VTA-NAc多巴胺能通路组蛋白修饰在老年小鼠全麻后苏醒延迟中作用的研究

Delayed recovery from general anesthesia tends to occur in aged patient. Except lower metabolism, aging-associated alteration in the brain is also one of the leading reasons. However, the underlying mechanisms are not well understood. Our preliminary data indicated that dopamine signaling in the ventral tegmental area (VTA)-nucleus accumbens (NAc) pathway plays a crucial role in modulating the process of arousal from general anesthesia. Moreover, aging-related alteration of histone modification can induce the changes of protein expression and function in the dopaminergic system in the brain. Therefore, we propose that the alteration of histone modification in the VTA-NAc dopamine pathway is a novel molecular mechanism underlying delayed recovery from general anesthesia in aged patient. To test this hypothesis, we will conduct a serial experiments in the animal model. ①We will observe the changes in dopamine-associated genes and proteins expression, and the modulation to propofol and isoflurane anesthesia in the VTA-NAc dopamine pathway in aged mice. ②Using in vivo CRISPR/Cas9 approaches, we will analyze the role of aging-related alterations of VTA-NAc dopaminergic system in the delayed recovery from general anesthesia. ③Using CRISPR/Cas9 to knock out the genes of histone-modifying enzymes, further study will be implemented to determine whether aging-related alterations in histone modification are the upstream molecular mechanism of the aforementioned alteration in the VTA-NAc dopaminergic system. Together, this work will stepwise probe the neural and molecular mechanisms underlying delayed recovery from general anesthesia in age mice, thereby providing important information for development of novel general anesthesia and safer strategies applicable to aged patients.

老年患者全身麻醉后常发生苏醒延迟，除代谢减慢外，大脑的老年化改变也是主要原因之一，但其中具体机制不明。我们的前期研究发现中脑腹侧被盖区（VTA）-伏隔核（NAc）多巴胺能通路对全麻觉醒发挥着重要的调控作用。而组蛋白修饰的老年化改变可导致多巴胺能系统蛋白表达及功能的变化，提示VTA-NAc多巴胺通路组蛋白修饰的改变可能是老年人全麻后苏醒延迟的重要分子机制。为验证这一假设，本研究拟：①观察老年小鼠VTA-NAc通路多巴胺能系统基因与蛋白表达、以及对丙泊酚和异氟醚全麻觉醒调控作用的变化；②通过在体CRISPR/dCas9分析通路中多巴胺能系统改变在老年小鼠全麻后苏醒延迟中的作用；③通过敲除组蛋白修饰酶基因，进一步明确组蛋白修饰的改变是否为上述多巴胺系统变化的上游分子机制。由此逐级探讨老年小鼠全麻后苏醒延迟的神经及分子机制，为开发适合老年人的全麻药以及寻求更加安全的老年全麻方案提供理论支持。

老年患者全身麻醉后常发生苏醒延迟，除代谢减慢外，大脑的老年化改变也是主要原因之一，但其中具体机制不明。中脑腹侧被盖区（VTA）-伏隔核（NAc）多巴胺能通路在睡眠-觉醒过程中发挥着重要的调控作用；同时，组蛋白修饰的老年化改变可导致多巴胺能系统蛋白表达及功能的变化，提示VTA-NAc多巴胺通路组蛋白修饰的老年化改变可能是老年人全麻后苏醒延迟的重要分子机制。为验证这一假设，本研究.1.首先通过光遗传学及化学遗传学技术，结合行为学和脑电记录，明确了VTA多巴胺能神经元、VTA-NAc多巴胺能通路以及该通路在NAc中释放的DA递质在全身麻醉中均发挥着重要的调控作用；.2.通过光纤钙信号、微注射、膜片钳技术，发现NAc中多巴胺D1受体的激活是VTA-NAc通路产生促进全身麻醉觉醒作用的重要下游途径;.3.结合微注射、行为学和脑电记录、Western blot等方法，发现老年小鼠NAc D1受体促全身麻醉觉醒的功能较青年小鼠显著降低，主要可能与老年小鼠D1受体表达下调有关；.4.由于老年化原因导致的D1受体表达水平下调可能与组蛋白乙酰化修饰相关，后续我们发现老年小鼠NAc内HDAC1和HDAC2较青年小鼠均显著升高，这可能是D1受体表达下调的重要表观遗传学机制；.5.为明确这一机制，我们在青年小鼠NAc中分别上调HDAC1和HDAC2表达，均发现小鼠诱导时间显著缩短，觉醒时间延长，在麻醉过程中发生了老年化趋势，但同时上调D1受体可消除该影响；在老年小鼠敲低NAc内HDAC1后觉醒时间明显缩短，说明了NAc中HDAC1水平在老年动物麻醉后苏醒延迟中发挥着重要作用。.本研究证明了脑内组蛋白乙酰化转移酶的老年化改变可导致NAc多巴胺D1受体水平下调，促全身麻醉觉醒功能减弱，可能是老年动物全麻后苏醒延迟的重要神经机制之一。该发现可为开发适合老年人的全麻药以及寻求更加安全的老年全麻方案提供理论支持。