| OBJECTIVES: To establish a model of acute cerebral ischemia-reperfusion in diabetic rats; To observe the expression of MMP-9, RECK and VEGF protein in the cerebral ischemic tissues; To observe the relationships between the expression of MMP-9, RECK and VEGF in the cerebral ischemic tissues; To investigate the involvement of MMP-9, RECK and VEGF in the diabetic rat brain after focal cerebral ischemia.METHODS:(1) The rat models of diabetes mellitus were made by high sucrose, fat diet and streptozotion injection, in which the successful ones were selected to be made into focal cerebral ischemia-reperfusion rats. The models of the middle cerebral artery occlusion were made by improved zealong's method. 120 male wistar rats were assigned randomly into normal control group ([control I ],6 rats), sham-treated group ([control II],6 rats), cerebral ischemia-reperfusion and diabetic rats([DM+CI group] ,54 rats) and cerebral ischemia-reperfusion rats([CI group],54 rats). Middle cerebral artery occlusion models were made by DM+CI group and CI group, and the two groups were randomly assigned to 7 subgroups according to the reperfusion time (1h,3h,6h,12h,24h,3d,7d);(2) The neural function of the rats after cerebral ischemia-reperfusion were assessed with longa method;(3) TTC staining and HE staining were applied to observe and mark the clinical features of infarction zones; The pathologic characteristics of cerebral ischemic tissues were observed by HE staining and immunohistochemistry through microscope; Positive cells of MMP-9, RECK and VEGF protein in the infarction cores and penumbra zones of brain tissues were observed and counted;(4) Statistical analyses were performed with the results by use of the SPSS 12.0 package. Mean values and standard error of mean were calculated for all studied areas, and statistical significance for intergroup was assessed by Student t test or ANOVA. The Spearman coefficient was used to study correlations between continuous variables. P<0.05 was considered statistically significant.RESULTS:(1) The assessment results of the rats neural functions The models of MCAO from CI group and DM+CI group appeared Homer sign; There were 46 rats with Horner sign, and the assessment score was 1. 54±0. 71; There were 36 rats with Horner sign from DM+CI group, and the score was2. 35±0. 55. A significant difference showed between the two groups (P<0.05). In sham-treated group, neural dysfunction sign wasn't found.(2) TTC staining results of rat brain The non-infarcted areas of brain tissues were stained red. The infracted zones appeared pale in accordance with the distribution of the middle cerebral artery. Among samples from diabetic ischemic strokes, the infarct zones were larger and the damages were more severe than euglycemic ischemic strokes.(3) Expression of MMP-9 In ischemic areas, MMP-9 was mainly located in the neuroglial, neurons and endothelial cells together with immunoreactive neutrophils. Positive cells are increased over time in diabetic and euglycemic ischemic rats but not in sham-treated or normal rats (P<0.05). However, at the same reperfusion time, the expression of MMP-9 was significantly higher in diabetic brains than in euglycemic ones (P<0.05). MMP-9 expression showed similar patterns in DM+CI and CI group: Increased at 3h, maximized at 24h, and gradually decreased after 3d.(4) Expression of RECK protein RECK protein expressed mainly in the neuroglial, neurons in the normal rat brains and peri-infarct zones, barely in the ischemic cores. RECK protein expression significantly decreased compared to normal and sham-treated rat brain (P<0.01). At the same reperfusion time, there were less RECK positive cells in DM+CI group than CI group (P<0.05). RECK protein expressed similarly in DM+CI and CI group: Decreased at 3h, minor at 24h, and gradually increased after 3d(5) Expression of VEGF VEGF mainly localized in the neurons, neuroglial and endothelial cells in the ischemic penumbra. It significantly increased in diabetic and euglycemic ischemic rats (P<0.01). At the same reperfusion time, VEGF expression in euglycemic ischemic rats is more than diabetic ischemic rats(P<0.01). In diabetic ischemic rats, VEGF expression peaked at 6h; in euglycemic ischemic rats, it peaked at 24h.(6) A correlation study in diabetic ischemic rats between MMP-9 and RECK, MMP and VEGF demonstrated a strong relation between MMP-9 and RECK protein(r=-0.880, P <0.01), MMP-9 and VEGF (r=0.758, P <0.05).CONCLUSIONS:(1) There were significantly larger infarction areas in DM+CI group after ischemia-reperfusion compared with CI group. The signs of neural dysfunction after ischemia in diabetic rats were more severe than euglycemic rats.(2) The categories of cells in which MMP-9, RECK and VEGF protein were expressed plentifully in the brain after cerebral ischemia in diabetic rats were the same with those in euglycemic rats. It indicated that the several cells mentioned above were likely to played roles in the courses after ischemia-reperfusion.(3) There were significantly higher expression of MMP-9 in the ischemic tissues after ischemia-reperfusion in diabetic rats paralleled with lower expressions of RECK and VEGF. It suggested MMP-9 might be involved in the regulation of RECK and VEGF expression.(4) The coherence between MMP-9 and RECK, MMP-9 and VEGF in the space distribution and the correlation between them along with the reperfusion time indicated that hyperglycemia caused increased MMP-9 and decreased VEGF and RECK protein ,while increased MMP-9 was involved in VEGF regulation. This mechanism probably was one of the reasons for the larger infarction areas and the worse outcome in cerebral ischemia of diabetic patients.
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