利用导管损伤制备钙化性主动脉瓣疾病小鼠模型的最优时间选择

Optimal time window for observation of calcific aortic valve disease in mice following catheter-induced valve injury

目的 探讨利用导管损伤制备钙化性主动脉瓣疾病小鼠模型的最优时间。方法 通过向8周龄小鼠右颈总动脉插入导管,在超声引导下将导管送至主动脉瓣口处损伤主动脉瓣膜,对导管损伤主动脉瓣膜后不同时间段的C57BL6小鼠主动脉瓣膜进行比较,将小鼠分为对照组、造模后4周、造模后8周与造模后16周组,从适应性喂养至造模结束取材均给予普通饲料饮食。使用超声检测小鼠心脏短轴缩短率、主动脉瓣膜峰值流速及主动脉瓣口面积,使用苏木素-伊红染色观察小鼠主动脉瓣膜厚度变化及瓣膜组织结构变化,利用茜素红染色检测小鼠主动脉瓣膜钙盐沉积情况,使用免疫荧光方法检测瓣膜成骨相关蛋白碱性磷酸酶表达。结果 小鼠从造模8周后开始出现心脏短轴缩短率明显下降。导管损伤4周、8周、16周后小鼠主动脉瓣膜明显增厚(P=0.002),4周、8周和16周比较无统计学差异;导管损伤4周、8周、16周后钙盐沉积明显增多(P<0.0001),4周、8周和16周比较无统计学差异;4周、8周、16周后成骨相关蛋白ALP指标表达量明显升高(P=0.0016)。导管损伤4周、8周和16周ALP表达量相比无统计学差异。结论 在保证小鼠生存率的情况下,导管损伤主动脉瓣膜4周后的小鼠模型即可模拟钙化性主动脉瓣膜疾病病理生理改变。

Objective To investigate the optimal time window for observation of catheter-induced valve injury that mimics calcified aortic valve disease in mice. Methods A catheter was inserted into the right common carotid artery of 8-week-old C57BL6 mice under ultrasound guidance, and aortic valve injury was induced using the guide wire. At 4, 8 and 16 weeks after modeling, the mice were subjected to ultrasound measurement of the heart short axial shortening rate, aortic valve peak velocity and aortic valve orifice area. Grain-Eosin staining was used to observe the changes in the thickness of the aortic valve,and calcium deposition in the aortic valve was assessed using Alizarin red staining. Immunofluorescence assay was performed to detect the expression of alkaline phosphatase(ALP) in the aortic valve. Results At 4, 8 and 16 weeks after modeling, valve thickness(P=0.002), calcium deposition(P<0.0001) and the expression of osteogenic protein ALP(P=0.0016) were significantly increased, but their increments were comparable at the 3 time points of observation. Conclusion In mouse models of calcific aortic valve disease induced by catheter valve injury, 4 weeks after the injury appears to be the optimal time window for observation of pathophysiological changes in the aortic valves to avoid further increase of the death rate of the mice over time.