The Effects Of Connexin 43 Phosphorylation On Cerebral Vasospasm

Objective:Gap junctions composed of proteins from the connexin family mediate intercellular communication between cells and play an important role in the pathogenesis of cerebral vasospasm(CVS). It has been known that connexin phosphorylation is implicated in the regulation of gap junctional communication and participates in the pathological processes of many diseases. This study is undertaken to investigate the changes of Connexin 43 (Cx43) phosphorylation and its potential roles in the pathogenesis of CVS after experimental subarachnoid hemorrhage (SAH) in rabbits.Methods:1. Experimental Design: 102 male New Zealand White rabbits weighing 2.5 to 3.5kg were randomly assigned to 4 groups: normal group (n=12), SAH-only group (n=30), SAH + Carbenoxolone (CBX) group (n=30), SAH + vehicle (normal saline) of CBX group (n=30).2. Models of CVS after SAH: Models of CVS after SAH were successfully made via double blood injection into the cisterna magna on two consecutive days. CBX was administered intracisternally on three consecutive days after CVS.3. Angiography and Morphological Study: Digital subtraction angiography was performed before and 7 days after SAH,and the diameter of the the basilar artery (BA) was measured. After the second angiogram, animals were euthanized to observe morphological changes of the BA.4. Western Blotting for expression of Cx43 phosphorylation (P-Cx43) and dephosphorylation (NP-Cx43) protein in BA after SAH: Animals were euthanized by an overdose of pentobarbital. Then the BAs removed and protein extracts were prepared in routine method. After protein quantification, Western Blotting was performed.Results: 1. Models of CVS after SAH was established successfully: Arterial narrowing and morphological changes in vascular structure after SAH were significant.2. Western Blotting for expression of P-Cx43 and NP-Cx43 protein in BA after SAH: Western Blotting with anti-P-Cx43 antibody or anti-NP-Cx43 antibody showed that P-Cx43 was detected as the slight band in the BAs in normal group animals but increased the density after SAH in a time-dependent manner, whereas NP-Cx43 was detected as the higher band than P-Cx43 in the BAs in normal group animals but decreased in a time-dependent manner in these groups after SAH.3. After administered intracisternally CBX, CVS was attenuated with the expression of P-Cx43 significantly decreased and the expression of NP-Cx43 significantly increased.Conclusion:1. The expression changes of P-Cx43 and NP-Cx43 play an important role in the pathogenesis of CVS.2. CBX reduces cerebral vasospasm after SAH by possibly modulating the expression of P-Cx43 and NP-Cx43.