| Transient global cerebral ischemia is a clinical outcome related to cardiac arrest,cardiopulmonary bypass surgery and other situations that deprives the oxygen and glucose inbrain during a short period. In the rat model simulating human transient global cerebralischemia, it was found that neurons in the hippocampus CA1region were severely damagedduring the period of reperfusion, which leaded to the impairment of rats’ memory or spatiallearning. Similarly, in human being, a clinical study showed that the lesion in CA1regioncould result in place learning deficit. These indicate that protection of neuronal injury or deathin the CA1region would improve the prognosis of the patients with transient ischemia andreperfusion.Protein aggregation is a biological phenomenon in which abnormal proteins accumulateand clump together either intra-or extracellularly. Until Hu et al found that abnormal proteinsaccumulated in the neurons destined to die after transient cerebral ischemia [6], proteinaggregation had been thought to play a pivotal role in neurodegenerative disease such asParkinson’s disease and Alzheimer’s disease. Currently, as well as other mechanisms such ascalcium overload and oxidative stress, protein aggregation is also regarded as one of theimportant pathological features of cerebral ischemia and reperfusion. Although theseaggregated proteins cannot function normally, they could stick to the intracellular membranestructures and result in cell losing its normal function and dying in the end. Therefore,aggregated proteins should be rescued or eliminated in order to keep neurons alive andfunction normally.Recently, it had been reported that ischemic postconditioning has protective effects onneuronal injury or death caused by focal or global ischemia and reperfusion[9,10]. Ischemicpostconditioning is defined as a series of rapid intermittent interruptions of blood flow in theearly phase of reperfusion that mechanically alters the hydrodynamics of reperfusion. It could be performed before re-establishment of blood supply to brain, which makes it become afeasible method used potentially in clinical practice. Despite studies showed that ischemicpostconditioning could rescue neuronal injury or death via inhibiting inflammation,suppressing oxidative stress or preventing apoptosis, the effects of ischemic postconditioningon protein aggregation are still unclear. Studying the alteration of protein aggregation wouldcontribute further to clarify the protective mechanism of ischemic postconditioning. Therefore,in the current study, we investigated the role of ischemic postconditioning in proteinaggregation by using rat model of transient global ischemia.Objective:To investigate the effect of ischemic postconditioning on protein aggregation caused bytransient ischemia and reperfusion and to clarify its underlying mechanism.Methods:Two-vessel-occluded transient global ischemia rat model was used. The rats in ischemicpostconditioning group were subjected to three cycles of30-s/30-s reperfusion/clamping after15min of ischemia. Neuronal death in the CA1region was observed by hematoxylin-eosinstaining, and number of live neurons was assessed by cell counting under a light microscope.Succinyl-LLVY-AMC was used as substrate to assay proteasome activity in vitro. Proteincarbonyl content was spectrophotometrically measured to analyze protein oxidization.Immunochemistry and laser scanning confocal microscopy were used to observe thedistribution of ubiquitin in the CA1neurons. Western Blot was used to analyze thequantitative alterations of protein aggregates, proteasome, Hsp70and Hsp40in cellularfractions under different ischemic conditions. ResultsHistological examination showed that the percentage of live neurons in the CA1regionwas elevated from5.21%±1.21%to55.32%±5.34%after administration of ischemicpostconditioning （P=0.0087）. Western Blot analysis showed that the protein aggregates inthe ischemia group was32.12±4.87,41.86±4.71and34.51±5.18times higher than that in thesham group at reperfusion12h,24h and48h, respectively. However, protein aggregates werealleviated significantly by ischemic postconditioning to2.84±0.97,13.72±2.13and14.37±2.42times at each indicated time point （P=0.000032,0.0000051and0.0000082）. Laser scanning confocal images showed ubiquitin labeled protein aggregates could not bediscerned in the ischemic postconditioning group. Further study showed that ischemicpostconditioning suppressed the production of carbonyl derivatives, elevated proteasomeactivity that was damaged by ischemia and reperfusion, increased the expression of chaperoneHsp70, and maintained the quantity of chaperone Hsp40.Conclusion:Ischemic postconditioning could rescue significantly neuronal death in the CA1regioncaused by transient ischemia and reperfusion, which is closely associated with suppressing theformation of protein aggregation. |
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