| ObjectiveGap junction between adjacent cells was the most direct and fastest communication between adjacent cells,which plays an important role to maintain the synergistic response between vascular cells.Connexins(Cxs),a large family of homologous membrane proteins,of which Cx 40,Cx37,Cx43,Cx45 are expressed in vascular cells.Cx43 and Cx45 were major expressed in Smooth muscle cells,Cx40 and Cx37 in Endothelial cells.In previous study,cerebral vasospasm(CVS)was associated with gap junction.Connexins was changed in expression and distribution after cerebral vasospasm.However,the mechanism and therapy of CVS remain unknown.Nitric Oxide(NO)is a vascular relaxation of the most important factor.Reduction of NO was induced CVS after subarachnoid hemorrhage(SAH).On the basis of this study in earlier research,the research of mechanism of NO in CVS and gap junction,was of theories foundation to cure CVS after SAH.Methods1.The SAH Model was builded by Cisterna Magna Double-Injection:Sprague-Dawley(SD)rats were euthanized by 10% chloral hydrate(0.3ml/100g).Then,SD rats were injected 0.3ml autologous arterial blood,An same surgical procedure is repeated 24 h after the initial SAH for induction of the second SAH.2.Identification of SAH model: the test were divided into SAH group and sham group.In the seventh day,FITC-Dextran was injected into caudal vein of rat.The diameter of the branches of middle cerebral artery were measured by living fluorescence microscope.3.Expression of connexin 40: All the groups of rats were executed and the brain was removed.The basilar artery(BA)was separate under under the microscope.The BAs were studied under control conditions with one or a combination of the following compounds: 100 ?M DETA-NO,100 ?M 8-Br-c GMP,100 ?M(D)-DT-2,100 ?M 40Gap27.The expression of connexin 40 was detected by western blotting.4.Basilar artery tone in vitro: Take out basilar artery rings of SD rats,then treated with the PSS solution that contains prostacyclin inhibitor indomethacin(Indo) and EDHF inhibitor apamin and charybdotoxin and saturated by mixed gas of 95%O2 and 5% CO2,pretreated with 60mmol/L Kcl KPSS making the artery rings contract,dealt with different concentrations of ACh(10-10-10-4mol/L),he NO-mediated relaxation was studied.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.Results:1.Cisterna Magna Double-Injection was able to build the model of SAH.A large number of contractive vessels were detected in SAH group by living fluorescence microscope,major was microvascular.(p<0.05 vs.Sham).Blood clot was saw in Willis circle,cisterna manga and ventral of brain stem.2.Expression of Cx40: In SAH group,The total protein level of Cx40 was no significant difference compared with that in normal control group(p>0.05);the total protein level of Cx40 treated with DETA-NO was significantly increased than that in SAH group;the total protein level of Cx40 was significantly increased after treat with PKG activator 8-Br-c GMP,mimic DETA-NO.PKG blocker(D)DT-2 and selective Cx40 blocker 40Gap27 could inhibit the increase of the total protein level of Cx40 induced by DETA-NO.3.Vascular tone experiment shows: In SAH group,NO-mediated dilatation was significantly decreased compared with that in normal control group.In SAH group treated with DETA-NO,NO-mediated dilatation was significantly increased compared with that in SAH group.NO-mediated dilatation was significantly increased after treat with PKG activator 8-Br-c GMP,mimic DETA-NO.PKG blocker(D)DT-2 and selective Cx40 blocker 40Gap27 could inhibit the increase of NO-mediated dilatation induced by DETA-NO.Conclusions1.NO could upregulation cerebrovascular expression of Cx40 to relieve CVS,changed ratio between Cx43 and Cx40.2.NO relieve CVS by NO-c GMP signaling pathways.NO may is mediated dilatation in part via connexin 40 dependent... |
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