The Expression Of P-p38 MAPK In The Hippocampus During The Induction Of Brain Ischemic Tolerance In Rats

Objective: Mitogen-activated protein kinase (MAPK) is one of the highly conservative protein kinases. It is a kind of intra-cellular signal transduction enzyme, which intimately related with cell multiplication, differentiation and apoptosis. It is a critical pathway that connected with cytoplasm and cell nucleus. Many studies have found that MAPKs signaling pathway maybe had an important role in brain ischemia and the inducing of brain ischemic tolerance. p38 MAPK is one subtype of MAPK family, which was found in 1993. It is composed of 360 amino acids and its molecular weight is 38KDa. Sublethal brain ischemia (2min global brain ischemia) enhanced ischemic tolerance of hippocampal pyramidal neurons in Mongolian gerbil through activated p38 MAPK, which made the pyramidal neurons survive severe cerebral ischemia (5min global brain ischemia) that usually led to severe injury. SB203580, a specific inhibitor of p38 MAPK, can block brain ischemic tolerance induced by 2 min global cerebral ischemia in dose-dependent manner. But it is still far from clear that the role and mechanism of p38 MAPK in the inducing of ischemia tolerance. Therefore, the present experiment is to investigate the expression of p38 MAPK in the hippocampus under the inducing of brain ischemic tolerance by cerebral ischemic preconditioning. The aim is to provide new clues and foundation for clinical cerebrovascular disease prevention and cure.Methods: One hundred and sixty adult male Wistar rats were randomly divided into 4 groups. All rats were permanently occluded bilateral vertebral arteries and recovered for 2 days before brain ischemia or sham operation, the details are as follows:①sham group (n=40): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow;②CIP group (n=40) the BCCA were clamped for 3 min then reperfused with the blood flow;③Ischemic insult (II) group (n=40): the BCCA were clamped for 8 min then reperfused with the blood flow (n=5);④CIP+II group (n=40): a CIP for 3 min was preformed then reperfused, 2 day-interval, a lethal ischemic insult for 8 min was given then reperfused with the blood flow. Each group was further divided into eight time points according to the different living period after the sham operation or the last operations: immediate, 15min, 30min, 3h, 1d, 2d, 4d and 7d (n=5 in each time point). At the determined time point, the rats were sacrificed by decapitation. Neuropathological evaluation was performed by thionine staining. The expression of p-p38 MAPK was observed by immunohistochemistry.Neuropathological evaluation of the CA1 hippocampus was examined under light microscope to determine delayed neuronal death (DND) by neuronal density (ND) and histological grade (HG) of the CA1 hippocampus. The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The average of the number in 3 areas of the CA1 hippocampus was calculated as value of ND. HG was divided into the following 4 grades: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, death of many neurons; gradeⅢ, death of almost complete neurons. The average HG of the bilateral hippocampus was counted as statistical data.Results:1 Neuropathological evaluationIn the hippocampal CA1 subfield of the sham rats, pyramidal neurons were arranged in order with 2~3 cell layers; the outline of the neurons was intact, the nucleus was full, and the nucleolus was clear. the HG was 0～Ⅰ. At each time points of CIP group, no significant change was observed in the hippocampal CA1 subfield by thionin staining. At 7d of the sham and CIP groups the values of ND were 204±7.21 and 194.67±8.08, respectively. Obvious DND in the hippocampal CA1 subfield was observed in the II group from the fourth day after 8min ischemia. At 7d of the II group HG was gradeⅡ～Ⅲ, which was much higher than that of the sham group, the value of ND was 17.33±11.37, which was much lower than that of the sham group. At each time point of CIP+II, there is no significant DND in hippocampal CA1 subfield. At 7d time point of the CIP+II group, HG (grade 0～Ⅰ) was significantly lower than that of the II group (P<0.01), and the value of ND, 190.67±4.16, was much higher than that of the II group (P<0.01). All the above indicates that the CIP protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult. In the hippocampal CA3 subfield, no obvious damage was observed at all time points of each group.2 The expression of p-p38 MAPK in the hippocampal CA1 subfield of rats2.1 The comparison of p-p38 MAPK between the different time points at the same groupIn the sham group, the immunoreactive cells in the hippocampal CA1 subfield were integrityand stained as light brown yellow. Neither total area among different time points nor the average optical density was significant different.In the CIP group, compared with the immediate time point, the total area of p-p38 MAPK immunoreactive particles was significantly increased from 15min to 4d, and the peak value was at 1d. The average optical density was significantly increased from 15min to 2d, and the peak value was at 3h that appeared brown (p <0.05).In the II group, the expression of immunoreactive particles was not obvious at immediate time point. Compared with the immediate time point, both the total area and the average optical density were significantly increased from 15min to 2d (p <0.05). The peak value of