| Background and ObjectiveThe ishcemia reperfusion injury is pathological phenomenon, after reperfusion the damage of brain will be worse than before.It is a complex pathophysiological process, including the injury of free radicle, destabilization of intracellular Ca2+, releasing of inflammatory cytokines, activation of apoptosis gene and increased releasing of excita-tory amino acid. In recent years, the role of immune inflammation factors in ischemia reperfusion injury, was concerned by people.As the core of nervous-immune-endocrine system, microglia is related to many pathologic process of nervous system diseases.Inhibiting the activation of MG and reducing the expiration of inflammatory mediators can relieve the injury of cerebral ischemia-reperfusion. INOS,produced in MG and relevanted to the activation of MG,is a key factors in ishcemia reperfusion injury.MG can produce a lot of nitric oxide after releasing of iNOS,which can destroy the myelin. Therefore,the intervention of MG can play protective effect in myelin by reducing of iNOS. At present the research in acute cerebral ischemia-reperfusion-injury is less reported. CsA is an effective immunosuppressive agent. But more and more experiments show that CsA have the effect of brain protection,they can inhibit activity of MG and calcium adjustable neural phosphatase (CaN), and close mitochondrial permeability transition.The study on MG and mylin protection in brain ischemia reperfusion is less reported.The focal cerebral ischemia-reperfusion-injury models were made by suture-occluded method.Through the intervention of CsA, the expressions of OX-42 and iNOS in the rats hippocampal CA1 and MBP in Corpus callosum were detected by immunohistochemistry staining. Chromotrope 2R-Brilliant green myelin staining was selected to observe the changes of myelin and the effect of CsA. The score of behavior obstacle in rats were rated at the same time.To explore the application of neural protectants provide theoretical basis and new ideas.Materials and methods1. Animals and groupsA total of 72 healthy, adult,Spgue-Dawley rats were randomralyne divided into 3 groups including sham-operative group, model group and cyclospori A group.There were 24 rats in each group. Each group was redistributed into 4 subgroups. Every subgroup had 6 rats. Cyclosporine A group was accepted CsA(20mg/kg) everyday after operation,the others groups were received same volume of saline.2 The establishment of model and the standard of successThe focal cerebral ischemia-reperfusion-injury models were made by suture-occluded method.Model group and cyclosporine A group were reperfused following ischemia 2h. Sham-operative group only separated the middle cerebral artery. Rats were classified when sobered according to the method of Zea Longa, exceed 1-3 record were successful model.3 The prepariation of the tissue sectionThe successful models were perfused and took tissue at the according time. Tissues of the every subgroups were dealt with Chromotrope2R-Brilliant green myelin staining and immunohistochemical staining of OX-42,iNOS and MBP.4. Examine parameter (1) The score of behavior obstacle in rats were rated,according to the method of Zea Longa s standard.(2) Chromotrope 2R-Brilliant green myelin staining was selected to observe the changes of myelin.(3) The expressions of OX-42 and iNOS in the rats hippocampal CA1 and MBP in Corpus callosum were detected by immunohistochemistry staining.5 Image analysisFive different territory with high power lens (×400) were randomly chosen in one section and imaging system was used to analyze the positive area rate. The average of five areas was considered as the measured values.6 Statistic analysisAll the experimental datas were expressed by±s and analyzed with Least Signifcant Difference(LSD) between two groups and one-way ANOVA among groups with SPSS12.0 statistic software. The significant testing standard wasα=0.05.Results1.The conditions of the animal and the score of behavior obstacle in ratsIn group sham-operative,all rats had no significant neuronal deficits.Model group had all severe neuronal deficits.Compared with model group, cyclosporine A group got better score of behavior obstacle.There was significant difference(P< 0.05).2 Chromotrope 2R-Brilliant green myelin stainChromotrope 2R-Brilliant green myelin stain:Melyin is green and red, similar to a fish bone spur shape and closely-arranged, myelin for red and neuraxial for green. Compared with sham-operative group, model group have no significant difference at 1d and 3d. The density of myelin in Callus is decreased obviously, appears empty bubble at 7d and 14d. The density of myelin in Callus in cyclosporine A group is decreased obviously at 7d and 14d too,but better than model group.3 The expressions of OX-42 in the rats hippocampal CA1OX-42 in sham-operative group had little expression in CA1. In the model group and the cyclosporine group, the number of OX-42 positived cells markedly appeared at 1d of reperfusion,then peaked at 3d of reperfusion, and it decreased nearly normal at 14d. Compared with model group, the expression of OX-42 and iNOS in Cyclosporine group were reduced, there was significant difference at 1d,3d,7d(P<0.05).4 The expressions of iNOS in the rats hippocampal CA1INOS in sham-operative group had little expression in CA1.INOS peaked at 1d, at 7d it closed to normal in the model group and the cyclosporine group. Compared with model group, the expression of iNOS in Cyclosporine group were reduced at 1d,3d. There was significant difference at 1d,3d(P<0.05).5 The expressions of MBP in the rats corpus callosumCompared with sham-operative group, the number of MBP in corpus callosum in model group and the cyclosporine group was reduced at 1d. It was still lower than sham-operative group at 14d.The results of CsA group was better than model group. There was significant difference (P< 0.05).Conclusions1.The activation of microglia and expression of iNOS are closely associated with cerebral ischemia-reperfusion-injury.2.CsA can significantly reduce the activation of microglia and expression of iNOS after ischemia-reperfusion-injury.It has White matter protection as well. |
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