Laboratory of Neurobiology, Kazan Federal University, Kazan, 420008, Russia.
INMED/INSERM UMR1249, Aix-Marseille University, 163 Avenue de Luminy BP13, 13273, Marseille Cedex 09, France.
Sci Rep. 2020 Nov 4;10(1):18970. doi: 10.1038/s41598-020-75975-w.
Anoxic spreading depolarization (aSD) has been hypothesized as a terminal event during oxygen-glucose deprivation (OGD) in submerged cortical slices in vitro. However, mechanical artifacts caused by aSD-triggered edema may introduce error in the assessment of neuronal viability. Here, using continuous patch-clamp recordings from submerged rat cortical slices, we first confirmed that vast majority of L4 neurons permanently lost their membrane potential during OGD-induced aSD. In some recordings, spontaneous transition from whole-cell to out-side out configuration occurred during or after aSD, and only a small fraction of neurons survived aSD with reperfusion started shortly after aSD. Secondly, to minimize artifacts caused by OGD-induced edema, cells were short-term patched following OGD episodes of various duration. Nearly half of L4 cells maintained membrane potential and showed the ability to spike-fire if reperfusion started less than 10 min after aSD. The probability of finding live neurons progressively decreased at longer reperfusion delays at a rate of about 2% per minute. We also found that neurons in L2/3 show nearly threefold higher resistance to OGD than neurons in L4. Our results suggest that in the OGD ischemia model, aSD is not a terminal event, and that the "commitment point" of irreversible damage occurs at variable delays, in the range of tens of minutes, after OGD-induced aSD in submerged cortical slices.
缺氧性弥散性去极化（aSD）被假设为体外浸没皮质切片缺氧-葡萄糖剥夺（OGD）期间的终末事件。然而，由 aSD 触发的水肿引起的机械伪影可能会在评估神经元活力时引入误差。在这里，我们使用来自浸没的大鼠皮质切片的连续膜片钳记录，首先证实，在 OGD 诱导的 aSD 期间，绝大多数 L4 神经元永久性地失去了其膜电位。在一些记录中，在 aSD 期间或之后自发地从全细胞转变为外侧向外配置，只有一小部分神经元在 aSD 后不久开始再灌注时幸存下来。其次，为了最小化 OGD 诱导的水肿引起的伪影，在 OGD 发作后对细胞进行短期贴片。如果再灌注在 aSD 后少于 10 分钟开始，近一半的 L4 细胞保持膜电位并显示出爆发的能力。如果再灌注延迟时间较长，找到存活神经元的概率以每分钟约 2%的速度逐渐降低。我们还发现 L2/3 中的神经元对 OGD 的抵抗力比 L4 中的神经元高近三倍。我们的结果表明，在 OGD 缺血模型中，aSD 不是终末事件，并且在 OGD 诱导的浸没皮质切片中的 aSD 之后，不可逆转损伤的“承诺点”以数十分钟的范围发生可变延迟。