the total area was 1d, and the peak value of the average optical density was 3h. The staining reduced obviously at 4d and 7d. Some glial cells were p-p38 MAPK immunoreactive positive. The staining of the glial cells was deeper then that of the hippocampal neurons. Compared with the immediate time point, no obvious change was observed in the total area and the average optical density.In the CIP+II group, there were a certain amount of the immunoreactive particles observed at the immediate time point. The expression decreased significantly at 15min. compared with the immediate time point, the total area decreased significantly (p<0.05), but the average optical density had no obvious change. At 30min time point, the immunoreactive cells became reverted to the level of the immediate time point. The immunostaining of the nucleus became deeper from 3h to 2d, and the peak value was at 2d. From 4d time point, the immunostaining decreased obviously to the level of the immediate time point.2.2 The comparison of p-p38 MAPK between the different groups at the same timeAt the immediate time point, neither total area nor the average optical density was significant difference among sham group, CIP group and II group. The total area of CIP+II group was much higher than that of II group. However, the average optical density had no significant difference between CIP+II group and II group.At 15min time point, compared with sham group, the total area and average optical density were increased significantly in both the CIP group and II group (p<0.05). The total area of II group was most outstanding. Compared with the II group, the total area and average optical density decreased significantly in the CIP+II group.At 30min time point, compared with sham group, the total area and average optical density in CIP group and II group were increased significantly (p<0.05). The total area and average optical density in II group was much higher than that of the CIP group. Compared with II group, the average optical density of CIP+II group decreased significantly.From 3h to 2d, the total area and average optical density increased significantly in both CIP group and II group compared with the same time point of sham group. And the II group was more outstanding. Compared with the same time point of II group, the total area of CIP+II group decreased significantly at 1d and 2d (p<0.05).At 4d time point, compared with sham group, the total area and average optical density of CIP group increased significantly (p<0.05); the total area of II group was decreased significantly because of delayed neuronal death, but average optical density of II group increased significantly (p<0.05). Compared with II group, the total area of CIP+II group increased significantly.At 7d time point, compared with sham group, the total area of CIP group increased significantly (p<0.05), no obvious difference of total area of II group was observed. Compared with II group, the total area of CIP+II group increased significantly.3 The expression of p-p38 MAPK in the hippocampal CA3 subfieldAt different time points of the sham group, the immunoreactive staining total area and average optical density of p-p38 MAPK had no significant difference in the hippocampal CA3 subfield.Compared with the immediate time point of CIP group, both the total area and the average optical density increased significantly at 1d time point of CIP group (p<0.05).Compared with the immediate time point of II group, the total area of p-p38 MAPK increased significantly from 30min to 2d (p<0.05), and the peak value was at 2d.At immediate time point of CIP+II group, the immunoreactive staining of nucleus were uniform, light brown yellow. Compared with the immediate time point, the staining of nucleus became deeper at 15min (p<0.05), but the total area decreased significantly (p<0.05). From 30min to 1d, the total area of immunoreactive cells was significant lower than that of the immediate time point. At 2d time point, the total area of immunoreactive cells reverted to the level of immediate time point, and the average optical density increased significantly (p<0.05). At 4d and 7d time points, immunoreactive cells decreased significantly and stained lightly, the total area and average optical density decreased significantly compared with the immediate time point (p<0.05).Conclusion:1 Lethal ischemic insult induces severe DND of the pyramidal neurons in the hippocampal CA1 subfield. The CIP for 3 min 2 days before the lethal ischemic insult protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult.2 CIP moderately activated p-p38 MAPK expression in the pyramidal neurons of hippocampal CA1 subfield. This change maybe protected hippocampal pyramidal neurons.3 Ischemic insult intensively activated p-p38 MAPK expression in pyramidal neurons of hippocampal CA1 subfield. Over-expression of p-p38 MAPK involved in the mechanisms of cerebral ischemic injury.4 The CIP for 3 min 2 days before the lethal ischemic insult prevented the over-expression of p-p38 MAPK induced by ischemic insult. That protected pyramidal neurons surviving ischemic injury.5 The expression of p-p38 MAPK in the hippocampal CA3 subfield was mild than that of CA1 subfield. The time of p-p38 MAPK activation in the hippocampal CA3 subfield was later than that of CA1 subfield. All the above showed that the response to ischemia of hippocampal CA1 subfield was intensive than that of CA3 subfield.