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Abstract SUMMARY AND CONCLUSION Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. It is the second one after alzheimer’s disease. It characterized by loss of dopamine producing cells of the substantia nigra pars compactica (SNpc), the loss of these neurons produces the motor features of the disease. The process is a slowly progressive and motor symptoms start to appear after degeneration of 50-60% of the SNpc, the pathological hallmarks of the disease are loss of nigrostriatal dopaminergic cell and the presence of intraneuronal eosiphilic proteinacious inclusions known as “Lewy bodies”, the cause of PD remains unknown. But may be due to genetic or familial Causes. About 5% of cases occur in young age and the remaining 95% of the cases are sporadic for which no specific etiology can be found. Pakinsonian symptoms can also occur as a side effect to dopamine blocking drugs eg., neuroleptics, especially the classic drugs eg, haloperidol, cinnarizine, may due to repeated head trauma, viral infection, or poisoning due to manganese or rotenone this is called secondary parkinsonism. The most common model of Parkinsonism which gives the same neuronal changes in animal as in human is rotenone. Rotenone is plant derived pesticide and a complex I (NADH-quinone oxidoreductase) inhibitor in oxidative phosphorylation chain of the mitochondrial respiration, the inhibition of complex I formation leads to ATP depletion, which induces oxidative stress in cells. Rotenone is also known to induce apoptosis, induces microglia activation and loss of dopaminergic neurons in model of Parkinson’s disease and also inhibits microtubule polymerization and arrest cell cycle progression at mitosis. L -dopa is a corner stone in treatment of parkinsonism but not used alone used in combination with carbi-dopa to prevent peripheral decarboxylase enzyme and increase L-dopa level, it used for prevention and treatment of parkinsonism. It is the main source of dopamine in brain as it pass blood brain barrier and turned to dopamine by decarboxylase enzyme . Colchicine is a plant alkaloid, which used in treatment of acute gouty attacks it reduces frequency of acute attacks and relieves pain. It has a suppressive and prophylactic effect. Colchicine inhibits the synthesis and release of leukotrienes, inhibits neutrophil activity and migration to the affected area leading to anti-inflammatory effect, beside its anti-mitotic effects it has anti-inflammatory, antioxidant and anti-apoptotic properties and difficulty in passing to blood brain barrier disappears in Pakinsonian patients due to depression of the p-glycoprotein efflux system. According to these clinical data we used colchicine as possibly neuroprotective drug in the rat rotenone model of Parkinson’s disease. Valproic acid (VPA) is used as an anti-epileptic drug but many studies suggest that valproic acid could be used as a neuroprotective in parkinsonism as it has many effects as anti inflammatory because VPA reduces polymerphonuclear cells migration, myeloperoxidase release, anti oxidant and anti apoptotic effects as it blocks the synthesis of pro-apoptotic factors.The aim of this work is: 1. Study the potential neuroprotective effects of L- dopa, colchicine and valproic acid on rotenone induced parkinsonism. 2. Compare effects of colchicine or valproic acid with L- dopa protective effect on rotenone induced parkinsonism Methods: This experiment was carried out using 48 male wistar rats each weighing (120-150) gm. They were kept under similar housing conditions and had free access to food and water. They were divided into 6 groups each group consist of 8 rats as follow: group (1): normal control group rats received distilled water orally. group (2): rats received carboxy methyl cellulose (CMC) 0.5% as a vehicle (1 ml /kg subcutaneous S.C. once daily for 28 days). group (3): rats received rotenone (2mg/kg S.C. daily for 28 days suspended in 0.5% CMC). group (4): rats received rotenone + L-dopa (10mg/kg intraperitoneal i.p. daily for 28 days suspended in 0.5% CMC) group (5): rats received rotenone + Colchicine (20 microgram/kg p.o. orally daily for 28 days suspended in 0.5% CMC). group (6): rats received rotenone + valproic acid (1.4 gram/kg p.o. orally daily for 28 days suspended in 0.5% CMC).All animals were evaluated throughout the experiment by the behavioral tests for monitoring PD development at 7th day,14th day 21st day and 28th day before sacrification of all rats twenty four hours after the last treatment (28th day) by: 1- Catalepsy test 2- The pole test 3- T maze spontaneous alternation test At the end of the work, all rats were sacrificed. The brain of each rat was immediately excised, washed with ice-cold saline and the basal ganglia of the two hemispheres were dissected. The left basal ganglia were immediately immersed in 10% formalin and used for light microscopic histopathological examination and Immunohistochemical evaluation of caspase-3.The right basal ganglia were homogenized in phosphate buffer saline (PBS)and centrifuged at 4000 rpm for 20 minutes. The