Injury of Hippocampal Interneurons following Neonatal Hypoxia-Ischemia and Therapeutic Hypothermia

项目概述：通过这个项目，我将实现我的职业目标，即在以下领域发展专业知识 发育神经可塑性将新的机械方法应用于脑损伤的靶方面和 目前的治疗方法没有解决新生儿的康复问题。我严格的指导，功课和 研究计划与我的具体知识差距保持一致，以确保我的职业发展 5年内独立资助临床医生-科学家。为此，拟议的实验将提供 掌握电生理学和高级神经病理学方法的机会。研究计划是基于 在一个强有力的科学前提下，出生后窒息后缺氧缺血(HI)脑损伤会持续改变 新生儿脑内突触可塑性的机制和治疗性低温(TH)不完全 预防这些影响，解释临床前模型中记录的持久性记忆障碍 TH的人类RCT。我假设海马区抑制性中间神经元(INS)的迟发性损伤， 参与记忆巩固的主要大脑区域，导致长期抑郁(LTD)的损害， 突触可塑性的基本机制。ErbB4表达和激活的中断，对 INS的存活和成熟，可能提供了机制上的联系。研究假设将通过以下方式进行检验 以下特定目标决定是否：1)新生儿缺氧缺血性脑病后海马区INS数量减少 2)HI后存活的海马INS的形态和功能在延迟期发生改变； 3)ErbB4激活的中断导致延迟阶段的海马INS的丢失和/或成熟停止 在HI之后。在这里，我将使用Vannucci程序在p10(完全等值)C57BL6和GAD67中诱导HI- 表达INS绿色荧光标记的绿色荧光蛋白转基因小鼠。小鼠将被随机接受 常温(36°C)或TH(31°C)4h。将使用电生理学方法评估突触活动 结合免疫染色和先进的神经病理学方法。此外，ErbB4的调制 体内系统将使用腺相关病毒(AAV)的转染方法进行诱导。拟议中的项目 它还具有很高的创新性，因为它涉及：i)研究对象，新生儿大脑；ii)研究领域，突触 损伤后的可塑性；iii)方法学、系统配对的电生理和免疫染色数据， 以及iv)统计计划，考虑到包括性别在内的生物变量。这一项目的成果和 由其衍生的未来研究将转化为新的神经元特异性治疗靶点，以减弱记忆 出生窒息后的缺陷。该项目将在5年内按顺序实现各项目标。 拟议的实验和时间表在我和实验室、动物的能力范围内 护理和外科设施。未来的研究将包括使用新的技术来传递小分子 和大脑的基因疗法，以性别和时间量身定做的方式调节细胞特有的通路，这 联合应用TH可改善新生儿窒息后的行为结局。

PROJECT SUMMARY: With this project I will achieve my career goal of developing expertise in the field of developmental neuroplasticity to apply new mechanistic approaches to target aspects of brain injury and recovery not addressed by the current therapies in neonates. My rigorous mentorship, coursework and research plans are aligned to address my specific knowledge gaps to ensure my career development as an independently funded clinician-scientist within 5 years. To that end, the proposed experiments will provide the opportunity to master methods of electrophysiology and advanced neuropathology. The research plan is based on a strong scientific premise that hypoxia-ischemia (HI) brain injury after birth asphyxia persistently alters mechanisms of synaptic plasticity in the neonatal brain and that therapeutic hypothermia (TH) does not fully prevent these effects explaining the persistent memory disabilities documented in pre-clinical models and human RCTs of TH. I hypothesize that delayed injury of inhibitory interneurons (INs) within the hippocampus, the prime brain region involved in memory consolidation, leads to impairment of long-term depression (LTD), an essential mechanism of synaptic plasticity. Disruption of ErbB4 expression and activation, crucial for survival and maturation of INs, may provide the mechanistic link. The research hypothesis will be tested with the following specific aims determining if: 1) the decreased number of hippocampal INs after neonatal HI alter LTD; 2) the morphology and function of surviving hippocampal INs are altered at delayed stages after HI; and 3) disruption in ErbB4 activation leads to loss and/or maturational arrest of hippocampal INs at delayed stages after HI. Here, I will use the Vannucci procedure to induce HI in p10 (full-term equivalent) C57BL6 and GAD67- EGFP transgenic mice expressing a green fluorescent tag for INs. Mice will be randomized to receive normothermia (36°C) or TH (31°C) for 4h. Synaptic activity will be evaluated using electrophysiology methods paired to immunostaining and advanced neuropathology methods. Additionally, modulation of the ErbB4 system in-vivo will be induced using adeno-associated virus (AAV) transfection methods. The proposed project is also highly innovative as it relates to: i) the subject of study, the neonatal brain; ii) the field of study, synaptic plasticity after injury; iii) the methodology, the systematic pairing electrophysiology and immunostaining data, and iv) the statistical plan, accounting for biological variables including sex. The results of this project and future research derived from it will translate into novel neuron-specific therapeutic targets to attenuate memory deficits after birth asphyxia. The project will be performed in a 5-year period addressing the aims sequentially. The proposed experiments and timeline are within my capabilities and the capabilities of the laboratory, animal care, and surgical facilities. Future research will include the use of novel techniques to deliver small molecules and gene therapy to the brain to modulate cell-specific pathways in a sex and time-tailored manner, which combined with TH will improve behavioral outcomes after birth asphyxia.