BACKGROUND:Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance.However,the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE:To dynamically measure erythropoietin and hypoxia-inducible factor-1α(HIF-1α) mRNA and protein expression at various times following preconditioning,and to investigate effects of erythropoietin and HIF-1αon cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN,TIME AND SETTING:The randomized,controlled study was performed at the Institute of Anatomy,Medical College,Qingdao University,China from March 2006 to March 2007. MATERIALS:Rabbit anti-rat HIF-1αmonoclonal antibody and biotinylated goat anti-rabbit IgG (Boster,China),rabbit anti-rat erythropoietin monoclonal antibody(Santa Cruz Biotechnology,USA), and one-step RT-PCR kit(Qiagen,Germany) were used in this study. METHODS:A total of 99 healthy,male,Wistar rats were randomly assigned to three groups:sham surgery(n = 9),non-ischemic preconditioning(n = 45),and ischemic preconditioning(n = 45).In the ischemic preconditioning group,rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method.In the non-ischemic preconditioning group,pre-ischemia was replaced by sham surgery.Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion,including 1-,3-,7-,14-,and 21-day subgroups.The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES:HIF-1αand erythropoietin protein expression was measured in the cerebral cortex,corpus striatum,and hippocampus of the ischemic hemisphere.HIF-1αand erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS:(1) Intergroup comparison:compared with the non-ischemic preconditioning group, HIF-1αprotein expression significantly increased in the rat cerebral cortex,corpus striatum,and hippocampus in the ischemic hemisphere at 1,3,and 7 days following reperfusion in the ischemic preconditioning group(P<0.05 or P<0.01).Erythropoietin protein expression significantly increased in the cerebral cortex,corpus striatum,and hippocampus,as well as HIF-1αand erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere,at 3 and 7 days following reperfusion in the ischemic preconditioning group(P<0.05).(2) Temporal expression:HIF-1αprotein expression in the rat cerebral cortex,corpus striatum,and hippocampus, as well as HIF-1αmRNA expression in the frontal and parietal cortex,in the ischemic hemisphere increased at 3 days,and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group.Temporal erythropoietin protein and mRNA expression was consistent with HIF-1αprotein expression.(3) Correlation:erythropoietin mRNA expression positively correlated with HIF-1αmRNA expression(r = 0.737,P<0.01). CONCLUSION:Ischemic preconditioning induced cerebral ischemic tolerance.Pre-ischemia-induced increase in endogenous HIF-1αexpression,as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance. BACKGROUND: Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. How the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE: To dynamically measure erythropoietin and hypoxia-inducible factor-1α (HIF-1α) mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1αon cerebral ischemic tolerance in a model of focal ischemia / reperfusion established using the twice suture method. Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS: Rabbit anti-rat HIF-1 αmonoclonal antibody and biotinylated goat anti-rabbit IgG (Boster, China), rabbit anti-rat erythropoietin monoclonal antibody Cruz Biotechnology, USA), and one-step RT-PCR kit (Qiagen, Germany) were used in this study. METHODS: A total of 9 Wistar rats were randomly assigned to three groups: sham surgery (n = 9), non-ischemic preconditioning (n = 45), and ischemic preconditioning (n = 45) .In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Published, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups.The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES: HIF- 1αand erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1αand erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS: (1) Intergro up comparison: compared with the non-ischemic preconditioning group, HIF-1αprotein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group (P < 0.05 or P <0.01) .Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1αand erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion (2) Temporal expression: HIF-1αprotein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1αmRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with Correlation: erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression (r = 0.737, P <0.01). CONCLUSION: Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemia-induced increase in endogenous HIF-1αexpression , as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.