| Objective:Preliminary studies show that gap junction involved in CVS after Spontaneous SAH. This subject of instruction moves forward to investigating CX which is the element of GJ. That is in order to show the expression of CX in the amount, type and distribution involved in cerebral vasospasm (CVS) changes and function after SAH, which is good for clarifying the pathogenesis of CVS and open up a new way for treatment.Motheds:1. Establishing rabbit's CVS model after secondary SAH: Cistern puncture inject fresh non-heparinized autologous arterial blood(0.8ml/kg) after using 3% Pentobarbital sodium(1ml/kg) to anaesthetize the News land White rabbit. We give it normal feeding when it is awaked, and then injecting blood repeatedly 24H's later.2. Observation of animal CVS model: setting up group according different phases. Doing two times'DSA angiography after and before establishing the model's 1d, 3d, 5d, 7d, 14d and comparing basilar artery diameter's changes in different phases. After the second angiography, the heart of total cerebral perfusion is fixed as HE staining without in the same phases (1d, 3d, 5d, 7d, 14d) and observes the morphological changes under light microscope. 0d is the normal group which does not make any treatment.3. Changes in different GJ protein expression: Animals in each group for extracting brain, separating brain arteries and extracting protein after DSA angiography. Western Blotting techniques were used to detect cerebral artery Cx37, Cx40, Cx43, Cx45 expression's phase changes.4. Distribution changes of cerebral artery Cx43: After the second angiography, we extract brain from heart cerebra perfusion with fixed, embedded, sliced and other steps to carry out immunohistochemistry techniques. This can detect small cerebral artery Cx43's distribution changes.Results:1. 1. Successfully establishing rabbit's CVS model after secondary SAH: DSA angiography demonstrates that artery stenosis appears in 1d, reaches to peak in 7d and relieves in14d. Under light microscope, we could discover that 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. Cerebral artery varies in different types of Cx protein number expression: vessel wall of cerebral artery (Cx43,Cx45) raises apparently in protein expression and shows changes in different phases. Among them, Cx45 get to peak in 5d (P<0.01), Cx43 reach peak in 7d (P<0.01); however, (Cx40, Cx 37) protein expression shows slightly changes: Cx40 reaches peak in 3d (P<0.05), cx37 reaches small peak in 7d (P<0.01). Cx45,Cx43 protein expression changes in phase with the basilar artery diameter changes in phase after establishment of model of cerebral vasospasm, the related coefficient is -0.847,-0.865,-0.934, which appears existence of a straight line with basilar artery diameter changes in phase.3. GJ distribution changes in cerebral arteries: In the normal cerebro-vascular adventitial fibroblasts and endothelial cells can see a small amount of Cx43 expression, yet the expression can not be seen in smooth muscle cells; after 7d of SAH, Cx43 expression changes apparently and a large amount of expressions can been found in smooth muscle cells, meanwhile expression increased rapidly in fibroblasts and endothelial cells.Conclution:1. Many types of GJ protein expressions have changed, which further indicates that GJ participated in the occurrence of CVS after SAH.2. GJ protein expression's types, number and distribution have transformed after SAH, which proved that when CVS happens, GJ remodeling happens. And GJ remodelling lead to the transmission of information in cells altered, which might make the pathological basis of CVS occur. |
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