The Expression Of P-p38MAPK In Astrocyte During The Induction Of Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning

Objective: Previous studies have proved that an advance, short, slightbrain ischemia which does not cause neuronal injury can induce neurons tosurvive severe brain ischemia that is usually lethal to the neurons. Thephenomenon is called brain ischemic tolerance, and the advance, short, slightbrain ischemia is named as cerebral ischemic preconditioning (CIP). Thephenomenon was first put forward by Kitagawa in1990. Many studies haveproved it from then on, however its mechanism has not been elucidated.Mitogen-activated protein kinase family (MAPKs) is a kind of proteinkinase, which widely exists in cells and contains serine/threonine residues.p38MAPK is a member of the family. Recently, many studies have confirmedthat MAPKs signaling pathway is closely related to cerebral ischemia injury,and maybe play an important role in the induction of brain ischemic tolerance.Our previous studies have demonstrated that CIP up-regulated the expressionof p-p38MAPK. Based on the above, the present study further observes thecell type expressed p-p38MAPK during the induction of brain ischemictolerance induced by CIP.Method: One hundred and eight adult male Wistar rats (280-320g) wereused. At first, bilateral vertebral arteries of each rat were occluded, after48hinterval, the rats were randomly divided into the following four groups:1Sham group: exposure bilateral common carotid artery withoutocclusion the blood flow.2CIP group: occlusion of bilateral common carotid arteries for3min,and then reperfusion.3Ischemic insult (II) group: occlusion of bilateral common carotidarteries for8min, and then reperfusion. 4CIP+II group: occlusion bilateral carotid arteries for3min as cerebralischemic preconditioning, reperfusion for two days, occlusion of bilateralcommon carotid arteries for8min as ischemic insult, and then reperfusionagain.Each group was further divided into9time points: immediate time point(0min),30min,15min,3h,6h,1d,2d,4d and7d after the sham operationor the last operations (n=3in each time point). At the determined time point,the rats were sacrificed by decapitation. Thionine staining was used forneuronal evaluation. Fluorescent immunohistochemical staining was used forthe expression of p-p38MAPK and glial fibrillary acidic protein (GFAP),which is the specific marker for astrocytes. The expression of p-p38MAPK inastrocytes in CA1hippocampus was observed during the induction of brainischemic tolerance induced by CIP.At least three sections from each block were studies, and at least threefields for each section were observed. The total number of cells for both p-p38MAPK expression and GFAP expression was counted by investigator withoutany knowledge of the group.Results:1Neuropathological evaluationThe neuropathological evaluation was studied by thionine staining. In thesham rats, pyramidal neurons in the CA1hippocampus were arranged in orderwith2to3cell layers, the outline of the neurons was intact, and nucleus wasfull. In the CA1hippocampus of CIP group, no significant neuronal damagewas observed at each time point. In ischemic insult group, a few injuryneurons appeared shrinking and hyperchromatic cell body were observed at2d time point; almost all neurons appeared delayed neuronal death at4d timepoint, which showed irregular shape, cell body shrinkage and nuclear pyknosis;almost all neurons died or missed at7d time point. ND of both4d time pointand7d time point were significantly lower than that of sham group (P <0.05).At each time point of CIP+II group, neurons in CA1hippocampus werearranged in orderliness, the outline of the neurons was intact and nucleus was full, the ND of7d time point was182.24±10.76, which was significantlyhigher than that of the same time point of II group (P <0.05).2The expression and modification of p-p38MAPK protein in astrocytesin CA1hippocampusFluorescence double labeling showed that some astrocytes expressedp-p38MAPK at0min time point of each group. Compared with the shamgroup, no significant difference in the expression of p-p38MAPK protein inastrocytes was observed in either CIP group or II group (p>0.05). Comparedwith II group, the number of astrocytes expressed p-p38MAPK in CA1hippocampus in CIP+II group increased significantly (p<0.05), and theamount of p-p38MAPK in each astrocyte is more. Meanwhile, astrocytesappeared more and longer processes and surrounding neurons in CA1hippocampus, which were in activated state.At30min time point, compared with sham group, the number ofastrocytes expressed p-p38MAPK in CA1hippocampus in CIP group wasunchanged. However, the more and longer processes of astrocytes wereobserved. In ischemic insult group, the number of astrocytes expressed p-p38MAPK in CA1hippocampus was decreased compared with sham group, andless and shorter processes of astrocytes were observed. Compared withischemic insult group, the number of astrocytes expressed p-p38MAPK inCIP+II group increased significantly (p<0.05). Astrocytes were in activatedstate with more and longer processes, and tightly surrounded pyramidalneurons in CA1hippocampus.At6h time point, compared with sham group, the number of astrocytesexpressed p-p38MAPK in CIP group increased significantly (p<0.05); nosignificant difference in the expression of p-p38MAPK protein in astrocyteswas observed in II group (p>0.05). Compared with ischemic insult group,the number of astrocytes expressed p-p38MAPK in CIP+II group increasedsignificantly (p<0.05), and the amount of p-p38MAPK in each astrocyte ismore.At2d time point, compared with sham group, the number of astrocytes expressed p-p38MAPK in CIP group increased significantly (p<0.05), and theamount of p-p38MAPK in each astrocyte is more; no significant difference inthe expression of p-p38MAPK protein in astrocytes was observed in II group(p>0.05). Compared with ischemic insult group, the number of astrocytesexpressed p-p38MAPK in CIP+II group increased significantly (p<0.05), butthe amount of p-p38MAPK in each astrocyte did not change significantly.At7d time point, compared with sham group, the number of astrocytesexpressed p-p38MAPK in CIP group increased significantly (p<0.05), butastrocytes processes short; no significant difference in the expression of p-p38MAPK protein in astrocytes was observed in II group (p>0.05), but astrocytemorphology is not clear. Compared with ischemic insult group, the number ofastrocytes expressed p-p38MAPK in CIP+II group increased significantly(p<0.05).Conclusion: Cerebral ischemic preconditioning induced theup-regulation of p-p38MAPK protein expressed in astrocytes.