Selenoprotein S Expression In The Rat Brain Following Focal Cerebral Ischemia

Objectives:Stroke has been the third leading cause of death after heart diseases and cancer, and also the most common cause of complex disability in adults around the world. Recent studies on cerebral ischemic stroke have demonstrated the importance of the inflammatory response. Ongoing inflammatory insults have been implicated as a secondary mechanism underlying neuronal injury-induced by ischemia, and anti-inflammatory strategics have gained considerable interest. Selenoprotein S (SelS), which is an endoplasmic reticulum (ER) resident protein, is known to promote cell survival by regulating inflammation. Moreover, SelS has been shown to be responsive to ischemia in cultured astrocytes. A Finnish report revealed that a variation in the SelS gene locus is associated with a higher predisposition to ischemic stroke in humans, suggesting a crucial role for SelS in protection against brain ischemia. However, the time-course of SelS expression following cerebral ischemia in vivo remains unclear. In the present study,we are to investigate the variation of SelS expression from3h to7d after reperfusion in rats with transient focal cerebral ischemia induced by1hour of middle cerebral artery occlusion.Materials and Methods:Male healthy Sprague-Dawley rats weighing250-3000g were randomly allocated into7groups, sham operation group, cerebral ischemia reperfusion3h,6h,12h,24h,3d,7d group (n=18). The transient focal cerebral ischemic stroke models were induced by middle cerebral artery occlusion (MCAO), following with the suture withdrawed60minutes later. Sham-operated rats underwent identical surgery, except for inserting the intraluminal filament. Neurological deficit scores of ischemia reperfusion groups were evaluated at defined time points, then removed the brains, compared with sham operation group.6brains in every group were sectioned coronally at2mm intervals for TTC staining;6brains in every group were perfused transcardiacally and taken out to compare the surface area of ischemic hemisphere with contralateral, then paraformaldehyde fixed and waxed for making paraffin section; For western blot analysis, The cortical tissues were obtained from3regions (infarct core, peri-infarct and contralateral regions) in the rest6brains of every group. During the experiment, the results including neurological deficit scores,changes of brain surface area, TTC staining and HE staining, were to confirm the success of producing cerebral ischemic stroke and the zones of cerebral infarction; Nissl’s staining immunohistochemistry together with western blot were to observe the changes of neurons, astrocytes and SelS expression at different times and places following cerebral ischemia, as well as the probable mechanism of SelS participating in cerebral ischemic stroke. Finally statistical analyses were performed.Results:1.Behavioral Observation:The neurological disfunctional symptoms appeared significantly in the rats suffered from the cerebral ischemia reperfusion injury,compared with the sham operation group, showing slow-moving,dragging circling to the contralateral side when walking on.76.1%(108/142) rats reached2-3scores showing contralateral circling if pulled by tail at3h after reperfusion following MCAO.and increased gradually after reaching maximum levels at24h showing spontaneously contralateral turning. The rats of whose neurological deficit scores was3points at3h after MCAO were recruited.2. Cerebral ischemic injury:Focal cerebral ischemia in rats caused a restricted infarction in the ipsilateral vascular territory supplied by the middle cerebral artery, including the sensorimotor cortex and striatum. Ipsilateral brain slice was divided into infarct core and peri-infarct regions according to the TTC staining; The surface area of the ischemic hemispheres was significantly increased at24h after reperfusion.and reduced at3d and7days; Swelling, shrunken and cavitation neurons were shown in the ischemic core, while in the boundary zone adjacent to the ischemic core, normal neurons and ischemic injured neurons coexisted; In the ischemic core, neuron density was not significantly decreased at3h or6h after reperfusion, but gradually decreased over12h,24h,3days and7days, which had almost completely disappeared at7days; In the boundary zone adjacent to the ischemic core, neurons was signifiantly decreased from24h to7d after reperfusion.3.1mmunohistochemistry showed the level of SelS protein was decreased in the ischemic core3d-7d after reperfusion, and in the ischemic penumbra adjacent to the ischemic core3d-7d after reperfusion,the SelS was upregulated,which results were significantly different with sham operation group.4.Western blot showed SelS expressions in ischemic core and peri-infarct were consistent with reactive astrocyte expression patterns.Conclusion:1.The technique of producing focal cerebral ischemic stroke model by middle cerebral artery occlusion with monofilament nylon suture was mature, resulting cerebral infarction zone was steady and reliable. Neurons were gradually necrotic and disappeared in the ischemic core, while astrocytes were increased in the boundary adjacent to ischemic core.2.SelS was upregulated in the ischemic penumbra adjacent to the ischemic core.3.SelS expression patterns were consistent with reactive astrocyte expression. It could be proposed that the anti-inflammatory properties displayed by SelS were constituents of astrocytes against inflammatory stimuli.4.SelS may play an important protecting role in the occurrence and development of cerebral ischemic stroke.