Dept. of Pediatrics, The University of Iowa, Iowa City, Iowa, USA; Iowa Neuroscience Institute, The University of Iowa, Iowa City, Iowa, USA.
Dept. of Biomedical Engineering, The University of Iowa, Iowa City, Iowa, USA.
Neurobiol Dis. 2023 Mar;178:106013. doi: 10.1016/j.nbd.2023.106013. Epub 2023 Jan 25.
Neonatal hypoxia causes cytotoxic neuronal swelling by the entry of ions and water. Multiple water pathways have been implicated in neurons because these cells lack water channels, and their membrane has a low water permeability. NKCC1 and KCC2 are cation-chloride cotransporters (CCCs) involved in water movement in various cell types. However, the role of CCCs in water movement in neonatal neurons during hypoxia is unknown. We studied the effects of modulating CCCs pharmacologically on neuronal swelling in the neocortex (layer IV/V) of neonatal mice (post-natal day 8-13) during prolonged and brief hypoxia. We used acute brain slices from Clomeleon mice which express a ratiometric fluorophore sensitive to Cl and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl] by multiphoton microscopy. Neurons were identified using a convolutional neural network algorithm, and changes in the somatic area and [Cl] were evaluated using a linear mixed model for repeated measures. We found that (1) neuronal swelling and Cl accumulation began after OGD, worsened during 20 min of OGD, or returned to baseline during reoxygenation if the exposure to OGD was brief (10 min). (2) Neuronal swelling did not occur when the extracellular Cl concentration was low. (3) Enhancing KCC2 activity did not alter OGD-induced neuronal swelling but prevented Cl accumulation; (4) blocking KCC2 led to an increase in Cl accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation; (5) blocking NKCC1 reduced neuronal swelling during early but not prolonged OGD and aggravated Cl accumulation during prolonged OGD; and (6) treatment with the "broad" CCC blocker furosemide reduced both swelling and Cl accumulation during prolonged and brief OGD, whereas simultaneous NKCC1 and KCC2 inhibition using specific pharmacological blockers aggravated neuronal swelling during prolonged OGD. We conclude that CCCs, and other non-CCCs, contribute to water movement in neocortical neurons during OGD in the neonatal period.
新生儿缺氧通过离子和水的进入导致细胞毒性神经元肿胀。由于这些细胞缺乏水通道，并且其膜的水通透性低，因此已经涉及到多种水途径。 NKCC1 和 KCC2 是参与各种细胞类型中水运动的阳离子-氯离子共转运体（CCCs）。然而，在缺氧期间 CCCs 在新生儿神经元中水运动中的作用尚不清楚。我们研究了在新生小鼠（出生后第 8-13 天）的大脑新皮层（IV/V 层）中通过药理学调节 CCCs 对神经元肿胀的影响，这些小鼠在长时间和短暂缺氧期间接受急性脑切片。我们使用表达对 Cl 敏感的比率荧光探针的 Clomeleon 小鼠进行急性脑切片，并在多光子显微镜下对神经元大小和 [Cl] 进行成像，同时进行氧葡萄糖剥夺（OGD）。使用卷积神经网络算法识别神经元，并使用重复测量的线性混合模型评估体细胞面积和 [Cl] 的变化。我们发现：（1）神经元肿胀和 Cl 积累在 OGD 后开始，在 20 分钟的 OGD 期间恶化，或在 OGD 短暂（10 分钟）期间重新复氧时恢复到基线。（2）当细胞外 Cl 浓度较低时，神经元肿胀不会发生。（3）增强 KCC2 活性不会改变 OGD 诱导的神经元肿胀，但可防止 Cl 积累；（4）阻断 KCC2 会导致在长时间 OGD 期间 Cl 积累增加，并在复氧期间加重神经元肿胀；（5）阻断 NKCC1 会减少早期但不会延长 OGD 期间的神经元肿胀，并在延长 OGD 期间加重 Cl 积累；（6）用“广谱” CCC 阻断剂呋塞米处理可减少延长和短暂 OGD 期间的肿胀和 Cl 积累，而同时使用特定的药理学阻断剂抑制 NKCC1 和 KCC2 会加重延长 OGD 期间的神经元肿胀。我们得出结论，在新生儿期 OGD 期间，CCCs 和其他非 CCCs 有助于新皮层神经元中的水运动。
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