Brainstem Cholinergic Circuitries in the Inflammatory Reflex during Sepsis

翻译后摘要：这项建议的目的是阐明脑干调节胆碱能电路参与 脓毒症时迷走神经为基础的炎症反射。败血症是一种复杂的致命疾病，定义为“生命- 由宿主对感染的反应失调引起的威胁性器官功能障碍”。败血症的发病率是 每年增加约170亿美元。此外，许多“败血症幸存者”发展免疫功能障碍， 慢性疾病和严重的功能性残疾，可能导致死亡。一个重要的未被充分研究的方面 脓毒症是神经系统受累。我们率先研究了神经 系统和炎症反射，并提供了重要的见解有关的作用，这一神经 在脓毒症和以免疫功能障碍为特征的其他病症中的免疫调节机制。这些 临床前的发现最近导致了通过迷走神经的炎症反射的成功临床探索 类风湿性关节炎的治疗方法尽管取得了这一进展，但我们在这方面仍存在重大差距。 了解迷走神经为基础的炎症反射及其与脓毒症的相关性。传出迷走神经 纤维起源于两个脑干核团，迷走神经背侧运动核（DMN）和疑核 （不适用）。实验证据支持传出迷走神经纤维与脾神经在神经节内相互作用 腹腔（太阳）神经丛的炎症反射。然而，传出迷走神经的脑干起源 对炎症反射至关重要的神经纤维以及这些回路在调节免疫反应中的特定作用 以及脓毒症期间的代谢指标目前是未知的。我们假设脑干DMN和NA 传出胆碱能纤维在脓毒症期间的炎症反射中具有不同的作用。这一假设将 通过以下具体目标进行测试： 具体目标1。阐明脑干胆碱能回路在迷走神经基础的 炎症反射工作假设：起源于脑干DMN的传出迷走神经纤维， NA差异有助于迷走神经为基础的炎症反射脾脏。我们将利用：1） 通过Cre重组酶（Cre-on）转录激活的重组腺相关病毒， 小鼠脑干胆碱能回路的精确分子定位; 2）选择性光遗传学胆碱能 刺激脑干回路以检查脾脏中乙酰胆碱的释放和抗炎作用 在内毒素血症小鼠中;和3）选择性迷走神经切断术以确认迷走神经的作用。 具体目标2。选择性激活脑干核团胆碱能神经元对脑缺血再灌注损伤的影响 炎症和代谢标志物与脓毒症后的存活率之间的关系。工作假设：选择性 DMN和NA的胆碱能光遗传学刺激差异性地阐明脓毒症紊乱。我们将 使用CLP后小鼠脑干核的选择性光遗传学刺激进行实验， 检查对以下方面的影响：1）急性和慢性阶段的炎症和代谢指标; 2）存活率。

Abstract: The objective of this proposal is to elucidate brainstem regulatory cholinergic circuitries involved in the vagus nerve-based inflammatory reflex during sepsis. Sepsis is a complex lethal condition defined as “life- threatening organ dysfunction caused by a dysregulated host response to infection”. The incidence of sepsis is increasing and costs approx $17B per year. Moreover, many “sepsis survivors” develop immune dysfunction, chronic illness, and severe functional disabilities, which may result in death. An essential understudied aspect of sepsis is the involvement of the nervous system. We have pioneered studies on the role of the nervous system and the inflammatory reflex and have provided important insight related to the role of this neural immunomodulatory mechanism in sepsis and other conditions characterized by immune dysfunction. These preclinical findings recently led to successful clinical exploration of the inflammatory reflex via vagus nerve stimulation in patients with rheumatoid arthritis. Despite this progress, there are major gaps in our understanding of the vagus nerve-based inflammatory reflex and its relevance to sepsis. Efferent vagus nerve fibers originate in two brainstem nuclei, the dorsal motor nucleus of the vagus (DMN) and nucleus ambiguus (NA). Experimental evidence supports that efferent vagus nerve fibers interact with the splenic nerve in ganglia of the celiac (solar) plexus within the inflammatory reflex. However, the brainstem origin of the efferent vagus nerve fibers critical to the inflammatory reflex and the specific role(s) of these circuitries in regulating immune and metabolic indices during sepsis are presently unknown. We hypothesize that brainstem DMN and NA efferent cholinergic fibers have differential roles in the inflammatory reflex during sepsis. This hypothesis will be tested by the following Specific Aims: Specific Aim 1. Elucidate the specific role of brainstem cholinergic circuitries in the vagus nerve-based inflammatory reflex. Working hypothesis: Efferent vagus nerve fibers originating in the brainstem DMN and NA differentially contribute to the vagus nerve-based inflammatory reflex to the spleen. We will utilize: 1) recombinant adeno-associated viruses transcriptionally activated by Cre recombinase (Cre-on) to achieve precise molecular mapping of brainstem cholinergic circuitries in mice; 2) selective optogenetic cholinergic stimulation of brainstem circuitries to examine acetylcholine release in the spleen and anti-inflammatory effects in endotoxemic mice; and 3) selective vagotomies to confirm the role of the vagus nerve. Specific Aim 2. Evaluate the effects of selective cholinergic activation of brainstem nuclei on inflammatory and metabolic markers and survival following sepsis. Working hypothesis: Selective cholinergic optogenetic stimulation of DMN and NA differentially alleviates sepsis derangements. We will perform experiments using selective optogenetic stimulation of brainstem nuclei of mice following CLP to examine effects on: 1) inflammatory and metabolic indices in acute and chronic stages; and 2) survival.