Brainstem Cholinergic Circuitries in the Inflammatory Reflex during Sepsis

摘要：本研究的目的是阐明脑干调节胆碱能回路。 败血症时以迷走神经为基础的炎症反射。败血症是一种复杂的致命性疾病，其定义为“生命-- 威胁着因宿主对感染的反应失调而导致的器官功能障碍“。败血症的发病率是 不断增加，每年耗资约170亿美元。此外，许多“败血症幸存者”会出现免疫功能障碍， 慢性病和可能导致死亡的严重功能残疾。一个基本的未被研究的方面 败血症的主要原因是神经系统受累。我们对神经质的作用进行了开创性的研究 系统和炎症反射，并提供了与该神经的作用相关的重要洞察力 脓毒症和其他以免疫功能障碍为特征的疾病的免疫调节机制。这些 最近临床前的发现导致了通过迷走神经的炎性反射的成功的临床探索 类风湿关节炎患者的刺激。尽管取得了这些进展，但在我们的 了解以迷走神经为基础的炎症反射及其与败血症的关系。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 (NA)。实验证据支持在神经节内传出迷走神经纤维与脾神经相互作用 炎性反射中的腹膜(太阳)神经丛。然而，传出迷走神经的脑干起源 炎性反射的关键神经纤维和这些回路在调节免疫中的特殊作用(S) 脓毒症期间的代谢指标目前尚不清楚。我们假设脑干DMN和NA 在脓毒症时，传出胆碱能纤维在炎症反射中具有不同的作用。这一假说将 通过以下具体目标进行测试： 具体目的1.阐明脑干胆碱能环路在迷走神经基础上的特殊作用 炎症反射。工作假设：迷走神经传出纤维起源于脑干DMN和 NA对迷走神经为基础的炎症反射到脾有不同的贡献。我们将利用：1) 由Cre重组酶(Cre-On)转录激活的重组腺相关病毒实现 小鼠脑干胆碱能回路的精确分子定位；2)选择性光遗传胆碱能 刺激脑干回路以检查脾中乙酰胆碱的释放和抗炎作用 内毒素血症小鼠；以及3)选择性迷走神经切断术，以确认迷走神经的作用。 特定目的2.评价选择性激活脑干核团胆碱能对大鼠脑内胆碱能神经元的影响 炎症和代谢标记物与脓毒症后的生存。工作假设：选择性 胆碱能光遗传刺激DMN和NA可不同程度地减轻脓毒症的紊乱。我们会 用选择性光遗传刺激小鼠脑干核团进行实验 检查对以下方面的影响：1)急性期和慢性期的炎症和代谢指标；2)生存期。

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.