The Effect Of Connexin, Arteriole Membrane Potential And Endoplasmic Reticulum Stress-induced Apoptosis Pathway On Delayed Cerebral Ischemia After Subarachnoid Hemorrhage

Prat1Expression of connexin and asymmetric dimethylarginine and the effect of their alteration on cerebral vasospasm after subarachnoid hemorrhageObjective:To investigate alteration of connexins in basilar artery, asymmetric dimethylarginine in cerebrospinal fluid and basilar artery diameter in subarachnoid hemorrhage(SAH) model rats, and study the effect of18β-glycyrrhetinic acid (18β-GA), an inhibitor of gap junction, on ADMA, connexins and CVS after SAH, and investigate the mechanism of connexins and ADMA in cerebral vasospasm.Methods:Sprague-Dawley rats (n=120) were divided into the control group, sham, SAH, and SAH+18β-GA groups. In the SAH group, blood was doubly injected into the prechiasmatic cistern of the rats. The neurological score, basilar artery diameter, connexin protein contents (Cx40, Cx43and Cx45), and ADMA were measured using Kaoutzanis scoring system, pressure myograph, Western blot and enzyme linked immunosorbent assay kit, respectively,1,3,5,7, and14days after SAH. Compared with the control group and the sham group, the various index of the phase change in the SAH group were summarized. After180-GA (10mg/kg) was intraperitoneally injected, change of above indicators were observed. Correlation between ADMA and connexins, and correlation between basilar artery diameter and connexins were evaluated using Pearson’s correlation analysis.Results:The neurological score significantly decreased3,5,7, and14on days after SAH. The basilar artery diameter significantly decreased on day1,3,5,7, and14, and the diameter was smallest on day7. Cx37was not detected in basilar artery at any time ponit. The Cx40expression in the SAH group did not significantly change on day1, substantially decreased compared with that of the other groups on days3,5,7and14, and was lowest on day7. The Cx43and Cx45expression significantly increased on all days, peaked on day7, and decreased on day14. The ADMA levels significantly increased at the five timepoints, and peaked on day5. Change of the above indicators in the SAH group were inhibited after injection18β-GA. Significant positive correlation was found between the basilar artery diameter and Cx40, and negative correlation was found between the basilar artery diameter and Cx43or Cx45. Significant correlation was found between ADMA and Cx43.Conclusion:Cx40, Cx43, Cx45and ADMA involve CVS after SAH, and18β-GA could alleviate CVS by inhibiting ADMA and affecting connexins. ADMA is probably involved in the pathophysiological events of CVS after SAH by affecting Cx43. Prat2Research of phase change of membrance potential in arteriole after subarachnoid hemorrhagObjective:To investigate alteration of membrance potential and membrane input conductance in arteriole (basilar artery branch) in SAH model rats, and study electrophysiological characteristics of arteriole for the research of microcirculation disorder and microthrombus after SAH.Methods:Sprague-Dawley rats (n=120) were divided into the control group, sham, SAH, and SAH+18P-GA groups. In the SAH group, blood was doubly injected into the prechiasmatic cistern of the rats. The basilar artery diameter, membrance potential and membrane input conductance in arteriole were measured using pressure myograph and whole cell patch clamp technique, respectively,1,3,5,7, and14days after SAH. Compared with the control group and the sham group, the various index of the phase change in the SAH group were summarized. After18β-GA (10mg/kg) was intraperitoneally injected, change of above indicators were observed. Correlation between basilar artery diameter and membrance potential in arteriole was evaluated using Pearson’s correlation analysis.Results:The basilar artery diameter significantly decreased on day1,3,5,7, and14, and the diameter was smallest on day7.The membrane potential in arteriole expression in the SAH group did not significantly change on day1, substantially increased compared with that of the other groups on days3,5,and7, and peaked on day7, and go back to normal on day14. The membrane input conductance in arteriole significantly increased at the five time-points, and peaked on day7, and decreased on day14. Change of the above indicators in the SAH group were inhibited in the SAH+18β-GA group. The negative correlation was found between the basilar artery diameter and the membrane potential in arteriole.Conclusion:Like the arteries, vasospasm of arterioles also occur after SAH, and vasospasm of arterioles may be correlated with enhancement of cell membrance permeability. Artery spasm may lead to the contraction of arteriole.18β-GA could alleviate alteration of electrophysiological characteristics of arteriole, which may provide evidence for clinical treatment of delayed cerebral ischemia. Prat3The role of endoplasmic reticulum stress-induced apoptosis pathway in neuronal apoptosis after subarachnoid hemorrhageObjective:To investigate alteration of GRP78and CHOP protein expression, caspase-12mRNA and neuron apoptosis in the hippocampus tissue, and confirm endoplasmic reticulum stress-induced apoptosis pathway play a role in neuron apoptosis, and investigate the role of GRP78and CHOP pathway in neuron apoptosis mechanism after SAH.Methods:Sprague-Dawley rats (n=72) were divided into the control group, sham, and SAH groups. In the SAH group, blood was injected into the prechiasmatic cistern of the rats. GRP78and CHOP protein contents, caspase-12mRNA, apoptosis and ultrastructure neuron cell in CA1region were measured using Western blot, Real Time-PCR, terminal deoxynucleotidy1transferase-mediated dUTP nick-end labeling staining (TUNEL) and electron microscopes, respectively,at1,6,24,48and72h after SAH. Pearson correlation coefficients were used to examine correlation.Results:The GRP78expression in the SAH group significantly increased at1h, substantially increased compared with that of the other groups at6,24,48and72h, and was highest at24h. The CHOP expression significantly increased at all time-points, peaked at24h. The caspase-12mRNA levels significantly increased at6,24and48h, and peaked at24h. In the SAH group, the metachromatic mass increased in the nucleus at1h and significantly increased at3h; moreover, disproportion of cell contents and edema was observed at6h; apoptosis change was observed at24h, including metachromatic mass increase, nuclear shrinkage, and denticulations in karyotheca; neurons were noticeably edematous, and cytoplasmic rarefaction was found at48h; significant expansion of ER was observed at72h. Significant positive correlation was found between the number of apoptotic neurons and GRP78, and between the number of apoptotic neurons and CHOP, and between the number of apoptotic neurons and caspase-12mRNA.Conclusion:Unfolded protein response and endoplasmic reticulum stress are found in neurons after SAH. Subsequently, endoplasmic reticulum stress-induced apoptosis pathway is activated and cause neurons apoptosis. In endoplasmic reticulum stress-induced apoptosis pathway, CHOP signaling and caspase signaling have demonstr-ated a direct involvement of neurons apoptosis after SAH.