| ObjectiveTo establish subarachnoid hemorrhage(SAH)rat model and detect Neurological impairment score,brain edema,apoptosis of hippocampal neurons and endoplasmic reticulum stress(ER stress)factor CAAT enhancer binding protein homologous protein expression related(CHOP),hippocampus glucose regulated protein 78(GRP78)after modeling 1h,6h,12h,24h,48h,72h.And compared with the blank control group and the sham operation group.To investigate the correlation and mechanism of ER stress with early brain injury(EBI)after SAH.Material and methods1.A total of 78 adult male SD rats with body mass(300 + 50)g were selected.The rats were divided into blank control group 6,sham operation group 36 and SAH group 36 by random number table.2.The prechiasmatic cistern SAH model.The puncture point of the rat was 5mm before the coronal suture and 3mm near the middle line.In sham operation group,0.3ml was injected into normal saline,and 0.3 ml of autologous arterial blood was injected into group SAH.3.According to the behavioral score of Kaoutzanis,the neurological deficit score was evaluated:eating,exercise response and eye opening reaction were observed.The total score is 11 points,and the lowest is 3.The neurobehavioral score of each group of rats was evaluated at each time point.4.Tissue fixation and treatment:The brain was removed by decapitation,and the hippocampus and frontal brain tissue were removed to detect the expression of Caspase-12,CHOP and GRP78,and the level of neuron apoptosis.The remaining brain tissues were used for dry and wet weight determination to assess brain edema.5.TUNEL assay was used to detect the apoptotic cells of hippocampal neurons in rats.6.Western blot method was used to detect the quantitative expression of CHOP,GRP78 and Caspase-12 protein.7.The water content of brain tissue samples was calculated by dry and wet weighing method to reflect the degree of brain edema:the calculation formula was:the water content of the brain tissue =(wet weight dry weight)/wet weight*100%.8.The morphology of cells was observed by electron microscopy,and the apoptosis of neurons was observed.9.Statistical treatment:the experimental data were all two factors analysis of variance,and the LSD method was used for multiple comparison.The Pearson correlation coefficient was calculated for each index 22,and a significant test was carried out.The difference was statistically significant in P<0.05.Results1.SAH model results:1 rat died during the group SAH modeling,3 rats died due to 72h after blood injection,the mortality rate was 11.11%.2 rats died during the sham modeling,1 rat died after normal saline injection in 72h,the mortality rate was 8.33%.No deaths in the blank control group,the mortality rate was 0.Mortality rate of three groups had no significant difference(P>0.05),timely fill do 7 experiments.After dissection of brain specimens,there was no subarachnoid hemorrhage or only a small number of subarachnoid hemorrhage in blank control group and sham operation group.In group SAH,there was a lot of hematoma near the Willis ring after craniotomy,and some blood clots were found in the anterior circulation.2.The results of neurological deficit score:(1)The rats were not fully awake 1h after anaesthesia with chloral hydrate,and the neurological deficit score was not possible.After 3h,the rats regained consciousness gradually,so they began to score from 6 h.(2)In the blank control group and sham operation group,there was no significant change in scores at each time point,which was close to full score.There was no significant difference between the two groups in the comparison between the internal and the group(P>0.05).(3)SAH score 6h began to decline,24h reached valley value,and then gradually increased.The 6h,12h,24h,48h,72h SAH group was significantly lower than the blank control group and sham operation group,and the difference between the two groups was statistically significant(P<0.05).(4)The SAH group had the most obvious decrease in the time point of 24h,and there was significant difference between the group and the other time points(P<0.05).3.Brain edema test:(1)There was no significant difference in brain edema between the blank control group and the sham operation group,and there was no significant difference in the time points between the groups(P>0.05).(2)Brain edema in group SAH was aggravated at 6h,24h reached its peak,and the brain edema of 48h and 72h was still heavy.12h,24h,48h,72h SAH group compared with the blank control group and the sham operation group,there was significant difference in brain edema(P<0.05).(3)In group SAH,brain edema was the most serious at 24h time point,and there was a significant difference in comparison with 1h,6h,12h and 72h group(P<0.05),but there was no significant difference in comparison with 48h time point(P>0.05).4.Detection of neuronal apoptosis by TUNEL:(1)There was no significant difference in hippocampal neuron apoptosis in the blank control group and sham operation group,and there was no significant difference in the difference between the groups(P>0.05).