缺血再灌注离体海马脑片活性的光通透性评价方法

Optical recording method for evaluation of neuronal damage in rat hippocampal slices during ischemia and reperfusion

目的 :探讨缺血再灌注离体海马脑片的光信号改变特征 ,以及 L型 Ca2 +通道在脑缺血急性期损害中的作用。方法 :以缺氧缺糖 (OGD)方法诱导离体海马脑片缺血模型 ,采用自行构建的脑片透光检测系统 ,结合图象处理软件 Photoshop中的灰度分析检测脑片活性 ,并观察尼莫地平的作用。结果 :体外缺血使海马 CA1区域对光通透性增加 ,(7.5 9± 1.4 2 ) min达峰值 ,再灌后 (6 .37± 1.89) min恢复至本底水平 (n=15 slices) ;L型 Ca2 + 通道阻断剂尼莫地平 (0 .5 μmol/ L,n=10 slices;5 μmol/ L,n=9slices)对缺血所致的海马脑片透光度增加具有抑制作用。而高剂量组 (5 0 μmol/ L,n=11slices)则引起脑片透光度的增加 ,给药后 (2 5 .83± 6 .32 ) min达峰值 ;尼莫地平使海马脑片透光度增加 ,并呈剂量依赖性。结论 :离体脑片光通透性检测是一种简易 ,无创性的脑片活性评价方法 ;L型Ca2 +通道对于维持正常脑片活性具有重要意义 ,同时参与脑缺血急性期损害 。

Objective: To develop a novel technique of optical recording and its validation for assessment of the neuroprotective effect of nimodipine, a L type calcium channel blocker. Methods: In vitro ischemia was induced by oxygen/glucose deprivation (OGD), the light transmittance (LT) of rat hippocampal slices undergoing OGD and reperfusion was quantitated using a simple apparatus relying on basic principles of light transmittance and a computerised image analysis system. Results: OGD was associated with increased LT in the stratum radiatum of CA1 area and the dentate gyrus in hippocampal slices. Peak LT occurred (7.59±1.42)min after OGD, followed by a marked decrease in LT ( n =15 slices). Nimodipine administration(0.5 μmol/L, n =10 slices, 5 μmol/L, n =9 slices) appeared to protect the tissue from OGD damage by inhibiting elevation of LT, However, 50 μmol/L nimodipine resulted in increased LT(25.83±6.32) min after administration( n =11 slices). Conclusion: LT signal measurement is a non invasive, reliable method for determination of neuronal damage in ischemic rat brain slices. Nimodipine is demonstrated opposite neuroprotective effects depending on its dose.