supernatants were collected and frozen at -80oC for further assay of the following parameters in basal ganglia: Tissue dopamine level. Tissue tumor necrosis factor alpha (TNFα) Tissue reduced glutathione level (GSH) Results: Injection of rotenone (2mg/kg S.C. daily for 28 days suspended in 0.5% CMC) for 28 days induce Parkinson model. There was significant difference between that group and control (receive distilled water), vehicle group (receive CMC0.5%) in behavioral tests as catalepsy test (grid test and bar test) there was increase in time for that catalepsy test which indicated rotenone toxicity and dopaminergic neurons degeneration and appearance of rigidity, bradykinsia and slow motion which started at 7th day and increased at 14th day, 21st day and 28th day. At the end of our study rats were suffered from severe neurodegeneration. In T maze spontaneous alternation test there was deterioration in memory started from 7th day, 14th day and reached severe impairment of memory and cognition at 21stday and 28thday. In the pole test which record time for locomotor activity, there was increase in time taken for rats to reach the base of wooden pole and there was sliding and dropping of many rats in the group, that was started at 7thday, 14th day and became severe and all rats of that group were slidded and dropped at 21st day and 28thday. There was significant changes in neurochemical parameters when compared to control group (received CMC), L-dopa treated group, colchicine treated group and valproic acid treated group as significant decrease in tissue dopamine level, significant decrease in tissue reduced glutathione level (GSH). Significant increase in tissue tumor necrosis factor α (TNFα). In histopathological features there was severe neuronal cell death, structural damage to the substantia nigra, there was distribution of lewy bodies and severe inflammation with huge edema and significant increase in vacuolated cells number in substantia nigra. In immunohistochemistery there was strong positive caspase 3 reactions and severe degree of apoptosis, so that group has high histopathological and immunohistochemistery scores than control group (received CMC), L-dopa treated group, colchicine treated group and valproic acid treated group. Regarding to L-dopa treated group, the current results showed significant improvement in behavioral tests as catalepsy test (grid test and bar test) there was significant decrease in time for that test which started at 7th day and improved at 14th day, 21st day and 28th day when compared with rotenone treated group and valproic acid treated group. But results nearly similar to colchicine treated group, significant results were recorded in T maze spontaneous alternation test there was deterioration in memory started at 7th day, 14th day, 21st day and 28th day but better than results in rotenone treated group and valproic acid treated group and there was improvement in memory and cognition at the end of our study than rotenone treated group, colchicine treated group and valproic acid treated group. In the pole test which record time for locomotor activity, there was significant decrease in time taken to rats to reach the base of wooden pole and there was very few rats were slidded and dropped in the group, that was started 7thday, 14th day and became improve in all rats of that group at 21st day and 28th day that were significant results in comparison to rotenone treated group, colchicine treated group and valproic acid treated group. There was significant improvement in neurochemical parameters when compared to rotenone treated group and valproic acid treated group as significant increase in tissue dopamine level, significant increase in tissue reduced glutathione level (GSH), with significant decrease in tissue tumor necrosis factor α level (TNFα). In histopathological features there was mild neuronal cell death, structural damage to the substantia nigra, no lewy bodies, mild inflammation with edema and very low number of vacuolated cellsin substantia nigra which more better than group (treated by rotenone) and valproic acid treated group and nearly similar to colchicine treated group. In immunohistochemistery there was nearly negative caspase 3 reaction and mild degree of apoptosis,so that group has very mild histopathological and immunohistochemistery scores than rotenone treated group and valproic acid treated group. Regarding to colchicine treated group, significant results were appeared in behavioral tests as catalepsy test (grid test and bar test) there was decrease in time for that test which started at 7th day and improved at 14th day, 21st day and 28th day when compared with rotenone treated group and valproic acid treated group, significant results were recorded in T maze spontaneous alternation test, there was deterioration in memory started at 7th day, 14th day,21st day and 28th day but colchicine got improvement of memory deterioration comparing to rotenone treated group and valproic acid treated group. Improvement in memory and cognition at the end of our study and nearly similar to L-dopa treated group. |