Effects Of Leg Ischemic Postconditioning On Focal Cerebral Ischemia-reperfusion Injury In Diabetic Rats

ObjectiveDiabetic patients have worse survival and recovery prospects after acute stroke than non-diabetic patients.The present study was to establish a model of focal cerebral ischemia-reperfusion(I/R) in streptozotocin-induced diabetic rat. To investigate whether leg ischemic postconditioning(remote postconditioning [RPostC]) could induce neuroprotective effects on focal cerebral ischemiareperfusion injury in diabetic rats.To investigate the effects of RPostC on neurologic dysfunction,cerebral infarction size,neuronal cell apoptosis,structure and function of mitochondria after focal cerebral I/R injury in diabetic rats.To explore the possible mechanism of RPostC in attenuating cerebral ischemia-reperfusion injury complicated with diabetes.Methods(1) A total of seventy-five healthy adult male SD rats weighing 270 to 320 g were used in this study.The diabetes mellitus rats models were induced by injecting streptozotocin into abdominal cavity 60 mg/kg body weight and confirmed by fasting blood glucose above 16.7 mmol/L with a symptom of polydipsia,polyphagia and polyuria 3 days after injection.Animals were subjected to MCAO as described by Longa et al with minor modification.The rats were anesthetized with chloral hydrate(300 mg/kg,i.p.).A midline neck incision was made and the left common carotid artery(CCA),the external carotid artery(ECA) and the internal carotid artery(ICA) were exposed and dissected away from adjacent nerves.The distal ECA was ligated and a thread with rounded tip was inserted into left ICA from the CCA and threaded cranially until resistance was felt(about 18 mm) which ensured the occlusion of the middle cerebral artery.The thread was gentally withdrawn from the ICA to allow MCA reperfusion.Sixty male SD rats were assigned to 4 groups:1) Control group;2) Sham group:animals were given the same procedure with the MCAO animals without inserting the thread;3) I/R group:the brains were undergone ischemia for 90 min followed by reperfusion for 6 hours;4) RPostC group.(2) Leg ischemic postconditioning(RPostC) protocal:The procedure was identical to I/R group during which animals were subjected to 3 cycles of 5min/5min legs ischemia/reperfusion,and the legs were binded tight enouph to ensure the femoral artery pulsation can not be touched.(3) Six hours after reperfusion,all animals were subjected to neurological examination blindly performed.Performance was scored using a commonly used scale(NDS) described by Longa et al.The animals were then killed and TTC staining was used to observe the morphous of infarction zone and determine the changes of brain infarct volume.Microscope was applied to detect the pathological morphous of cerebral ischemic tissues.The changes of neuronal cells apoptosis were observed through TUNEL staining method and the ultrastructure of mitochondria were observed by electron microscope.Rat brain mitochondria were isolated and the level of malondialdehyde(MDA),Superoxide dismutase(SOD),Na⁺,K⁺-ATPase,Ca²⁺-ATPase,and glutathione peroxidase (GSH-Px) in mitochondria was measured according to the commercial kit manual.(4) The data were expressed as mean±stand error while the NDS was expressed as median.Data analysis was performed using the SPSS 11.0. statistical package programme.P＜0.05 was considered as statistically significant.Results(1) Effects of RPostC on NDS The fasting blood glucose of rats was above 16.7 mmol/L with symptom of athrepsy,polydipsia,polyphagia and polyuria 3 days after injection.Compared with control group and sham group,the NDS was significantly increased in I/R group and RPostC group.There was no significant difference in NDS between I/R and RPostC group(P＞0.05).(2) Effects of RPostC on cerebral infarction size determined by TTC staining The infarction zone appeared white and distributed in accordance with the distribution of the middle cerebral artery.Compared with control group and sham group,the cerebral infarction size was significantly increased in I/R group and RPostC group(P＜0.01).Compared with I/R group,the cerebral infarction size was significantly smaller in RPostC group(P＜0.05).(3) Effects of RPostC on pathological morphous stained by HE In the control and sham groups,the nucleoli were distinct,the tissue structure was clear and the pathological changes were not notable.The ischemic pathological changes in I/R group were more serious,where cytoplasma concentration, pyknosis and karyorrhexis were common.In RPostC group the pathological changes were attenuated and few neurons were morphologically altered.(4) Effects of RPostC on neuronal cells apoptosis labelled by TUNEL staining Most of the apoptic neurons showed the following typical characteristics:aggregation of chromatin in the edge and the concentration of nucleus.Apoptic neurons scarcely dispersed in the control group and sham group,and 6 hours after reperfusion,the apoptic neurons in I/R group was significantly increased.After remote postconditioning,the apoptic neurons were markedly reduced compared with I/R group(P＜0.01).(5) Effects of RPostC on ultrastructure of mitochondria In control and sham groups,mitochondrial ultrastructure was basically normal.Mitochondria was orderly ranged,and the diastem was slightly swollen with nearly intact membrane. 6 h after reperfusion,the mitchondria in I/R group swelled obviously and the cristae was disrupted,dissovoled or disappeared.Mitochondrial membrane was vague and a lot of vacuoluses could be seen.RPostC reduced these changes in I/R group.Mitochondria was orderly arranged,with mitochondrial membrane basically intact,and there was little vacuolization. (6) Effects of RPostC on MDA level and SOD,ATPase,GSH-Px activities in mitochondria MDA level was markedly elevated and SOD,ATPase,GSH-Px activities was significantly reduced in I/R group and RPostC group compared with those in the control group and sham group(P＜0.05 or P＜0.01).Compared with the I/R group,the level of MDA was significantly reduced and the mitchondria SOD,ATPase and GSH-Px activities in RPostC group was markedly enhanced(P＜0.05 or P＜0.01).Conclusion(1) Leg ischemic postconditioning is a neuroprotective treatment in attenuating focal cerebral I/R injury in diabetic rats,demonstrating a new way which shares similar protective effects with ischemic postconditioning:RPostC.(2) RPostC can be induced by leg ischemic postconditioning and has an obvious protective effect on reduction of cerebral infarction size,neuronal cells apoptosis,brain and mitochondria damage after focal cerebral I/R in streptozotocin- induced diabetic rats.(3) RPostC is able to protect the brain from I/R injury in diabetic rats and its mechanism may be partly attributed to reduction of the mitochondrial dysfunction and destruction,increase of the activities of SOD,ATPase and GSH-Px,scavenging free radicals by antioxidant enzymes,decreasing the neurons apoptosis,and attenuating the cerebral infarction size.(4) RPostC might promise clear clinical potency as a simple neuroprotective technique but the present study showed no obvious improvement in NDS.It is important to investigate the optimal proposal of RPostC in the future such as when to start and how long to persist in order to improve the cerebral function.