(2)The neuron apoptosis in SAH group was more obvious.The apoptotic cells in 12h increased significantly,and the apoptotic cells of 24h reached the peak.The number of apoptotic cells at 48 h began to decrease gradually,and the number of 72h apoptotic cells decreased significantly.At 6h,12h,24h,48h,72h time point,there was significant difference between the SAH group and the blank control group and the sham operation group(P<0.05).However,there was no significant difference in the 1h time point compared SAH group with the blank control group and the sham operation group(P>0.05).(3)In group SAH,apoptosis was the most serious in 24h time point,and there was significant difference compared with other time points(P<0.05).5.Detection of CHOP protein expression results by Western blot:(1)The expression of CHOP protein in the blank control group and the sham operation group was weak,and there was no significant difference between the groups at different time points(P>0.05).(2)The expression of SAH protein in CHOP group increased significantly compared with the blank control group.The expression of 6h CHOP protein in group SAH began to rise and reached the peak at 24h,then gradually decreased,and 72h was still more clearly expressed.At 12h,24h,48h,72h time point,there was significant difference between the SAH group and the control group and the sham operation group(P<0.05).At 1h,6h time point,There was no significant difference(P>0.05).(3)In group SAH,the expression of CHOP protein at the time point of 24h was the most obvious,and there was significant difference compared with the other time points in the group(P<0.05).6.Detection of GRP78 protein expression results by Western blot:(1)The expression of GRP78 protein in the blank control group and the sham operation group was weak,and there was no significant difference between the groups at different time points(P>0.05).(2)The expression of GRP78 protein in group SAH began to rise at 6h and reached the peak at 24 h,then gradually decreased,and the expression of 72 h was still more obvious.At 12h,24h,48h,72h time point,there was significant difference between the SAH group and the control group and the sham operation group(P<0.05).At 1h,6h time point,there was no significant difference(P>0.05).(3)In group SAH,the expression of GRP78 protein at the time point of 24h was the most obvious,and there was significant difference compared with the other time points in the group(P<0.05).7.Detection of Caspase-12 expression results by Western blot:(1)The expression of Caspase-12 in the blank control group and the sham operation group was weak,and there was no significant difference between the groups at different time points(P>0.05).(2)The expression Caspase-12 in SAH group began to rise obviously at 6h,reached the peak at 24h,then gradually decreased,and there was still a small amount of expression in 72h.At 6h,12h,24h,48h,72h time point,there was significant difference between the SAH group and the control group and the sham operation group(P<0.05).But at 1h time point,there was no significant difference(P>0.05).(3)In group SAH,the expression of Caspase-12 protein at the time point of 24h was the most obvious,and there was significant difference compared with the other time points in the group(P<0.05).8.Observation of electron microscope:Apoptosis of hippocampal neurons in SAH group is more serious.After 6h,cell shrinkage and volume decreased.When 12h,there were more vacuoles in the cytoplasm and smaller nuclear volume.The chromatin in the nuclear membrane was concentrated and showed apoptosis.24h was the most severe,and the nucleus was broken into fragments and apoptotic bodies.In the blank control group and the sham operation group,the hippocampal neurons and organelles were in good shape.9.According to Spearman correlation analysis:The expression of CHOP,GRP78 and Caspase-12 protein in SAH group was significantly positively correlated with the severity of brain edema and neuronal apoptosis,and negatively correlated with the scores of neurological deficits(P<0.05).The 24h time point was the most obvious.ConclusionsThis study showed that:(1)The expression of CHOP,GRP78 and Caspase-12 increased significantly in 72h after SAH,indicating that ER stress did occur after SAH.(2)By analyzing the neurological deficit score,the degree of brain edema and the difference in neuronal apoptosis between the blank control group,the sham operation group and the SAH group,and the cell and organelle morphology under electron microscope.It showed that EBI did happen in the early stage of SAH.(3)It is confirmed that there is a positive correlation between the expression of CHOP,GRP78 and Caspase-12 and the severity of EBI.It is preliminarily revealed that CHOP,GRP78 and Caspase-12 expression play an important role in the pathological process of EBI after SAH.The main mechanism is ER stress overactivated after SAH,which is responsible for the apoptosis of cerebral cortex neurons through endoplasmic reticulum associated death(ERAD)pathway.(4)It will provide a theoretical basis for further study of the pathogenesis of EBI after SAH,and find targeted therapeutic strategies and potential therapeutic targets. |
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