The Study On Degeneration Of Axon In Dopaminergic Neuron During Establishing Mice Models Of Parkinson's Disease

Parkinson's disease(PD) is a common progressive and neurodegenerative disease in central nervous system. The primary pathology of PD is selective loss of dopaminergic neurons in the substantia nigra pars compacta. Previous studies mainly focused on the mechanisms of dopaminergic neurona apoptosis. Axonal degeneration of dopaminergic neurons is thought to follow of neurons'loss for a long time.Recently, some findings have demonstrated that simply inhibiting process of dopaminergic neurons apotosis do not postpone the onset and development of PD efficiently. Dopaminergic axonal degeneration might be a key feature and target for PD. Although we have gradually convinced of the important role of axonal degeneration in PD, the mechanisms and the role of axonal degeneration remain incompletely understand. And, we have little special methods to treatment of axonal degeneration in PD. So the study of mechanisms and the role of axonal degeneration in PD will not only bring us new theories, but also bring us new strategies and target to treat PD.Part 1. Establishment of chronic old C57BL mice model for Parkinson's disease Objective To investigate dopaminergic axonal degeneration induced by MPTP and establish chronic old C57BL mice model for PD research in vivo. Methods The group of mice are randomly divided 30 mice into experimental group and 10 into control group by the method of using simple random. Experimental group are injected intraperitoneally MPTP30mg/kg, 3.5d once, for 10 times. Control group are injected the same equivalent normal saline with experimental group by intraperitoneal injection (ip) and at the same time. We extract 3 mice from the experimental group after intraperitoneal injection 12h and extract 1 mouse from control group, so the mice are divide into 10 groups according to the number of administration. Behavioral experiments include Pole test and Traction test. Results Mice showed the performance of irritability such as severe trembling, reduction of activities, the pace of inconsistent positions, straight tail and pelage, etc. and individual characteristics of epilepsy may occur within 0.5 ~ 1h after administration of 1 to 3 times (4 ~ 10d).The acute performance gradually disappeared after 2h and basically back to normal after 24h. But with the increase in the times of administration (11 ~ 21d), although mice show acute manifestations but disappeared slowly and gradual reduction of spontaneous movements, body tremors, arched back and stiff tail, the slow movement of limbs clumsy, crawling slowly, PD symptoms gradually stabilized. From the 8th (28.5d) administration 12h later, the score of Pole test of mice model is 1 point, but from the 9th(31.5d) administration 12h later, the score of Traction test is 1 ,and until the 10th (35d) administration 12h later, both of score of two behavioral tests are 0, the features of PD symptoms are typical, stable, and not restored. The performance of control group mice are normal. Conclusion The age of aged mice are similar to old age people , prone to stability of damage and reduction of DA content in the brain SNc neurons and axon terminals without the risk of a significant trend of natural recovery, and chronic models of reduction of DAG neurons show apoptosis in morphology, is a slow, gradual process of development which is similar to the pathogenesis of PD pathology and human physiological and biochemical processes, symptoms are stability, the phenomenon of spontaneous recovery is not significant, which is more suitable for research of mechanism DAG neuronal degeneration in Parkinson's disease. Therefore, this study investigates the control group and group of PD mice model of different number of injections (1 to 10) by behavioral testing, to observe the process of behavior change in chronic PD aged mice model. The next step is based on the behavioral testing of different injections of PD model., and can compared with staining of tissue sections and quantitative analysis of protein in the next step.Part 2. Identified dopaminergic axonal degeneration in Parkinson's disease Objective To observe the change of number and damage of dopaminergic neurons and their axons in chronic PD aged mice model, certify dopaminergic axonal degeneration from the morphological description. To verify dopaminergic axonal degeneration with quantitative analysis of protein by detecting the change of cytoskeleton proteinβ-TubulinⅢand microtubule associated protein CRMP2. Methods In accordance with the first part of the experimental group, and then the animals are perfused and the brains are frozen section, using TH immunohistochemistry staining, FJC staining, western blot analysis show that there are time-effect relationship between dopaminergic axonal degeneration and dopaminergic neurons degeneration in PD model. Results TH staining shows that the model group after the 2nd administration has the number of TH-ir cells reduced but not obviously, the cell's structure is clear and in order. However, axon and dendrite become thin and length is significantly shorter, cancellated and interlaced axons are rarefaction, the surface is not smooth, and some are swollen and beaded or axonal interruption and lost. OD of TH-ir fibers show: after 1st administration in PD mice model, OD of bilateral striatum TH-ir nerve fibers compared with the control group, ,there are no significant difference (P> 0.05), suggest that there are reduction of axonal and dendritic fibers in dopaminergic neurons, although not obviously.Therefore,its density has no statistically significant. However,after the 2nd administration, OD of TH-ir nerve fibers in striatum compared with the control group, there are significant difference (P <0.05). They suggest that axon,dendrite, axon terminal and synapse are decreased in striatum from SNc dopaminergic neurons or the function of transport and re-uptake of dopamine is decline and even disappearance, the level of DA in striatum by axonal anterograde transport is decreased. That also is a sign of axonal degeneration.FJC staining: after 1st and 2nd times administration, we have detected any FJC-ir cells. However, after the 3rd dose ,we detect FJC-ir cells, and some positive cells have only cell bodies but axonal roots and axon completely disappear. Relative content of proteinsβ-TubulinⅢ:after the 2nd dose ,there are significant difference(P <0.05) in comparison with the control group but after the 1st administration there are no significant difference (P> 0.05).Relative content of proteins CRMP2: after the 2nd dose ,there are significant difference(P <0.05) in comparison with the control group but after the 1st administration there are no significant difference (P> 0.05).Conclusion Axonal degeneration in the event of time is earlier than apoptosis in MPTP-induced PD aged mice model, suggesting that axonal degeneration may be a separate mechanism which is independent or earlier than apoptosis in neuronal cell bodies, and play an important role in the pathogenesis of Parkinson's disease.