Research On The Pathogenesis Of Cerebral Vasospasm Related To EDHF

VObjectiveEndothelium derived relaxing factor mainly contais three factors: NO, PGI2 and EDHF. Along with the vascular diameter decreasing,EDHF becomes more important in regulating vasodilation. EDHF regulates vascular diastole depending on the normal function of GJ channel,performing a complete EDHF reaction depends on intercellular communication function that is mediated by the normal gap junction between cells,Cx40 and its correlated GJ channels have crucial role on EDHF relaxing blood vessels.When Cx40 expression is silenced, ACh makes EDHF regulating vasodilation weak.Previous studies have shown that:first,cerebral vasospasm happens in SAH;Second, Cx43 expression is obviusly higher in the SAH than the normal,but Cx40 expression isn’t;third,downregulate the expression of Cx43 in SAH by exploiting Cx43 RNA interfering adenovirus and cerebral vasospasm will be improved,gap junction blocker carbenoxolone(CBX) can also relieve cerebral vasospasm in SAH. There are no reports about EDHF involving in cerebral vasospasm in SAH,we speculate that the reason of EDHF regulating cerebral vascular diastolic dysfunction is Cx remodeling,that is to say,Cx43 and Cx40 is imbalanced,changing the GJ channels′ function,and impair relaxing cerebral vasodilatation function of EDHF,then cerebral vasospasm occurs.There are two purposes of this experiment :first,to test cerebral vessels’ reaction on EDHF in the normal and SAH;second,after transducing Cx43 si RNA adenovirus which can reduce expression of Cx43 into SAH and treating with CBX which can block abnormal GJ channel in SAH model,then we test blood vessels’ reaction on EDHF in cerebral blood vessel. The study further confirms EDHF ’s mechanism in regulating cerebral vasospasm in SAH, looking for a new and effective therapy to improve the clinical prognosis of SAH. Methods1.Establishment and identification of the subarachnoid hemorrhage(SAH) model:Take about 0.3ml caudal vein blood of the general healthy SD rats that received intraperitoneal anesthesia, then the blood is slowly injected into the cisterna magna under microscope.The model was established by reinjecting blood into the cisterna magna after 24 h. SAH model was identified by performing HE staining of basilar artery in nomal group,SAH model 3d group and SAH model 7d group, respectively.2.Test the basilar artery’s reaction on EDHF and the expression of Cx43 of the normal group and SAH model group:1)Take out basilar artery rings of SD rats,then treated with the PSS solution that contains prostacyclin inhibitor indomethacin(Indo) and nitric oxide synthase inhibitor N- nitro-L- nitro arginine(L-NAME) and saturated by mixed gas of 95% O2 and 5% CO2 about 30 minutes,;next,treated with 60mmol/L Kcl KPSS making the artery rings contract;finally dealt with different concentrations of ACh(10-9-10-5mol/L), the basilar artery rings will react on EDHF.Record the tension of artery rings in different concentration of ACh by adopting the powerlab physiological information acquisition and processing system and calculate the diastolic rate; 2)The normal SD rats and SAH rats are fixed by paraformaldehyde, basilar artery rings are detached from the fixed and fixed in vitro and embedded by paraffin, then evaluate the Cx43 expression in the basilar artery rings by immunochemistry.3.Observe the basilar aretery rings’ reaction on EDHF and the Cx43 expression in the SAH models which have been processed by Cx43 si RNA adenoviruses:1)To test the interfering effect of Cx43 si RNA adenovirus:Cx43 si RNA adenoviruses and empty adenoviruses are injected into the cisterna magna of normal healthy SD rats,the Cx43 si RNA adenoviruses group and the empty adenoviruses group are killed after 0(normal group), 3, 7, 14 days,respectively,we take out the basilar artery rings, and detect the Cx43 expression to calculate the silencing effects of Cx43 si RNA adenoviruses by Western blotting.2)The Cx43 si RNA adenoviruses and the empty adenoviruses are injected into the cisterna magna of normal rats.A week later,SAH models are established.At last,we test the basilar artery rings’ reaction on EDHF in the two SAH models and watch the Cx43 protein expression in SAH model that have been pre-treated by Cx43 si RNA adenoviruses.4. Detect the basilar artery rings’ s reaction on EDHF in SAH models that are pre-treated by carbenoxolone:take ot the basiler artery rings in SAH modes,then treated with carbenoxolone 30 minutes,then we confirm the basilar artery rings’ s reaction on EDHF. Results1,The basilar arteries are luminal narrowing,endothelial layers thickening and shrinking significantly in SAH models by HEstaining,suggesting that the SAH models are successfully established.2.The Western blotting shows: 1) Cx43 proeins expression in SAH models that have been injected with si RNA adenovirus 0 days(normal),3days,7days,14 days is 0.796±0.02, 0.604±0.03,0.369±0.04, 0.394±0.01,respectively. The Cx43 expression decreases in SAH models pretreated with Cx43 si RNA adenoviruses 3 days(P<0.05), there is statistical significance.The Cx43 expression decreases in SAH models pretreated with Cx43 si RNA adenoviruses 7 days and 14 days reduces significantly(P<0.01). 2) Cx43 proeins expression in SAH models that have been pretreated with empty adenoviruses 0 days,3days,7days,14 days is 0.68±0.04, 0.67±0.01, 0.69±0.03, 0.70±0.02,,respectively. The Cx43 expression doesn’t decrease in SAH models pretreated with empty adenoviruses 0days,3days,7days,14days(P>0.05).3,Immunohistochemical results show:Cx43 proteins are more positive in endothelial cells, smooth muscle cells, adventitial cells that are dyed brown in SAH;instead,in SAH models pre-treated with Cx43 si RNA adenoviruses.4, Vascular tension experiment shows: the maximum diastolic rate aroused by the basilar artery rings’ reaction on EDHF in the normal SD rats that have been treated with Ach is 41.084±5.459%;7.096±5.573%,5.142±2.579% in SAH 3days and 7days respetively,there is obviously difference between the two groups and the normal group(P<0.05);7.722±4.516%, 8.714±2.641% in SAH 3days and 7days that have been pre-treated with empty adenoviruses,respectively,there’s no difference between the two group and SAH models(P>0.05); 51.268±14.072%, 50.284±5.57% in SAH 3days and 7days that have been pre-treated with Cx43 si RNA adenoviruses, respectively,there’s apparently difference between the two group and SAH models(P<0.05);41.924±6.246%、42.50±13.693% in SAH 3days and 7days that have been pre-treated with CBX,respectively,there’s apparently difference between the two group and SAH models(P<0.05). Conclusions1,The occurrence and development of cerebral vasospasm are related to EDHF impairment in regulating diastolic function of blood vessels.2, In SAH,EDHF impairment in regulating diastolic function of blood vessels is related to the increase of Cx43,resulting in imbalance of Cx43 protein and Cx40 protein,remodeling of GJ and dysfuntion of GJ channels.3, Carbenoxolone inhibited abnormal GJ channel of SAH,EDHF regulates diastolic function of blood vessels will restore,cerebral vasospasm will relieve4,Expression of Cx43 protein is downregulated by transducting Cx43 si RNA adenoviruses into the SAH to rectify the imbalance of Cx43 and Cx40 and improve the function of GJ,the function that EDHF regulates diastolic function of blood vessels will restore.Thereby cerebral vasospasm will relieve.