A submersion method to induce hypoxic damage in organotypic hippocampal cultures
A submersion method to induce hypoxic damage in organotypic hippocampal cultures
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DOI：
10.1016/s0165-0270(99)00030-8
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发表时间：
1999-07-01
影响因子：
3
通讯作者：
El-Beheiry, H
中科院分区：
文献类型：
--
作者：
Frantseva, MV;Carlen, PL;El-Beheiry, H
半孔膜培养的器官型脑切片是研究缺血性脑损伤机制的一种越来越受欢迎的体外制备方法。为了模拟体内缺氧，将培养的脑切片置于一个特殊的培养箱中，使其暴露于无氧环境中。这一要求将体外缺血模型的使用限制在高度专业化的实验室。在这里，我们描述了一种简单的方法来复制缺氧损伤，将培养的海马切片浸入无葡萄糖的脱氧培养基中1小时。通过这种处理获得的海马神经元损失的程度和分布类似于原位活组织缺氧引起的损失，即CA1锥体细胞层最脆弱，齿状颗粒细胞层最不容易缺氧，通过荧光活性标记碘化丙啶（PI）测量。CAI锥体神经元的电生理功能损伤在时间上与神经元死亡的程度一致。此外，已知的神经保护治疗，如低温和苯妥英应用，以类似于先前发表的报告的模式改善神经元损伤。因此，目前的体外缺血模型简单、可靠、成本低。它非常适合于对缺氧脑损伤机制的短期和长期研究。(C) 1999 Elsevier Science B.V.版权所有
Organotypic brain slices cultured on semi-porous membranes is an increasingly popular in vitro preparation for studying mechanisms of ischemic brain damage. To model in vivo hypoxia, cultured brain slices are exposed to anaerobic atmosphere by placing them into a special incubator. This requirement limits the use of in vitro ischemic models to highly specialized laboratories. Here, we describe a simple method that reproduces hypoxic injury, where cultured hippocampal slices are submerged into glucose-free deoxygenated medium for 1 h. The extent and distribution of hippocampal neuronal loss obtained with this treatment resembled that caused by hypoxia in living tissue in situ, i.e. CA1 pyramidal cell layer was most vulnerable and dentate granular cell layer was least susceptible to hypoxia as measured with fluorescence of the viability marker propidium iodide (PI). Electrophysiologic functional impairment determined by field recordings of CAI pyramidal neurones temporally coincided with the extent of neuronal death. In addition, known neuroprotective treatments, such as hypothermia and phenytoin application ameliorated neuronal damage in a pattern similar to previously published reports. Therefore, the present in vitro model of ischemia is simple, reliable and of low cost. It is well suited for short and long-term studies of the mechanisms of hypoxic brain damage. (C) 1999 Elsevier Science B.V. All rights reserved.