The Biological Role Of Ca²⁺-activated K⁺ Channel （KCa3.1） On Hemodynamic Induced Cerebral Aneurysm Initiation Mechanism

Objective:Aneurysmal subarachnoid hemorrhage is one of the main causes of death and disability in the worldwide. An epidemiological survey conducted by World Health Organization revealed that the prevalence of aSAH in China is 2/100000, while it’s 9.7/10000 according to the nationwide epidemiological data of US in 2003. aSAH carries a high mortality rate.12%-15% patients would lost the opportunity for treatment by the onset of aSAH.50% of the survived patients without clipping or endovascular embolization would die for rebleeding in 6 months after aSAH, even as high as 60%-80% in the next 10 years. Before 1992, the medical cost for the treatment of aSAH had been 1,755,600,000 dollars per year in US. That being said, aSAH not only causes life-threatening injury but also brings devastating complication to the patient and his family. The research should focus on the pathology behind the initiation and development of aSAH and emphasize on the molecular mechanism to discover the possible treatment options and reduce the progression of aSAH. The treatment will ultimately reduce the morbidity and mortality of aSAH and improve the overall quality of life for patient. Therefore, this study aims to elucidate the hemodynamic and biological mechanisms of cerebral aneurysm initiation; to investigate whether KCa3.1 is the biological target that sensing the harmful mechanic stress and responding to it. This will be helpful to stop aneurysm initiation, even to reverse aneurysm progression. The present study will be helpful to the future noninvasive or drug therapy development of cerebral aneurysm management.Methods:We established the hemodynamic-induced cerebral aneurysm model of rat with the ligation of one common carotid artery and contralateral external carotid artery and pterygopalatine artery. Clotrimazole was used as the specific blocker of KCa3.1. Batson’s #17 was used for vascular corrosion casting technique to obtain the arterial cast of the circle of Willis. The aneurysmal changes of the vessel were examined with SEM scanning analysis. The aneurysmal changes were classified to 4 stages:Stage 0:normal vascular cast; Stage 1:homogeneously dilated vessel and/or roughened and irregularly shaped endothelium imprints; Stage 2:shallow fusiform elevation; Stage 3:saccular aneurysm or focal aneurysm-like dilation. The dilation of anterior communicating complex were assessed by its volume, which was calculated by the equation v=m/p. Both the aneurysmal changes and the dilation anterior communicating complex were used to present the inhibition of the aneurysm initiation by the blockade of KCa3.1. The artery of the circle of Willis was separated from the rat brain and was immersed with RNALater to prevent the RNA degradation. The expression level of KCa3.1 mRNA was tested with rt-PCR. The paraformaldehyde-fixed and paraffin-embedded rat brain with artery of the circle of Willis was sliced and the Alexa 488 labeled second antibody were used for immunofluorescence. The expression of the protein of KCa3.1 channel were tested with confocal laser scanning microscope. The key downstream target NF-κB and iNOS were detected with immunohistochemical staining. Categorical data for the classification for aneurysmal remodeling between groups were tested for differences using Kruskal-Wallis test and Nemenyi Test; Volume data were test for differences between groups using one-way ANOVA with Bonferroni correction. P value less than 0.05 would be considered as statistically significant.Results:The hemodynamic-induced cerebral aneurysm model of rat we established is stable and feasible for the study of cerebral aneurysm initiation. SEM scanning results demonstrated that The aneurysmal change incidence was decreased by the blockade of KCa3.1 with clotrimazole. The most rats in CLT group were at Stage 1 and there were no Stage 2 or 3 changes found. However, aneurysm changes were significantly obvious in aneurysm group and sham group (p<0.05):shallow fusiform elevation and tortuous dilated Acom Complex with focal aneurysmal dilation could be observed. Lamina shear stress results from hemodynamic alteration upregulated the KCa3.1 mRNA and itself within the artery wall of the circle of Willis. The result of rt-PCR revealed that the expression levels of the KCa3.1 mRNA in the CLT groups of different time were significantly lower than those in aneurysm groups correspondingly (1D Group p<0.001; 1M Group p<0.001; 3M Group p<0.001) and the expression level in aneurysm groups were significantly higher than control group (p<0.001). Alexa 488 labeled second antibody glowed bright green fluorescence under the confocal laser scanning microscope. The fluorescence in the control group was at the base level and became intensified with the time of hemodynamics change increasing. KCa3.1 in the 1M aneurysm group were distribute in ECs as points and flakes and was inhibited and distribute as points in smooth muscle layer in the CLT group. In the 3M aneurysm group, upregulated expression of KCa3.1 altered spatially over time, which migrated from ECs to the subendothelial layer. The specific blockade of KCa3.1 also decreased the NF-κB and iNOS expression level in the artery wall, which respectively inferred the decrease of the inflammatory reaction and oxidative stress.Conclusion:KCa3.1 might be the key converter to transduce the hemodynamic stress to the biological reaction signal. The blockade of KCa3.1 could decrease the inflammatory reaction, oxidative stress in the artery wall and aneurysmal changes.