| ObjectiveTo establish an model of focal cerebral ischemia-reperfusion in streptozotocin-induced diabetic rats;To explore the effect of ischemic postconditioning(I-Post)on cortical neurons apoptosis in focal cerebral ischemia of diabetic rats;To investigate the effect of I-Post on the structure and function of mitochondria after focal cerebral ischemia-reperfusion in streptozotocin-induced diabetic rats;To debate the mechanism of I-Post in acute cerebral ischemia complicated with diabetes.Methods(1)The models of experimental diabetes rats were established by a single intraperitoneal injection of STZ(60 mg/kg body weight),and the successful models were selected to establish the models of focal cerebral ischemia-reperfusion rats by Longa's technique.Sixty-five rats were successfully made into diabetic models and randomly divided into 4 groups:1)control group: with no other intervention;2)Sham-operated group:the surgical procedure was identical to other groups,but the MCA was not ligated;3)I/R group:the brain was undergone ischemia for 90 min followed by reperfusion for 6h;4)I-Post group:rats were subjected to 3 cycles of 15s/15s reperfusion/reocclusion after 90 min of ischemia.(2)Postconditioning protocols:the brain was subjected to middle cerebral artery occlusion for 90 min,then treated for 15 sec,15 sec,15 sec by brief reperfusion consecutively,each was separated with occlusion for 15 sec,followed by a persistent reperfusion,which made the total reperfusion time the same as the other three groups.(3)The assessment standard of Longa' 5 scores was applied to assess the neural function of the rats after cerebral ischemia-reperfusion.(4)TTC staining was applied to observe and note the formation of infarction zone;The pathologic characteristics of cerebral ischemic tissues were observed through microscope;The TUNEL was used to detect the change of TUNEL positive cells in every group;The ultrastructure of mitochondria was observed by electron microscope 6 hours later.Rat brain mitochondria were isolated in a medium of 0.01 mol/L sucrose,0.01mol/L Tris-HCl,0.0001mol/L EDTA-2Na, 0.8%Sodium Chloride,pH 7.4,by differential centrifugation of brain homogenates essentially as described previously.The level of malondialdehyde (MDA),Na+,K+-ATPase,Ca2+-ATPase,Superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in mitochondria were measured 6 hours later, according to the commercial kit manual.(5)The data were expressed as mean±SD,the NDS was expressed as median.Data analysis was performed using the SPSS 13.0 statistical package programme.P＜0.05 was considered as statistically significant.Results(1)The diabetic rats appeared the diabetic symptoms such as eat more,drink more and urine more after the model was made.Plasma glucose in diabetic rats was routinely 24.6±3.5mmol/L.There was no significant difference in neurologic deficit scores between the I/R group and I-Post group(P＞0.05).(2)The TTC staining results of the rat's brain The brain tissues without infarction were stained as red,the infarction zone could not be stained and appeared as white and the white zones distributed in accordance with the distribution of the middle cerebral artery.There was a larger white areas in I/R than in I-Post after the same reperfusion time(P＜0.05). (3)Observing the results of pathological sections stained by HE Under the optical microscopy,we found the ischemic changes in I/R group were more serious than I-Post group,which manifested the tissues became loose,the neurons nuclus atrophy,the staining became light,the cell morphous altered.But in the control and sham-operated groups,the tissue structure was clear,the nucleoli was distinct,the pathological changes was not remarkable.(4)TUNEL The apoptosis cells were characterized by body shrinkage, nuclear pyknosis,peripheral margination of chromatin crescents or chromatin clumps.Compared with I/R group,the apoptosis cells were less after reperfusion in I-Post group(P＜0.01).(5)The ultrastructure of mitochondria Compared with control and sham-operated groups,the changes of the mitochondria of brain tissue in I/R group were obvious.The cristae of mitochondria were less and some of them became indistinct or disappeare;Some double membrane were not distinguishable clearly.The changes ultrastructure of mitochondria in cortex were significantly decreased in I-Post group compared with I/R group.(6)The level of malondialdehyde(MDA),Na+,K+ -ATPase,Ca2+-ATPase, Superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in mitochondria The content of MDA in mitochondria was greatly higher in I/R group than it in control group and sham-operated group(P＜0.01),Na+,K+ -ATPase,Ca2+-ATPase,SOD and GSH-Px activities in mitochondria were greatly higher in control group and sham-operated group than those in I/R group(all P＜0.01).The content of MDA in mitochondria was markedly decreased and the activities of Na+,K+ -ATPase,Ca2+-ATPase,SOD and GSH-Px in mitochondria were increased in I-Post group compared with the I/R group(P＜0.05 or P＜0.01).Conclusion(1)I-Post could protect brain against reperfusion injury as well;(2)I-Post could not attenuate behavioral deficits after focal cerebral ischemia in diabetic rats; (3)I-Post could reduce infarct size and the number of apoptotic cells of cortical neurons after focal cerebral ischemia-reperfusion in streptozotocininduced diabetic rats;(4)I-Post could protect diabetic rats against brain I/R injury.The brain protective effects of I-Post may be mediated,in part,by inhibiting the production of oxygen free radical,increasing antioxidation,ameliorating energy metabolism, and beneficially improving the integrity of structure and function of mitochondria in brain tissue. |
