| When the peripheral nerve injured, the precondition of recovery of function is the regeneration of axon. There are many factors can influence the growth of axon. The change of activity, the extent of necrosis, the regeneration after necrosis and the function after the regeneration of neuron connected with peripheral nerve are the prerequisite of the axon growth and recovery of function. The neuron apoptosis is connected with axon regeneration and repair. So, it has an important meaning to identify clearly the pathological change of the axon after injury. The research found that some neurotrophic factors, neurotransmitters and hormones have an important effect on the nerve growth, development, cell proliferation, apoptosis and regeneration. The species of extracellular ATP can participate the effect of nerve system not only as the neurotransmitter and modulator but also has a trophic action to glial cell and neuron. It participates in many courses, e.g. cell differentiation and apoptosis, the morphologic change of chondriosome, to stimulate the synthesis and release of cytokine and nerve growth factor. It has a great influence to neurological growth and plasticity. The found of receptor P2Y on the neuron and schwann cell and the change of it mediated on electrophysiology variation and cell proliferation, these improved that the extracellular ATP can protect the injured neuron, and this effect has been mediated with receptor P2Y. When the reparation start after injury, we found the MAPK system been activated, the ERK1/2 been activated, and the course concerned with receptor P2Y. In recent years, people found that Na-K-ATPase can not only as a ion pump, but also have the feature of as a signal transduction molecule. The ion Ca in cell is an important signal molecule in many physiological and biochemical event of cell living. The abundant Ca- ATPase in arcoplasmic reticulum can balance the ion Ca in the cell. The ATPase through the interaction with other proteins in the cell can transfer the extracellular signal into the cell and regulate the cell functions. The Glycogen synthase kinase3(GSK3) is a conservative kinase in the signal transmission system, it can produce a marked effect in many input signal path, and its subtype GSK—3β is an important factor of apoptosis. In order to understand the change of neuron following peripheral injury, what the effect of extracellular ATP and what's the effect of ATPase and receptor P2Y in the course, we design the experiment. The experiment is divided into three parts as following.Part 1Effects of extracellular ATP on the ATPase activity of denervated skeletal muscle and motor neurons of anterior spinal cordObjective To investigate the effect of extracellular ATP on ATPase activity of denervated gastrocnemius and motor neurons of anterior spinal cord following sciatic nerve injury in SD rat. Method Sprague-Dawley rats were used, randomly divided into three groups: The right side sciatic nerves were transected but not sutured group (injury group), the right side sciatic nerves were transected and sutured immediately, and the ATP (experimental group) and normal sodium (control group) were injected into the right side gastrocnemius with same volume. After 12hour, 1day, 3day, 7day, 14day, 28day and 56day of the operation, measure the muscles wet weight and Na+-K+-ATPase and Ca2+ -ATPase activities with both of the two tissues. Result Compared with the three groups, The ATPase bioactivity in experimental and control groups were superior to that in the injury group at some times, and this change was the same with the change of the muscle wet weight. Statistically significant significance was found. Conclusions The effect of extracellular ATP can improve the ATPase activity of denervated muscles and motor neurons of anterior spinal cord at SD rat, which suggested it has certain protective and promote regeneration effects on denervated muscle and motor neurons of anterior spinal cord, and these effects may produce through ATPase.Part 2Experimental Study of Change of motor neurons of anterior spinal cord following sciatic nerve injury and the effect of extracellular ATPObjective To explor the Change of motor neurons of anterior spinal cord following sciatic nerve injury and the effect of extracellular ATP on the motor neurons SD rat. Method Sprague-Dawley rats were used, randomly divided into three groups: The right side sciatic nerves were transected but not sutured group (injury group), the right side sciatic nerves were transected and sutured immediately, and the ATP (experimental group) and normal sodium (control group) were injected into the right side gastrocnemius with same volume. After 12hour, 1day, 3day, 7day, 14day, 28day and 56day of the operation, observe the motor neuron's change on immunohistochemistry and measure the change of protein of p—GSK—3β(ser9). Result After the injury of sciatic nerve, the main change of motor neurons of anterior spinal cord is apoptosis. Use extracellular ATP and ripair sciatic nerve can decrease neuron apoptosis and influence the activity of protein GSK—3β. Conclusion The main change of motor neuron of anterior spinal cord following sciatic nerve injury is apoptosis, after inject with ATP, it can protect the motor neuron and this effection may concern with protein GSK—3β.Part 3Experimental Study of the Mechanism of Action of Extracellular ATP on Motor Neurons of Anterior Spinal Cord in Vitro Following Machinery Injury Objective To explore the mechanism of action of extracellular ATP on motor neurons in vitro following machinery injury. Method Get the neonate Sprague-Dawley rat's medulla spinalis, through the conventional primary cell culture procedure, we use the method of difference adherence time and get the motor neurons of anterior spinal cord. When the neuron is in maturity, we make the mechanical injury model in vitro. All the models were divided into four groups: group A is control group; group B is 100μM ATP group; group C is 100μM ATP+20μg/ml suramin group and group D is 100μM ATP+10μM ouabain+10μg/ml Thapsigargin group. Culture the four groups neurons, after one day, we count the motor neuron, observe the survival and activity of neurons through MTT shade selection experiment, use flow cytometry to analyze the percentage of apoptosis of motor neurons of anterior spinal cord and detect the expression of protein p-GSK-3β (ser9) through Western-Blot technology. Result After we cultured the neuron one day, we found the counting of motor neuron and the survival and activity of neurons of group A was the lowest. They had no significant deviation controlling with group D, and had significant deviation controlling with group A and C. The percentage of apoptosis of motor neurons in group A was the highest, it had significant deviation controlling with others. The counting of motor neuron and the survival and activity of neurons of group B was the highest. They had no significant deviation controlling with group C, and had significant deviation controlling with group A and D. The percentage of apoptosis of motor neurons in group B was the lowest, it had significant deviation controlling with others. The percentage of apoptosis of neurons of group C was better than group A and worth than the others, the counting of living cells was no significant deviation controlling with group B, but better than group A and D. The percentage of apoptosis of neurons of group D was lower than group A, but had no significant deviation with others. The counting of living cells had significant deviation controlling with group B and C, and had no significant deviation controlling with group A in it. In group A, we can see the lower expression of p-GSK-3β (ser9) , and in group B and D, we only can see a amounts of it, it was significant. In the group C had a few expression of p-GSK-3β (ser9) . Conclusion The cultured machinery injured motor neuron of anterior spinal cord will apoptosis. In the apoptosis, the GSK-3β may in the course of signal conduction. Extracellular ATP can protect the injured neurons, decrease the count of apoptosis and this effect may mediate with receptor P2Y, and ATPase can influence the signal conduction, produce a marked effect of protecting the neuron and lightening the damage. |
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