Anesthesia-on-chip: Modelling the impact of fetal anesthesia on neural development

越来越多的手术是在胎儿出生前进行的。其中包括在当地大奥蒙德街医院进行的脊柱裂复杂手术。对于这些手术，母亲必须接受全身麻醉。许多使用的麻醉剂会穿过胎盘影响胎儿。麻醉药、镇痛药和用药前药物对胎儿大脑发育的影响在很大程度上是未知的，并且很难在可能有延迟其发育里程碑的合并症的患者中直接调查。动物实验表明，一些麻醉剂，如诱导剂异丙酚，具有神经毒性。其他药物，如镇静剂右美托咪定，可能具有神经保护作用。这项应用旨在用人类诱导多能干细胞（iPSC）衍生的皮质神经元代替动物用于麻醉研究，特别是在胎儿时期。我们现在开发了一种“芯片麻醉”方案，我们可以看到培养中的神经祖细胞的生长，并通过麻醉剂减少它们的兴奋性。已经报道了几种复杂的人类“迷你大脑”类器官模型，但这些系统固有的可变性、低通量和高成本限制了它们的实用性。相反，我们使用一种完善的二维神经元分化系统，使细胞能够在治疗组之间进行动态分析和定量比较。这种方法非常适合博士研究生。由于动物麻醉研究的可翻译性有限，它将被广泛推广为一种动物替代技术，可能会被该领域迅速采用。我们未发表的数据表明，神经元在体外麻醉暴露下存活并继续生长，但它们的分化受到损害。这证实了胎儿麻醉可能比产后麻醉更有神经毒性的担忧，因为胎儿生长过程中有大量的祖细胞，如果在未来几年内无法获得完全的人体替代品，预计将刺激动物实验的迅速增加。为了验证和应用这一平台，我们建议：比较在多能干细胞分化的人类神经元和不同阶段的人类胎儿脑组织中常见麻醉剂受体的表达。定义芯片麻醉方案的直接影响，使用人类干细胞来源的神经元来建立不同分化阶段的麻醉敏感性。评估在分化过程中不同时间点麻醉的培养物的体外神经毒性，用临床相关的药物如异丙酚和右美托咪定联合治疗，或反复麻醉。

An increasing number of surgical procedures are performed on the fetus before birth. These include complex surgery for spina bifida, performed locally at Great Ormond Street Hospital. For these procedures, the mother must receive a general anesthetic. Many of the anesthetic agents used will cross the placenta and affect the fetus. The impact of anesthetics, analgesics and pre-medication agents on fetal brain development are largely unknown, and difficult to investigate directly in patients who may have co-morbidities delaying their developmental milestones. Animal studies suggest some anesthetic agents, such as the induction agent propofol, are neurotoxic. Others agents, such as the sedative dexmedetomidine, may be neuroprotective. This application aims to replace animal use with human induced pluripotent stem cell (iPSC)-derived cortical neurons in the study of anesthesia, particularly during the fetal period.We have now developed an "Anesthesia-on-chip" protocol in which we can visualize growing neural progenitor cells in culture and diminish their excitability with anesthetics. Several complex models of human "mini brain" organoids have been reported, but the inherent variability in those systems, low throughput, and high cost limit their utility. Instead, we use a well-established 2D neuronal differentiation system which enables cells to be dynamically analysed and quantitatively compared between treatment groups. This method is ideally suited for a PhD studentship. It would be broadly generalizable as an animal-replacing technology likely to be rapidly adopted by the field due to the limited translatability of anesthesia research in animals. Our unpublished data shows that neurons survive in vitro anesthetic exposure and continue to grow, but their differentiation is impaired. This corroborates concerns that fetal anesthesia may be more neurotoxic that post-natal anesthesia due to the abundance of progenitor cells during fetal growth, and is expected to stimulate a rapid increase in animal experimentation if fully human alternatives are not made available within the next few years. In order to validate and apply this platform, we propose to:Compare expression of receptors for common anesthetics during human neuron differentiation from pluripotent stem cells, and in human fetal brain tissue at different stages.Define the immediate effects of anesthesia-on-chip protocols using human stem cell-derived neurons to establish susceptibility to anesthesia at different stages of differentiation.Assess in vitro neurotoxicity in cultures anesthetized at different time points during differentiation, treated with clinically-relevant combinations of agents such as propofol and dexmedetomidine, or anesthetized repeatedly.