准备急性脑切片会产生模拟严重穿透性脑损伤的创伤。在新生儿急性脑切片中,创伤诱导的神经元钙动力学的时空特征及其对网络活动的影响相对未知。使用多光子激光扫描显微镜对急性新生小鼠脑片(P8-12)中的体感新皮层进行了观察,我们同时对神经元Ca2动力学(GCaMP6s)和细胞毒性(碘化丙啶或PI)进行成像,以确定细胞毒性Ca2负载神经元(GCaMP填充)与不同深度和孵育时间的细胞活力之间的关系。切片表面富含PI+细胞和充满GCaMP的神经元,随深度呈指数下降。具有高PI+细胞的区域与升高的神经元和神经纤维Ca2+相关。随着长时间的孵育,PI+细胞和GCaMP填充的神经元的数量增加。GCaMP填充的神经元不参与刺激诱发或癫痫诱发的网络活动。重要的是,浅表组织,创伤引起的损伤程度更高,显示癫痫相关的神经元Ca2+反应减弱。钙蛋白酶抑制阻止了深层组织中PI细胞和GCaMP填充的神经元的增加以及延长的孵育时间。同种型特异性药理学抑制暗示钙蛋白酶-2是急性切片中创伤诱发损伤的重要因素。我们的结果表明,急性新生儿脑切片中细胞死亡与异常神经元Ca2负荷之间存在钙蛋白酶介导的时空关系。此外,我们证明,急性脑片中的神经元表现出改变的生理学,这取决于创伤诱导的损伤程度。阻断钙蛋白酶可能是预防年轻大脑创伤性脑损伤期间急性神经元死亡的治疗选择。意义陈述这是首次描述急性新生儿切片中新皮质损伤的时空动力学的研究,模仿严重的穿透性创伤性脑损伤,使用PI标记和升高的神经元Ca2+负荷作为细胞毒性的标志物。我们发现深度和时间依赖性神经元损伤,导致神经元反应改变。通过药理学抑制钙蛋白酶减轻了神经元Ca2升高和细胞毒性,参与多种细胞死亡机制的Ca2+依赖性蛋白酶家族。我们的研究为新生儿急性脑切片中损伤依赖性神经元和回路功能改变提供了证据。钙蛋白酶抑制减少了新生儿大脑中创伤诱导的细胞死亡,将它们确定为这个年龄段的潜在治疗目标。
Preparing acute brain slices produces trauma that mimics severe penetrating brain injury. In neonatal acute brain slices, the spatiotemporal characteristics of trauma-induced calcium dynamics in neurons and its effect on network activity are relatively unknown. Using multiphoton laser scanning microscopy of the somatosensory neocortex in acute neonatal mouse brain slices (P8-12), we simultaneously imaged neuronal Ca2+ dynamics (GCaMP6s) and cytotoxicity (propidium iodide or PI) to determine the relationship between cytotoxic Ca2+ loaded neurons (
GCaMP-filled) and cell viability at different depths and incubation times. PI+ cells and
GCaMP-filled neurons were abundant at the surface of the slices, with an exponential decrease with depth. Regions with high PI+ cells correlated with elevated neuronal and neuropil Ca2+ The number of PI+ cells and
GCaMP-filled neurons increased with prolonged incubation.
GCaMP-filled neurons did not participate in stimulus-evoked or seizure-evoked network activity. Significantly, the superficial tissue, with a higher degree of trauma-induced injury, showed attenuated seizure-related neuronal Ca2+ responses. Calpain inhibition prevented the increase in PI+ cells and
GCaMP-filled neurons in the deep tissue and during prolonged incubation times. Isoform-specific pharmacological inhibition implicated calpain-2 as a significant contributor to trauma-induced injury in acute slices. Our results show a calpain-mediated spatiotemporal relationship between cell death and aberrant neuronal Ca2+ load in acute neonatal brain slices. Also, we demonstrate that neurons in acute brain slices exhibit altered physiology depending on the degree of trauma-induced injury. Blocking calpains may be a therapeutic option to prevent acute neuronal death during traumatic brain injury in the young brain.