Study On The Protection Of Noninvasive Delayed Limb Ischemic Preconditioning Against Cerebral Ischemia-Reperfusion Injury In Rats

Objective:To study the protection of noninvasive delayed limb ischemicpreconditioning (NDLIP)against cerebral ischemia reperfusion (I/R)injury inrats.Method:Healthy male Wistar rats were divided randomly into 4 groups.(1)Sham group:Left common carotid artery was dissociated,while was notoccluded.(2)I/R group:rats were subjected to 1h occlusion of middle cerebrealartery followed by 24h ofreperfusion.(3)ECIP+I/R group:rats were subjected to3 cycles of 5 min of ischemia and reperfusion on the common carotid artery beforelh of ischemia and 24h of reperfusion.(4)NDLIP+I/R group:rats weresubjected to 3 cycles of 5 min of ischemia and reperfusion on the left hind limbfor 3 days to induce NDLIP.On the forth day,l h of ischemia and 24h ofreperfusion was performed.Delayed protections of NDLIP were estimated inthe light of the changes of neural behavior mark,energy metabolism,cerebral celldeath and apoptosis,cerebral tissue antioxidative ability,blood vessel endothelialfunction and fibrinolysis system function.Neurological functions were studiedthrough observing neural behaviors.Determinations of ATP,ADP and AMP werecarried out by HPLC.TAN and EC were calculated.Cerebral infarct size wasdetermined based on 2,3,5-triphenyltetrazolium chloride staining.Cerebral cellapoptosis was detected using the TUNEL method.Expressions ofapoptosis-associated genes Fas and FasL were measured using Real-time PCR andexpressions of apoptosis-associated proteins Fas and FasL were measured usingWestern blot method.Changes of cerebral cortex and hippocampus morphologywere observed after HE staining.Activity of myeloperoxidase (MPO),total-superoxide dismutase (T-SOD),manganese-superoxide dismutase (Mn-SOD),glutathione peroxidase (GSH-PX)and xanthine oxidase (XOD)in cerebral tissue,content of malonaldehyde (MDA)and concentration of nitric oxide (NO)inserum were detected by spectrophotometer.Mn-SOD mRNA was measured byRT-PCR method.Content of endothelin-l(ET-1)and activities of tissueplasminogen activator(t-PA)and plasminogen activator inhibitor-1(PAI-1)were measured by ELISA.Results:1 Effects of NDLIP on neural behaviors and energy metabolism inducedby cerebral I/R injuryCompared with I/R group,the neural behaviors marks of ECIP+I/R groupand NDLIP+I/R group decreased (P＜0.01),the contents of ATP (P＜0.01),ADP (P＜0.01)and AMP (P＜0.05)in ECIP+I/R group and NDLIP+I/R groupincreased.The calculation value of TAN (P＜0.01)and EC (P＜0.01)inECIP+I/R group and NDLIP+I/R group increased,too.2 Effects of NDLIP on cerebral cell death and apoptosis induced by cerebral I/RinjuryCompared with I/R group,cerebral infarct size were diminished (P＜0.05)in ECIP+I/R group and NDLIP+I/R group.Activities of MPO were decreased(P＜0.01),apoptosis of cortex cell (TUNEL:P＜0.01)and hippocampus cell(TUNEL:P＜0.01)in ECIP+I/R group and NDLIP+I/R group decreased.Theanti-death protection of NDLIP was as effective as that of ECIP.3 Apoptosis mechanism of prevention of NDLIP against cerebral I/RinjuryCompared with I/R group,expressions of apoptosis gene Fas (P＜0.01)and FasL (P＜0.01)in cortex in ECIP+I/R group and NDLIP+I/R groupdecreased,expressions of Fas (P＜0.01)and FasL (P＜0.01)in hippocampus ofECIP+I/R group and NDLIP+I/R group decreased.Compared with I/R group,expressions of apoptosis protein Fas (P＜0.05)and FasL (P＜0.05)in cortex ofECIP+I/R group and NDLIP+I/R group decreased.4.Effects of NDLIP on cerebral antioxidative activity after cerebral I/R injuryCompared with I/R group,the activities of T-SOD (P＜0.01),Mn-SOD(P＜0.01)and GSH-PX (P＜0.01)in cortex and activities of T-SOD (P＜0.01)andMn-SOD mRNA(P＜0.01)in hippocampus of ECIP+I/R group and NDLIP+I/Rgroup increased,the expression of Mn-SOD in cortex (P＜0.01)and inhippocampus (P＜0.01)of ECIP+I/R group and NDLIP+I/R group increased.The activities of XOD (P＜0.01)and MDA content in cortex (P＜0.01)and in hippocampus (P＜0.01)of ECIP+I/R group and NDLIP+I/R group decreased.5.Effects of NDLIP on blood vessel endothelial function before and aftercerebral I/R injuryBefore ischemia,there was no difference on the concentration of NO inserum among I/R group,ECIP+I/R group and NDLIP+I/R group.But comparedwith I/R group,NO in serum increased (P＜0.05)and ratio of ET-1 and NOdecreased (P＜0.05)in ECIP+I/R group and NDLIP+I/R group.After 24h of reperfusion,compared with I/R group,the concentration ofET-1 in serum decreased (P＜0.01),the concentration of NO in serum increased(P＜0.01)and the ratio of ET-1 and NO decreased (P＜0.05)in ECIP+I/R groupand NDLIP+I/R group.The endothelial protection of NDLIP was as effective asthat of ECIP.6 Effects of NDLIP on fibrinolysis system before and after cerebral I/R injuryBefore ischemia,there was no any difference on fibrinolytic indicatorsamong the groups.After 24h of reperfusion,compared with I/R group,theactivities of t-PA (P＜0.01)increased and the activities of PAI-1 decreased(P＜0.01)in ECIP+I/R group and NDLIP+I/R group.Conclusion:1.NDLIP decreased neural behavior marks and improved neurologicaldeficits of rats after cerebral I/R.NDLIP increased contents of ATP,ADP andAMP and calculation value of TAN and EC,improve energy metabolism aftercerebral I/R.2.NDLIP decreased cerebral infract size and cell apoptosis,attenuatedcortex and hippocampus morphology injury.The anti-cell death effect ofNDLIP was as effective as that of ECIP.The mechanism was involved in thedecrease of MPO activity and the down regulation of expressions of apoptosisassociated protein Fas and FasL.3.The mechanism of the prevention of NDLIP on cerebral I/R was involvedin the down regulation of expressions of apoptosis associated protein Fas andFasL.4.NDLIP increased cerebral antioxidative ability after I/R injury, decreased peroxidative damage.The antioxidative protection of NDLIP was aseffective as that of ECIP.5.In normal condition,NDLIP increased the concentration of NO inserum,slanted the balance between ET-1 and NO toward NO.After I/R injury,NDLIP decreased the disturbance of endothelial function,increased NO inserum,decreased ET-1 concentration and maintained the balance betweenET-1 and NO to approach normal.The endothelial protection of NDLIP was aseffective as that of ECIP.6.NDLIP increased t-PA activities and decreased PAI-1 activities.Theeffect on fibrinolysis system of NDLIP was as effective as that of ECIP.