Study Of The Role Of Gap Junctions In The Pathological Mechanism Of Cerebral Vasospasm

Objective:To Study the relationship between the change of gap junction (GJ) channels in rats basilararteries and cerebral vasospasm (CVS) at different time points after SAH, the electrophysiologicalchange of gap junction channels in basilar arteries (BAs) were observed and tested the relationshipbetween the changes of gap junction protein (connexins Cxs) and CVS, and observed the effect ofgap junction inhibitor18-β blockers glycyrrhetinic acid on the regulation of the channel. Toresearch deeply the mechanism and provide new drug targets and treatment methods for theclinical prevention and treatment of the CVS after SAH.Methods:The experimental group: Totally120adult Sprague Dawley rats were divided into4groups byrandom number table method.: control group (n=30) and sham group (n=30), surgical group (n=30), treatment group (n=30), each group was divided into five subgroups by random numbertable method, namely, on day1,3,5,7,14(n=6).The model establishment and drug treatment: Isform male Sprague Dawley rats were used toestablish SAH model by secondary injection of blood crossing front pool method. The treatmentgroup was established by intraperitoneal injection of gap junction blockers18-β glycyrrhetinicacid (10mg/kg) intervention everyday day, after1day，second operation, and continue injected18-β glycyrrhetinic acid (10mg/kg) until the rats to death.Neurobehavioral scores: Kaoutzanis M neurobehavioral evaluation method was used for eachgroup respectively on day1,3,5,7,14according to movement, open eyes and diet.neurobehavioral scores:3points was the worst,11points the normal, ranging from3to11points.The model identification: The brains were taken out after anesthesia and soaked them in the10%formalin solution for24hours, and HE staining for model identification. Subarachnoidhemorrhage blood and vessels morphological changes were observed under light microscopy.Cerebral vascular diameter measurement: Each group model rats were stripped2-3mm basilararteries after anesthesia then put them into perfusion fixed groove. Intravascular pressure andtemperature was stabilized60mmHg and37℃respectively by Myoview software, then measuredbasal artery diameter.Cerebrovascular Cxs expression detection: The rat brains put in0.9%saline after anesthesia,stripped cerebrovascular under dissecting microscope and put them into liquid nitrogen, extractedtotal protein, and detected the expression of Cx37, Cx40，Cx43and Cx45by Western blot.Observe GJ conductivity changes: Each group model rats were stripped diameter40-80um and0.4mm long cerebrovascular at different times after anesthesia, digested adventitia and exposedvascular smooth muscle. Gap junction electrophysiological changes were recorded by whole cellpatch clamp technique. Results:SAH SD rats model establishment and identification: Subarachnoid hemorrhage was observedobviously in HE staining under microscope. The diameter of basal arteries decrease and VSMCmorphological disorder after SAH, however, they have no much more change after intraperitonealinjection18β glycyrrhetinic acid.The change of neurobehavioral score: Neurobehavioral score began to decline on the third day,fell to the lowest on the7thday, and recovered on the14thday after SAH, however, they improvedsignificantly after intraperitoneal injection18β glycyrrhetinic acid.The results of basal arteries diameters: Basal arteries diameters became narrow on the first day,the worst on the seventh day, and recovered on the14thday after SAH. The treatment groupimproved significantly after intraperitoneal injection18β glycyrrhetinic acid.The expression of gap junction protein in cerebrovascular:A.Cx37was not detected.B.Cx40detection: The expression of Cx40decresed from the3thday to the7thday, andincreased on the14thdayin operation group compare with the treatment group. Other groups nochanges.C.Cx43, Cx45detection: Cx43and Cx45increased from first day to7thday, and decreased onthe14thday in operation group compared with treatment group. Other groups no changes.Gap junction channel conductivity changes: Gap junction channels conductivity increased fromfirst day to7thday after SAH, and reduced on the14thday in operation group compared withtreatment group. Other groups no changes.Conclusions：The SAH model had been established by secondary injection of blood crossing front poolmethod.The occurrence of CVS associated with changes of morphological structure andelectrophysiological of cerebrovascular GJ after SAH.CVS could be relieved by intraperitoneal injection18β glycyrrhetinic acid after SAH, and itsmechanism may be associated with the changes in cerebrovascular GJ.