻋﺔ اﻟﻤﻨﺨﻔﻀﺔ ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ (١٫٥ ﺟﺮاي) ﻋﻠﻰ اﻟﺤﻤﺎﯾﺔ ﻣﻦ اﻷﻋﺮاض اﻟﻤﺘﺮﺗﺒﺔ ﻋﻠﻰ ﺣﻘﻦ اﻟﺮﯾﺰرﺑﯿﻦ ﻓﻲ اﻟﺠﺮذان و اﻟﻤﺘﻤﺜﻠﺔ ﻓﻲ اﻟﺘﻮﺗﺮ اﻟﺘﺄﻛﺴﺪي، اﻋﺘﻼل ﻋﻤﻞ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ و اﻟﻤﻮت اﻟﻤﺒﺮﻣﺞ ﻟﻠﺨﻼﯾﺎ. ﯾﻤﻜﻦ اﻹﺳﺘﻨﺘﺎج أن إﻋﺎدة ﻣﺴﺘﻮى اﻟﺠﻠﻮﺗﺎﺛﯿﻮن إﻟﻰ ﻣﺴﺘﻮﯾﺎﺗﮫ اﻟﻄﺒﯿﻌﯿﺔ ﻗﺪ ﯾﻌﺘﺒﺮ ًﻋﺎﻣﻼ ھﺎﻣًﺎ ﻓﻲ ﺗﺤﺴﯿﻦ اﻟﺸﻠﻞ اﻟﺮﻋﺎش. إن ﻛﻞ ﻣﻦ ﻣﺴﺘﺨﻠﺺ اﻟﺠﻨﻜﻮﺑﯿﻠﻮﺑﺎ-٧٦١ و اﻟﺠﺮﻋﺔ اﻟﻤﻨﺨﻔﻀﺔ ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ (١٫٥ ﺟﺮاي) ﻗﺪ أﻇﮭﺮا ﺗﺄﺛﯿ ًﺮا ًواﻗﯿﺎ ﻟﻸﻋﺼﺎب ﺿﺪ اﻟﺸﻠﻞ اﻟﺮﻋﺎش اﻟﻤﺤﺪث ﺑﺎﻟﺮﯾﺰرﺑﯿﻦ ﻋﻠﻰ اﻟﻌﺪﯾﺪ ﻣﻦ اﻟﻤﺴﺘﻮﯾﺎت ﺑﻌﺪة آﻟﯿﺎت ﻟﻠﻌﻤﻞ؛ و اﻟﺘﻲ ﺗﺘﻀﻤﻦ ﺗﺤﺴﻦ ﻛﻞ ﻣﻦ اﻟﺤﺎﻟﺔ اﻟﺘﺄﻛﺴﺪﯾﺔ و ﻋﻤﻞ اﻟﻤﯿﺘﻮﻛﻮﻧﺪرﯾﺎ و ﺗﺜﺒﯿﻂ اﻟﻤﻮت اﻟﻤﺒﺮﻣﺞ ﻟﻠﺨﻼﯾﺎ. و ﯾﻤﻜﻦ ﻟﻸﺑﺤﺎث اﻟﻤﺴﺘﻘﺒﻠﯿﺔ ﻋﻠﻰ ﻣﻀﺎدات اﻟﻤﻮت اﻟﻤﺒﺮﻣﺞ ﻟﻠﺨﻼﯾﺎ أن ﺗﻔﺘﺢ ًأﻓﺎﻗﺎ ﺟﺪﯾﺪة ﻓﻲ ﻋﻼج اﻟﺸﻠﻞ اﻟﺮﻋﺎش.

Page 151 of 152 ﺘﺄﺜﻴﺭ ﻤﺴﺘﺨﻠﺹ ﺍﻟﺠﻨﻜﻭ ﺒﻴﻠﻭﺒﺎ ﻋﻠﻰ ﺍﻟﻤﺘﻐﻴﺭﺍﺕ ﺍﻟﺒﻴﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﺍﻟﻤﺤﺩﺜﺔ ﻤﻥ ﺍﻟﺸﻠل ﺍﻟﺭﻋﺎﺵ ﻓﻲ ﻤﺦ ﺍﻟﺠﺭﺫﺍﻥ ﺍﻟﻤﺸﻌﻌﺔ ﻣﻘﺪﻣﺔ ﻣﻦ اﻟﺼﯿﺪﻻﻧﯿﺔ / إﻧﺠﻰ رﻓﻌﺖ ﻋﺒﺪ اﻟﻌﺰﯾﺰ راﺷﺪ (ﻣﺎﺟﺴﺘﯿﺮ اﻟﻌﻠﻮم اﻟﺼﯿﺪﻟﯿﺔ – ﺟﺎﻣﻌﺔ اﻟﻘﺎھﺮة) ﺻﯿﺪﻻﻧﯿﺔ ﺑﻘﺴﻢ اﻟﺒﺤﻮث اﻟﺪواﺋﯿﺔ اﻹﺷﻌﺎﻋﯿﺔ - اﻟﻤﺮﻛﺰ اﻟﻘﻮﻣﻲ ﻟﺒﺤﻮث و ﺗﻜﻨﻮﻟﻮﺟﯿﺎ اﻹﺷﻌﺎع ھﯿﺌﺔ اﻟﻄﺎﻗﺔ اﻟﺬرﯾﺔ

ﻟﻠﺤﺼﻮل ﻋﻠﻰ درﺟﺔ دﻛﺘﻮر اﻟﻔﻠﺴﻔﺔ ﻓﻲ اﻟﻌﻠﻮم اﻟﺼﯿﺪﻟﯿﺔ (ﻛﯿﻤﯿﺎء ﺣﯿﻮﯾﺔ) ﺗﺤﺖ إﺷﺮاف

ﺃ.ﺩ/ ﺁﻤﺎل ﺃﺤﻤﺩ ﻋﺒﺩ ﺍﻟﻔﺘﺎﺡ ﺃ.ﺩ./ ﻤﻨﻰ ﻋﺒﺩ ﺍﻟﻠﻁﻴﻑ ﺍﻟﻐﺯﺍﻟﻲ ﺃﺴﺘﺎﺫ ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﺤﻴﻭﻴﺔ ﺃﺴﺘﺎﺫ ﻋﻠﻡ ﺍﻷﺩﻭﻴﺔ ﻭﺍﻟﺴﻤﻭﻡ ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ - ﺠﺎﻤﻌﺔ ﺍﻟﻘﺎﻫﺭﺓ ﺭﺌﻴﺱ ﺍﻟﻤﺭﻜﺯ ﺍﻟﻘﻭﻤﻲ ﻟﺒﺤﻭﺙ ﻭ ﺘﻜﻨﻭﻟﻭﺠﻴﺎ ﺍﻹﺸﻌﺎﻉ ﻫﻴﺌﺔ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺫﺭﻴﺔ

ﺃ.ﻡ.ﺩ./ ﻨﺭﻤﻴﻥ ﻋﺒﺩ ﺍﻟﺤﻤﻴﺩ ﺼﺎﺩﻕ ﺃﺴﺘﺎﺫ ﻤﺴﺎﻋﺩ ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﺤﻴﻭﻴﺔ ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ- ﺠﺎﻤﻌﺔ ﺍﻟﻘﺎﻫﺭﺓ

ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩﻟﺔ ﺠﺎﻤﻌﺔ ﺍﻟﻘﺎﻫﺭﺓ

۲۰١٢

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