Possible Role Of Gap Junction Channels Changed In Experimental Subarachnoid Hemorrhage Cerebral Vasospasm Models

Objective:Gap junction(GJ) are widely kept in cardio-cerebral vascular system, while changes of gap junction channel function plays an important role in the number of cardio-cerebral vascular diseases. Based on the preliminary studies, we have studied the the changes of the GJ channel type of cerebral vascular after subarachnoid hemorrhage (SAH) through the techique of co-immunoprecipitation, to lay the ground work for more in-depth study on the mechanism of GJ channels's involving in the form of CVS in the cerebral blood vessels after subarachnoid hemorrhage , which may provide more effective way to prevent CVS.Method:1. Establishing the second rabbit model of CVS after SAH: White rabbits from New Zealand were anesthetized with 3% pentobarbital sodium (1ml/kg), puncture the cistern magna and inject with fresh heparinized autologous arterial blood heparin (0.5ml/kg), We give it normal feeding when it is awaked, and then the process repeatedly 24h later.2. The detection of the animal CVS model: In every groups(the normal group and SAH group), DSA angiography was conducted before establishing and 7d after the model established, and the changes of basilar artery diameter were compared. After the second angiography, the heart of total cerebral perfusion is fixed as HE staining without in different groups and observes the morphological changes under light microscope.3. Changes of type expression of GJ protein: After the second DSA examination in each group,animals were enthasizd and the brain arteries were sparated ,The changes of Cx37, Cx40, Cx43, Cx45 protein expression were detected by the co-immunoprecipitation and the Western Blotting techniques, detect the changes of the interaction between the cerebral artery Cx37, Cx40, Cx43, Cx45 protein expression, to suggest the changes of the composition of the homotypic GJ channel or heterotypic GJ channel from the connexins. Results:1. Successfully establishing rabbit's CVS model after secondary SAH: DSA angiography demonstrates that the proportion of basilar artery diameter of the normal control group before and after the model established was 97.2%±1.5%, whereas the SAH-7d group before and after the model established 63.7%±7.1%.The difference is significantly between the above two groups. Under light microscope, we could discover that the cavity of vessel tube was seen narrow and its wall was seen thickening. the vascular endothelial cell nuclear chromatins under spasm circumstance gathered, internal elastic membrane is significantly wavy, smooth muscle cell is sparse distribution, organizations around the red dye increased in the nuclear spindle length, Edema shows between the internal elastic membrane and smooth muscle layer.2.Since the Cx43 was taken for the precipitation protein, the Cx43,Cx45,Cx37 of the SAH group were apparently more increased than that of the normal group;Similarly, the Cx40 of the SAH group was apparently less than that of the normal group;since the Cx45 was taken for the precipitation protein, the Cx45,Cx43,Cx37 of the SAH group were apparently more increased than that of the normal group, the Cx40 of the SAH group was apparently less than that of the normal injection group; Since the Cx40 was taken for the precipitation protein, the Cx40,Cx43,Cx45,Cx37 of the SAH group were apparently reduced more than that of the normal group; Since the Cx37 was taken for the precipitation protein, the Cx45,Cx43,Cx37 of the SAH group were apparently more increased than that of the normal group,The Cx40 of the SAH group were apparently reduced more than that of the normal group.3. The gap junction channels of homotypic type formed by the Cx43 and Cx43, Cx45 and Cx45, Cx37 and Cx37 after SAH increase significantly compared with that of the normal group. The gap junction channels of the homotypic type formed by the Cx40 and Cx40 after the SAH are less than that of normal group. The heterotypic gap junction channels formed by Cx37 and Cx45, Cx37 and Cx43, Cx45 and Cx43 after SAH are much more compared with that of the normal group. The heterotypic gap junction channels formed by Cx37 and Cx40, Cx40 and Cx43, Cx40 and Cx45 are much less compared with that of the normal group . Conclution:During the process of CVS, the interaction between different Connexins (Cx37, Cx40, Cx43, Cx45) changes, which accordingly causes to form a variety of gap junction channels of the same type or changes of the number of heterotypic gap junction channels. Which indicats the changes of GJ channels may be involved in the occurrence of CVS after SAH.