| Objective To explore the neuroprotective effect of hypobaric hypoxia preconditioning on middle cerebral artery occlusion?MCAO?rats and the relationship between the ischemic tolerance and the expression of TLR4 and MyD88,we observed the volume of cerebral infarction.The neuroprotective effect of hypoxia preconditioning was studied from the macroscopic view,and the damage of neurons was observed by Nissl stain.The effects of hypoxia preconditioning on the injured neurons in the affected side of MCAO rats were observed microscopically at the same time.in order to evaluate the role of TLR4/MyD88 signal pathway on the ischemic tolerance induced by hyoparic hypoxia preconditioning,we tested the expression of TLR4,MyD88 and NF-?B from different degree?transcriptional and translational level?.In this way,we want to explore that whether hypoxia preconditioning change the expression of the key protein in TLR4/MyD88/NF-?B pathway in rats'brain and the relationship between the change and the protective effect.It will provide new ideas for the mechanism of brain protection in cerebral ischemia,and also will help us to seek new directions for the prevention and treatment of cerebral ischemic diseases.Methods One hundred and sixty adult male SD rats of SPF grade were randomly divided into control group?n=30?,sham operation group?n=30?,hypoxic treatment group?n=30?,MCAO group?n=35?and the hypoxia preconditioning group?n=35?,in group MCAO,the experimental rats were embolized with unilateral middle cerebral artery via left common carotid artery.The respiration,heart rate and body temperature were closely monitored during the operation.The rats were kept warm after operation and kept in another cage after operation.rats in the hypoxia preconditioning group and hypoxic treatment group were treated with hypobaric hypoxia in the hypobaric chamber for 7 days and 3 hours a day,and group of preconditioning rats were treated by unilateral middle cerebral artery embolization via left common carotid artery at the same time on 8th day.Neurologic damage in rats'brain was observed by Longa Neurologic function score test.The size of cerebral infarction and the degree of neuronal injury were observed from the perspective of Nissl staining and the size of cerebral infarction show by TTC stain.We use the immunohistochemical stain method to observe the expression of TLR4 and MyD88on brain cell,and Elisa method to determine the expression of TLR4-MyD88 and its down-stream transcription factor NF-?B.Use Westernblot method to make Semi-quantitative determination of TLR4 MyD88 protein expression in left cerebral cortex and hippocampus.Detect the mRNA expression of TLR4 and MyD88 by q-PCR.Results?1?By TTC staining,it was found that the hypobaric hypoxia stimulation at altitude of 5000m/day for one week could not induce the decrease of brain cell activity in normal rats,and the sham-operation would not cause brain cell death.After 24 hours of unilateral middle cerebral artery ischemia,a large area of infarct appeared in the blood supply area,and the activity decreased or disappeared,and there was also infarction in the affected brain tissue of rats with unilateral middle cerebral artery ischemia after hypoxia preconditioning.However,the infarct volume was significantly decreased in the middle cerebral artery ischemia group than that in the unilateral middle cerebral artery ischemia group?t=2.42,P<0.05?.?2?The neurological function score of group HM rats was lower than that in group M?t=2.283 P<0.05?.?3?It was found that the neurons in cortex and hippocampus were well formed,no edema,no rupture,no dissolution or disappearance of Nissl bodies in the cortex and hippocampal CA1 area of rats in group M,and the loss of neurons in the affected cortex and hippocampal CA1 area was serious in group M under 40×times microscope.The damage degree of neurons in the CA1 area of the affected cortex and hippocampus in HM group was less than that in M group.?4?Immunohistochemical staining showed that TLR4 and MyD88 were mainly expressed in neurons,but only in sham operation group?S?treatment did not affect the expression of TLR4 MyD88 in rat cerebral cortex and hippocampus.Hypoxia stimulation alone?H group?had no significant effect on the expression of TLR4-MyD88 in hippocampal CA1 cells,but the number of TLR4+cells and MyD88+cells was up-regulated and the cells were deeply stained in the damaged cortex of M group,and the cell overlap in hippocampus could not be counted accurately.In HM group,TLR4 and MyD88 in cortex and hippocampus were increased.The expression of seed protein was significantly down-regulated than that of M group.?5?Enzyme linked immunosorbent assay?Elisa?was used to detect the difference of TLR4 MyD88 and its downstream transcription factor?B protein in the dominated region of left middle cerebral artery?MCA?of rats in each group.It was found that the difference of TLR4expression was statistically significant?P<0.05?and that of MyD88 was significantly different?P<0.05?.?6?The expression of TLR4,MyD88 protein in damaged cortex and hippocampal CA1 was detected by semi-quantitative analysis by Western blotting.It was found that there were significant differences in the expression of MyD88protein in cortex of different groups?P<0.01,P<0.01?.The expression of TLR4 and MyD88 in hippocampus was significantly different?P<0.01,P<0.01?.?7?The expression of MyD88 in the damaged cortex of each group was confirmed by fluorescence quantitative PCR method from the gene level.There was a significant difference in the expression of TLR4 and MyD88 between different groups?P<0.001,P<0.001?.Conclusions?1?The hypoxic preconditioning can reduce the injury of neurons and the degree of cerebral edema in the rats with ischemic brain injury,reduce the degree of nerve function injury,thereby improving the ischemic tolerance of the brain tissue and playing the role of resisting damage;?2?At an altitude of5000m,intermittent hypobaric hypoxia stimulation for 3 hours per day could not cause obvious brain tissue damage in rats.?3?Both TLR4 and MyD88 were expressed in nerve cells,and the expression level was the same in each group.Both ischemic and hypoxic stimuli could up-regulate the expression of TLR4 and MyD88,and the expression of TLR4/MyD88 cells was down-regulated in ischemic tissues after preconditioning.?4?Hypobaric hypoxia alone could induce the up-regulation of the expression of MyD88 and TLR4 molecules in cerebral cortex.The changes of MyD88 and TLR4 molecules in each group were in the same direction.Both hypoxia and ischemic stimulation could up-regulate the expression of these three molecules.After preconditioning,the expression was down-regulated,but still higher than that in normal control group.?5?The changes of TLR4 and MyD88 protein levels were basically consistent with the changes of gene levels. |
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