借助POMC-EGFP转基因小鼠开展全身麻醉药物选择性诱导脑内神经元细胞凋亡的机制研究

General anaesthesia facilitates surgical operations and painful interventions in millions of patients every year. Recent observations found that exposure to anaesthetics dramatically increases the rate of neuroapoptosis in developing animals and subsequent neurocognitive impairment. Also epidemiological human studies have associated early childhood anesthesia with long-term neurobehavioral abnormalities, raising substantial concerns that anesthetics may cause similar cell death in young children. Importantly, although the phenomenon’s mechanism is unknown, but understanding the selectivity of cellular loss following anesthetic exposure may be important in elucidating the phenomenon’s mechanism. Combined, those previous findings was felt to represent an inherent vulnerability of the developing brain and to be limited by the age of the organism, for example, only occurring between 4 and 14 days of age (P4–P14) in small rodents. But recently, our findings suggest that anesthetic vulnerability reflects the age of neuron, not the age of organism. The differential windows of vulnerability among brain regions is closely 1–2 weeks after these regions’ peak in neurogenesis. Moreover, BrdU birthdating of affected neurons confirmed their immature state, indicating that cells <15 days old were most vulnerable. And, let us see what will happen during this 1-2weeks after neuron cell was born. First, there is a switch from GABAergic to glutamatergic excitatory input to immature newborn granule cells. Second, Moreover, concurrently a change occurs in the chloride gradient, mediated by a switch from predominant NKCCl cotransporter expression to KCC2 expression, implying that the former might be excited by the anesthetic’s GABA agonistic effects, whereas the latter inhibited. And because POMC- EGFP transgenic mice have these characters as follows: First, POMC is a transgenic marker for newly born granule cells in dentate gyrus and the average postmitotic age of EGFP-labeled granule cells are around 2 weeks. Second, EGFP-labeled cells are at a similar functional stage regardless of the age of the animal. So newborn granule cells in adults and neonates all have similar synaptic connectivity. Third, they all lack glutamatergic synaptic input but all have depolarized GABAergic responses. Because all commonly used sedatives and anesthetics act at N-Methyl-D-aspartic acid glutamate (NMDA) and/or Υ-aminobutyric acid type A (GABAA) receptors of neurons to have their effects on CNS. So it is conceivable that anesthetics induce apoptosis by interfering with the balance of neuronal activity when they are used in general anesthesia. Thus in the future anesthesia neurotoxicity’s mechanistic studies, by using transgenic mice (POMC), we try to find how general anesthetics selectively interfere with the Excitory/Inhibitory balance of neuronal activity, thereby inducing neuroapoptosis.

许多幼年动物模型中发现全身麻醉诱导广泛的脑神经元细胞凋亡。证据指出在啮齿类动物、人类，幼年时暴露于全麻可引起长期神经行为缺陷。全麻药物脑毒性作用机制并不清楚，但是了解其诱导神经元凋亡的选择性对于机制研究至关重要。过去观点：该凋亡现象仅限于新生期动物。但我们发现：该选择性不是简单由动物年龄决定，而是取决于神经元细胞年龄；即脑内各不同区域的神经发生高峰窗后1周-2周、年龄为13-15天的不成熟神经元细胞。该时期是神经元上GABA/NMDA受体功能转换的关键期。POMC-EGFP转基因小鼠可选择性标识该年龄段神经元，且这些神经元只接受GABAergic、不接受NMDAergic的兴奋性传入。因此借助该小鼠探究神经毒性机制：从体内和体外、在神经元细胞的GABA/NMDA受体层面上，探究全身麻醉药物在发挥其全麻效应的同时、是如何选择性地干扰那些尚未成熟神经元细胞的兴奋/抑制平衡，从而诱导了凋亡。

全身麻醉药，无论是 GABAA受体激动剂（如：七氟烷、丙泊酚、异氟烷）还是NMDA 受体拮抗剂（如：氯胺酮）在许多物种的神经发育高峰期都能诱导超过正常生理水平的、广泛的大脑神经细胞凋亡。目前对于全身麻醉药物脑神经毒性作用的易感时间窗，已经从最初提出的“动物新生期”更精确地定位于“脑内不同区域各自的神经发生高峰窗后的1-2周，即细胞年龄为13-15天的神经细胞”（我们前期研究成果）。但是，为什么处于这一特定年龄段的不成熟神经细胞对全麻药诱导凋亡作用表现敏感？本研究借助POMC-EGFP转基因小鼠进一步探究了这种细胞年龄选择性的潜在机制。首先，我们的研究明确并且定量分析了持续吸入3%七氟烷6h后，尽管诱导了相当比例的POMC-EGFP标记的不成熟神经细胞的凋亡数量增加，但这种诱导凋亡的选择性并非完全和单一地集中于这类具有特定受体功能状态（只接受GABAergic兴奋性传入、而不接受glutamatergic兴奋性传入）的不成熟神经细胞；其次，本研究发现NeuroD1标记的晚期神经祖细胞、calretinin标记的不成熟颗粒神经元细胞，对持续6h吸入3% 七氟烷诱导的神经细胞凋亡最为敏感，而早期神经祖细胞和成熟颗粒神经元细胞几乎未受到影响；证实POMC-EGFP转基因小鼠特异性绿色荧光标记的不成熟神经细胞主要与NeuroD1、calretinin共表达；最后，通过对不同年龄段小鼠持续吸入3%七氟烷6h，定量分析发现选择性诱导小鼠海马DG区神经细胞凋亡在P21小鼠达高峰，此结果与我们之前异氟烷的研究结果一致，从而从另一方面证实两种不同的吸入全身麻醉药物——异氟烷和七氟烷在诱导发育中动物大脑神经细胞凋亡的选择性上是一致的。总之，本研究借助POMC- EGFP转基因小鼠，更进一步地探究了七氟烷诱导凋亡的不成熟神经细胞的细胞年龄／细胞受体功能定位，即七氟烷可能选择性诱导那些可同时接受GABAergic 和glutamatergic兴奋性传入的不成熟神经细胞发生凋亡。因此，本研究结果为全身麻醉药物脑神经毒性作用的选择性机制研究开辟了新的方向和思路。