Cellular and molecular mechanisms underlying anesthetic actions and neurotoxicity

有证据表明，最常用的局部用药(如异丙酚)吸入麻醉剂(七氟醚、地氟烷等)可能会产生长期的副作用，导致动物的神经毒性和学习记忆障碍，但仍缺乏直接证据。我的研究项目得到了NSERC的支持，一直专注于确定麻醉剂诱导的神经毒性的细胞和分子机制-在蜗牛和大鼠模型中。然而，尽管有这些进展，我们在以下方面的知识仍然存在重大差距：a)麻醉剂诱导的神经毒性的潜在靶点，b)如何最好地防止麻醉剂诱导的对神经元的伤害，以及c)开发毒性最小且更有效的新型麻醉剂化合物。我们有四个明确的目标，将直接解决这些问题。 目的#1：确定p110肽是否减轻麻醉诱导的淋巴和大鼠神经元毒性。 我们已经证明了麻醉药诱导的神经毒性涉及突触蛋白以及线粒体的结构和功能的扰动。然而，线粒体融合抑制剂Mdivi-1可以逆转这种神经毒性作用，但这种化合物是非特异性的，如果用于活体动物，本身可能是神经毒性的。我们现在希望证明，预先将培养的神经元暴露于p110肽将防止麻醉诱导的细胞死亡、抑制突起生长和突触蛋白的结构和功能。 目的#2：观察麻醉诱导的完整大鼠脑片毒性。 我们将使用我们新开发并获得专利的3-D芯片(NSERC)，分离的海马脑片将被连接到CA3-CA1突触的突触可塑性监测。这项研究将提供第一个直接证据，证明麻醉剂确实扰乱了完整脑片中的神经元通讯和突触可塑性。然后，我们将使用p110肽来阻断麻醉剂诱导的毒性。 目的#3：探讨孕期雌性大鼠暴露对新生仔鼠学习记忆的影响。 我们将使怀孕的大鼠或其新生的幼鼠(P2-P12)暴露在七氟醚、地氟烷、异丙酚等环境中20-30分钟，并将全程监测它们的生命体征。暴露后4-8周，我们将对这些动物进行各种学习和记忆任务(新奇物体识别、水迷宫、恐惧条件反射、Morris水迷宫等)测试。对照组注射空气或生理盐水的动物将作为对照组。 目的#4：应用p110多肽减轻麻醉性学习记忆障碍。 我们将寻求通过用p110预处理幼鼠来减轻麻醉剂对认知和学习记忆的影响。我们预计该多肽将使动物免于麻醉剂引起的细胞毒性。总之，这些研究将对麻醉剂诱导毒性的潜在机制形成新的理解，并揭示缓解策略。

Evidence suggests that most commonly used local (Propofol e.g.) and inhalation (sevoflurane, desflurane etc) anesthetic agents may exert long-term side effects leading to neurotoxicity and learning and memory deficits in animals, however a direct evidence is still lacking. My research program, supported by NSERC has been focused at defining the cellular and molecular mechanisms underlying anesthetic-induced neurotoxicity - in both snail and rat models. Notwithstanding these advances, however there remain significant gaps in our knowledge vis-à-vis:a) the potential target sites for anesthetic-induced neurotoxicity, b) how best to prevent anesthetic-induced insult to neurons, and c) to develop novel anesthetic compounds that are least toxic and more efficacious. We have four specific aims with well-defined objectives that will address these issues directly. Aim #1: To determine whether P110 peptide mitigates anesthetic-induced neuronal toxicity in cultured Lymnaea and rat neurons. We have demonstrated that anesthetics-induced neurotoxicity involves perturbation of synaptic proteins as well as mitochondrial structure and function. Whereas the neurotoxic effects were reversed with a mitochondrial fusion inhibitor Mdivi-1, this compound is however non-specific and may itself be neurotoxic if used in live animals. We now wish to demonstrate that prior exposure of cultured neurons to P110 peptide will prevent anesthetic induced cell death, suppression of neurite outgrowth and synaptic proteins structure and function. Aim #2: To demonstrate anesthetic-induced toxicity in the intact rat brain slices. We will use our newly developed and patented 3-D chip (NSERC), and the isolated hippocampal brain slices will be interfaced to monitor synaptic plasticity at the CA3-CA1 synapse. This study will provide the first direct evidence that indeed anesthetics perturb neuronal communications and synaptic plasticity in the intact brain slices. We will then use P110 peptide to block anesthetic induced toxicity. Aim #3: To demonstrate whether exposure of pregnant female rats affects learning and memory in the new born pups. We will expose either pregnant rats or their new born pups (P2-P12) to sevoflurane, desflurane, propofol etc. for 20-30 minutes and their vital signs will be monitored throughout. 4-8 weeks post exposure, we will test these animals with various learning and memory tasks (novel object recognition, water maze, fear conditioning, Morris Water maze etc). Control air or saline injected animals will be used as controls. Aim #4: To mitigate anesthetic-induced deficit in learning and memory with P110 peptide. We will seek to mitigate anesthetic-induced effects on cognition and learning and memory by pre-treating the pups with P110. We anticipate that the peptide will rescue animals from anesthetic induced cytotoxicity. Together these studies will forge novel understanding of mechanisms underlying anesthetic-induced toxicity and reveal mitigation strategies.