Ischemic Postconditioning Protects Brain From Ischemia/Reperfusion Injury By Attenuating Endoplasmic Reticulum Stress

Endoplasmic reticulum (ER) stress has been implicated in the pathology of cerebral ischemia. During prolonged period of stress or when the adaptive response fails, apoptotic cell death ensues. Ischemic postconditioning is defined as a series of rapid intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of reperfusion. Recently, cerebral ischemic postconditioning (Postcond) has been shown to reduce cerebral ischemia/reperfusion (I/R) injury in cerebral ischemia model. As Postcond could be undertaken before reperfusion, it was practical and could be widely used in the clinical work in the future. However, few studies focused on the mechanism of Postcond-induced neuroprotection, the mechanism remains to be understood.In our study, a focal cerebral ischemia model was used. We aimed to investigate the protective effect of Postcond against I/R injury, to elucidate whether Postcond attenuates brain I/R damage by suppressing ER stress-induced apoptosis and if the phosphatidylinositol-3kinase/Akt (PI3K/Akt) pathway is involved. Our study include the following three parts:1. Postcond protects cerebral against I/R injury.2. Postcond protects cerebral against I/R injury by attenuating endoplasmic reticulum stress.3. Postcond suppression of ER stress-induced apoptosis is mediated by PI3K/Akt pathway.Part I:ischemic postconditioning protects cerebral against ischemia/reperfusion injuryObjective:A permanent focal cerebral ischemia rat model was used in the study, we investigated the protective effect of ischemic postconditioning against ischemia/reperfusion injury.Methods:Adult male Sprague-Dawley rats (350-450 g). Animals were randomly divided into three groups for the experiment:Sham group, Ischemia/reperfusion (I/R) group, Ischemic postconditioning (Postcond) group. The left distal middle cerebral artery(MCA)was exposed and cauterized permanently. The bilateral common carotid arteries (CCA) were occluded by aneurysms clips for 30 min after MCA occlusion and then released, then Postcond was performed:after 30 sec of CCA reperfusion, the CCAs were occluded again by aneurysms clips for 10 secs, followed with another 2 cycles of 30 sees reperfusion and 10 secs occlusion (total of 3 cycles). Physiological parameters (PH, PCO2 and PO2) were measured using blood gas analyzer 20 mins before cerebral ischemia,20 mins after cerebral ischemia,20 mins after reperfusion. At 24 h of reperfusion, neurological behavior evaluation and cerebral infarction size were evaluated by the methods of Bederson scoring and TTC staining.Results:There were no statistically differences in any of the parameters between the groups at a specific period or within a group at different periods. Compared with I/R group, Postcond improved the neurological functions and reduced infarct size at 24 h of reperfusion (P<0.05).Conclusions:Postcond could induce neuroprotection against cerebral I/R injury.Part II:Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stressObjective:A permanent focal cerebral ischemia rat model was used in the study. To elucidate the mechanism of neuroprotection induced by ischemic postconditioning, we investigated the effect of ischemic postconditioning on the expression of ER stress proteins.Methods:Adult male Sprague-Dawley rats(350-450 g). Animals were randomly divided into three groups for the experiment:Sham group, Ischemia/reperfusion (I/R) group, Ischemic postconditioning (Postcond) group. The left distal middle cerebral artery(MCA)was exposed and cauterized permanently. The bilateral common carotid arteries (CCA) were occluded by aneurysms clips for 30 min after MCA occlusion and then released, then Postcond was performed:after 30 sec of CCA reperfusion, the CCAs were occluded again by aneurysms clips for 10 secs, followed with another 2 cycles of 30 sec reperfusion and 10 sec occlusion (total of 3 cycles). At 6 h,12 h and 24 h of reperfusion, the expression of CHOP, caspase-12 and GRP78 in the penumbra cortex were measured with immunohistochemical analysis and Western blot analysis, while the neuronal apoptosis in the penumbra cortex was detected by the method of terminal deoxynucleotidyl transferase mediated Biotin-dUTP niek-end labeling (TUNEL).Results:Compared with the I/R group, Postcond significantly reduced the positive cells of both CHOP and caspase-12 at 24 h of reperfusion, while significantly increased the positive cells of GRP78 from 12 h to 24 h of reperfusion(P<0.05). Compared with the sham group, in the I/R group the expression of CHOP, caspase-12 and GRP78 in the penumbra cortex had gradually increased from 6 h to 24 h and peaked at 24 h of reperfusion (P<0.05). Compared with the I/R group, Postcond significantly inhibited the expressions of CHOP and cleaved-caspase-12 at 24 h of reperfusion (P<0.05), while up-regulated GRP78 protein level from 12 h to 24 h of reperfusion (P<0.05). TUNEL staining in the penumbra cortex was barely detectable in the sham group. In the I/R group TUNEL-positive cells in the penumbra cortex were slight positive at 6 h of reperfusion, and were markedly increased from 12 h of reperfusion, then were strong positive at 24 h of reperfusion. Compared with the I/R group, Postcond significantly reduced the number of TUNEL-positive cells in the penumbra cortex at 24 h of reperfusion.Conclusions:Cerebral I/R causes ER stress. Postcond inhibited the expression of CHOP and caspase-12 activition, while increased the expression of GRP78. Postcond protecte against cerebral I/R injury by reducing ER stress-induced apoptosis.PartⅢIschemic postconditioning attenuate endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathwayObjective:A permanent focal cerebral ischemia rat model was used in the study. To elucidate whether the phosphatidylinositol-3kinase/Akt (PI3K/Akt) pathway is involved in suppressing ER stress induced apoptosis by Postcond.Methods:Adult male Sprague-Dawley rats(350-450 g). Animals were randomly divided into four groups for the experiment:Sham group, Ischemia/reperfusion (I/R) group, Postcond+DMSO group, Postcond +LY294002 (Postcond+LY) group. The left distal middle cerebral artery(MCA)was exposed and cauterized permanently. The bilateral common carotid arteries (CCA) were occluded by aneurysms clips for 30 min after MCA occlusion and then released, then Postcond was performed:after 30 sec of CCA reperfusion, the CCAs were occluded again by aneurysms clips for 10 sec, followed with another 2 cycles of 30 sec reperfusion and 10 sec occlusion (total of 3 cycles). Postcond with coinjection of LY294002(10μl,10 mM, in 3% dimethyl sulfoxide, icv, 15 mins before Postcond). Cerebral infarction size were evaluated by TTC staining. The expression of CHOP, caspase-12 and GRP78 in the penumbra cortex were measured with immunohistochemical analysis and Western blot analysis at 24 h of reperfusion. The neuronal apoptosis in the penumbra cortex was detected by the method of terminal deoxynucleotidyl transferase mediated Biotin-dUTP niek-end labeling (TUNEL).Results:Compared with I/R group, Postcond significantly reduced infarct size at 24 h of reperfusion, LY294002 treatment partly inhibited Postcond’neuroprotection(P<0.05). Compared with the sham group, in the I/R group the expression of CHOP, caspase-12 and GRP78 in the penumbra cortex significantly increased (P<0.05). Compared with I/R group, Postcond significantly inhibited the expressions of CHOP and cleaved-caspase-12, while up-regulated GRP78 and P-Akt protein level (P<0.05). Compared with Postcond+DMSO group, LY294002 treatment significantly increased the expressions CHOP and caspase-12 while down-regulated GRP78 expression in the penumbra cortex (P<0.05). TUNEL staining in the penumbra cortex was barely detectable in the sham group. Compared with the sham group, in the I/R group the positive cells of CHOP、caspase-12 and GRP78 in the penumbra cortex were significantly increased, also the number of TUNEL-positive cells were significantly increased (P<0.05). compared with the I/R group, Postcond significantly decreased the positive cells of CHOP and caspase-12 and the number of TUNEL-positive cells, while increased the positive cells of GRP78(P<0.05). Compared with Postcond+DMSO group, LY294002 treatment significantly increased the positive cells of CHOP and caspase-12 and the number of TUNEL-positive cells, while decreased the positive cells of GRP78(P<0.05).Conclusions:Postcond protects brain from I/R injury by attenuating endoplasmic reticulum stress-induced apoptosis, and PI3K/Akt pathway is involved.