| Spinal cord injury(SCI)causes catastrophic consequences and white matter damage is one of key factors to block the motor function recovery of paraplegic patients.Main pathological changes of white matter injury include oligodendrocytic cell death and axonal degeneration.Our pre-test showed that the expression of ryanodine receptor(RyR),the most important channel regulating intracellular Ca2+ release,was significantly increased and located in oligodendrocytes and axons after SCI,strongly indicating that RyR-mediated Ca2+ release from intracellular stores may be involved in spinal cord white matter injury.As a result,using traumatic and ischemic animal models,we will investigate the the cellualr location of RyR subunits in spinal cord white matter and the change of their expression after SCI;Using the methods of pharmacologic intervention we will explore the effects of RyR on spinal cord white matter injury;using animal behavior test,we will study the effects of RyR on rat functional recovery.The expected results will provide an experimental evidence for a novel mechanism underlying white matter injury and new strategy of prevention and treatment for SCI.Part 1 Cellualr location of RyR subunits in spinal cord white matter and the change of their expression after SCI in rats Objective: To investigate the cellualr location of RyR subunits in spinal cord white matter and the change of their expression after SCI in rats.Methods: Immunohistochemical staining was performed to detect the location of RyRs in CNPase+ oligodendrocytes,NF200+ nerve fibers,GFAP+ astrocytes and NeuN+ neurons;The SCI model was established by clamping method,and the rats were sacrificed at 0 h,4 h,12 h,24 h,48 h and 72 h after SCI.Immunohistochemistry and RT-PCR were used to examine the change of RyRs expression in the injured white matter after SCI.Results: 1)Immunohistochemical double-staining results showed that The three RyR subunitswere expressed in the spinal cord,and located in CNPase+ oligodendrocytes,NF200+ nerve fibers of the white matter.Meantime,we found RyRs were also expressed in GFAP+ astrocytes and NeuN+ neurons;2)Immunostaining and RT-PCR results showed that the expression of RyRs in the injured white matter was gradually increased over time after SCI,and the peak of it's expression was at 24 h after SCI.Conclusion: RyRs were expressed in oligodendrocytes and nerve fibers;the expression of RyRs was significantly increased,indicating that RyRs may be involved in the process of white matter injury after SCI.Part 2 Effects of RyR activity on white matter injury after SCI in rats Objective: To investigate the effects of RyR on white matter injury after SCI.Methods: After injected with RyR activator caffeine and RyR inhibitor datrolene,the SCI rats were randomly divided into 4 groups: the Sham group,the SCI group,the CA group and the DA group.CNPase and TUNEL staining were used to examine the number changes of oligodendrocytes in the injured white matter;NF200 staining and Western blot were used to detect axonal damage;Electron microscope was used to examine morphological changes of myelin sheath,meantime,we detect the the changes of myelin-related proteins(MBP and CNPase)in the injured site.Results: 1)Compared to the SCI group(7.56 ±1.19),RyR activator caffeine could evidently increase the number of oligodendrocytes apoptosis in the injured site(15.27 ± 2.40,P< 0.05).Oppositely,RyR inhibitor datrolene could decrease it's apoptosis(the SCI group 7.56 ±1.19 vs.the DA group 2.35 ± 0.48,P< 0.05);2)Compared to the Sham group,axonal fibers were broken,arranged randomly and NF200 protein was decreased in the SCI group(P< 0.05).Compared to the Sham group,the length of axnal fibers was further shortened,and the protein levels of NF200 was further decreased(P< 0.05).However,in the DA group,the appearance of NF200+ fibers was longer and more regular in arrangement than that in the SCI group,and NF200 protein was evidently upregulated(P< 0.05);3)Scanning electron microcopy images showed that the myelin was thinning and dissolved in morphology,and the typical changes of demyelination was observed as well after SCI.After treatment with caffeine,the myelin thickness was further thinner and the demyelination was much more deteriorated than that in the SCI group.Nevertheless,datrolene could ameliorate the demyelination and attenuate reduced myelin thickness.Furthermore,Western blot results showed that the expression of myelin-related proteins(MBP and CNPase)was decreased after SCI(the Sham group vs.the SCI group,P< 0.05),and further decreased by caffeine(the SCI group vs.the CA group,P< 0.05).Which,however,were upregulated by datrolene(the SCI group vs.the DA group,P< 0.05).Conclusion: increasing the activity of RyR could aggravated whiter matter injury,however,decreasing the activity of RyR could alleviate the damage degree of white matter,indicating that RyR played an important role in the white matter injury after SCI.Part 3 Effects of RyR activity on motor functional recovery after SCI in rats Objective: To investigate the effects of RyR on rats motor functional recovery.Methods: After making models,rats were randomly divided into 3 groups: the SCI group,the CA group and the DA group.Observers were required to evaluate rats hind limbs movement functional recovery and record scores according to BBB motor rating scale at 3 d,7 d and 14 d and 21 d and 28 d after SCI.Results: Rats movement function in the three groups were all gradually recovered with time after SCI.Compared to the SCI group,BBB scores were evdently decreased at different time point in the CA group(P< 0.05).However,BBB scores in the DA group were significantly higher than that in the SCI group at each time point(P< 0.05).Conclusion: RyR activity could influence rats BBB scores after SCI,proving that inhibiting RyR activity promoted rats functional recovery. |
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