Protective Of Fasudil In White Matter Of Rats Following Acute Cerebral Ischemia-reperfusion

Background and Objective:Recent years, neurological deficit which caused by the ischemic cerebrovascular disease has influenced the quality of life in elder.With the development of imageology technology, white matter injury has been found and understood more and more. The most common reason of white matter injury was ischemic cerebral injury, secondly still include inflammation,neurodegenerative disease,toxic encephalopathy,and trauma and nutrition metabolism disorders etc.The injury of white matter results in a degeneration of fasciculus, which leads to neurological disorders that caused by the interruption of neuronal activity. The white matter mainly constitutes with the axons and the myelin surrounding the axons, so the research on the protection of white matter focuses on axon and myelin regeneration. The blood-supply of white matter were mainly provided by the deeply arterial perforator, it is easily known that white matter are injuried by cerebral ischemia, but whether the axon and myelin are regenerated after cerebral ischemia?Nogo-A is a myelin-derived inhibitory protein, which exists in the central nervous system neurons and oligodendrocytes, and shows a strong inhibition in the axon growth. Nogo-A combines together with acceptor NgR (a kind of surface protein on axon), which can activate the second messengers cAMP and Ca2+,then activate Rho kinase downstream(Rho-associated kinase, ROCK).Rho kinase is a small molecular GTP kinase, sometimes which plays an important role in a molecular switcher in the signaling pathway in cells, such as cell migration, contraction, proliferation and apoptosis. Thus, it is a protective effect of brain to inhibiting activation of Rho kinase.Fasudil is a new type of Rho kinase selective inhibitor as an isoquinoline sulfonamide derivatives, it is used to relive the cerebral vascular spasm caused by SAH at first. Fasudil participates in a variety molecules function activities through inhibiting the activity of Rho kinase specifically, such as increasing regional cerebral blood flow, increasing expression of eNOS to improve endothelial function, inhibiting the migration of neutrophils to produce a protective effect on cerebral infarction, producing a facilitation on nerve regeneration and recovery of motor function.In this study the model of middle cerebral ischemia-reperfusion was used to observe the neurological function defect scale, chromotropic acid 2R-brilliant green myelin staining and the effects of Fasudil on the expressions of MBP,Nogo-A and Rho kinase, and to explore the mechanism of Fasudil in the injury of white matter after cerebral ischemia-reperfusion in SD rats. Methods:A total of 72 healthy male adult SD (Spragne-Dawley)rats were divided into sham-operated group, ischemia-reperfusion group (control group) and Fasudil-treatment group (Fasudil group) randomly, each group with 24 rats were divided into 4 points by time again(1 day,3 days,7 days,14 days). Adopting Zea-Longa thread embolism method to establish the middle cerebral artery ischemia-reperfusion (MCAO) model, then the rats were killed at corresponding time point,brains were removed, paraffin embedded, sliced, applying myelin staining and immunohistochemical staining respectively.Motor function in rats of every group was evaluated using nerve function defect scale;the morphological changes of myelin were observed by chromo tropic acid 2R-light green myelin staining method;and the expressions of MBP,RhoA and Nogo-A in hippocampal CA1 area of rats were observed throught the method of Immunohistochemical technique.Results: 1 nerve function defect scale:compared with sham-operated group, the neural function defect symptoms in control group and Fasudil group were obviously at the same time points, scores of neural function defect scale were increased, there were statistically significant differences (p<0.05); the score of neural function defect scales in Fasudil group compared with control group were lower at the same time points, neural function defect symptoms were recovered, scoring compared with each other made a statistical significance differences at each time points corresponding.2 the myelin staining comparison:arrangement of myelin in sham-operated group were arranged in neat shape, the myelin fibers arranged closely; The control group myelin fibers were disconnected, arranged disorderly, loose, free bubble formed. The myelin fibers in Fasudil group compared with the control group arranged considerably neatly at corresponding time point, tissue of empty bubble were disappeared, there are still a few fracture fibers.3 immunohistologic result3.1 The changes of corpus callosum tissue of MBP-positive fibers:the sham-operated group on the left corpus callosum shows a higher density of MBP-positive fibers, arranged compactly; MBP-positive fibers in control and Fasudil Group displayed loose and disordered. As the time of reperfusion prolonged, MBP-positive fiber expression compared with the previous expression increased gradually and became arranged neatly; MBP-positive fibers and staining in Fasudil group after reperfusion enhanced more than the control group at different time points.3.2 Changes of expressions of Nogo-A protein:expressions of Nogo-A at the left hippocampal area in sham-operated group distributed a few positive cells; expressions of Nogo-A positive cells after reperfusion at the control and Fasudil group increased at 1 day, increased at 3 days and 7 days continuously, and reduced at 14 days, but still higher than the expressions of sham-operated group; the expression of Nogo-A in Fasudil group were lower compare with control group after reperfusion at the corresponding time points, there were statistical significance about all of the data above(P<0.05).3.3 changes of expressions of Rho:there were a few expressions of Rho positive cells distributed at the left hippocampal tissue in sham-operated group; after reperfusion one day, the expression of Rho positive cells had increased in the control group and the Fasudil group, the expression raised in a submit at 3 day, the expression of Nogo-A in Fasudil group were lower compare with control group after reperfusion at the corresponding time points, there were statistical significance about all of the data above(P<0.05).Conclusions:1 Fasudil could reduce the scales of neurological deficit and improve the symptoms of neurological deficit.2 Fasudil could reduce the lost of meylin after acute ischemic in white matter, inhibit the axon regeneration inhibitors, and play a protective effect on white matter after damages.