Enteric glial ETBR signaling in postoperative ileus

尽管实施了GI手术的增强恢复方案， 术后肠梗阻（POI）和术后胃肠道功能障碍（POGD） 住院、发病率和医疗保健费用增加到数十亿美元。正在研发的新型疗法包括 有限公司需要更好地理解POI的致病机制，以开发更好的治疗方法。 战略布局新出现的证据表明，肠神经胶质细胞在人类的健康和疾病中起着重要作用。 胃肠道内皮素（ET-1）信号在肠道反应性肠胶质细胞中的作用是一个新的靶点， 为我们研究POI和POGD的发病机制（Gulbrensan & Christofi，Gastro，2018）。我们最近 工作，强大的试点，可行性和初步数据（图1-16），摘要和出版物支持新的 假设肠神经胶质中的神经胶质ET-1 / ETBR信号传导破坏运动性，并且其有助于 在肠道炎症背景下POI和POGD的发病机制。致病机制 涉及内皮素的包括神经胶质对ET-1/ETBR信号传导的超敏性，反应性神经胶质细胞的诱导， 表型、肠神经胶质增生、肠神经病和神经运动通路的改变。我们的研究 还将研究靶向ETBR在POI和POGD中的治疗潜力。我们将测试我们的 在小鼠和人类中的3个具体目标的一般假设：目标1将集中于ET-1/ETBR的机制 运动抑制。采用多层次方法研究ETBR对小鼠和人运动性的影响，包括 肌肉收缩、蠕动波、颗粒推进、上升和下降反射以及GI传输。 神经胶质、神经元和运动性中的ETBR信号传导在神经胶质、神经元的细胞特异性Ca 2+报告小鼠中进行研究， 兴奋性和抑制性神经元通路，胶质细胞条件性ETBR消融或ET-1上调的转基因模型 调节和ETBR-EGFP报告基因小鼠。测试了从小鼠到人类的发现的可转化性， 人EGC网络中的Ca 2+波，“人类首次"肌间神经节网络中的膜片钳记录”， 和ETBR研究上升和下降反射。目标2将重点探讨 POI中肠神经胶质细胞中的ETBR信号传导-致病机制包括ET-1/ETBR中的超敏反应 在RiboTag小鼠中通过RNAseq分析的反应性神经胶质表型的诱导，以及通过 将RiboTag与神经胶质特异性ETBR敲除交叉。在hEGCs中测试了结果对人类的可转化性 或神经节网络的钙离子或膜片钳记录。在小鼠中测试对肠神经病变的影响， 神经胶质消融ETBR或ET-1上调Ca 2+报告小鼠神经元。目标3将重点关注胶质ET-1 和ETBR作为POI的治疗靶点，使用POI和结肠切除术的肠道手术操作模型 POI患者，和药理学拮抗剂在小鼠中，或测试在小鼠中与神经胶质消融ETBR或以上 ET-1的调节。总体而言，术后肠梗阻的一种新的胶质ET-1 / ETBR致病机制可能是 被鉴定为预防性治疗的相关治疗靶点。

Despite the implementation of enhanced recovery protocols for GI surgeries, there is still significant postoperative ileus (POI) and postoperative gastrointestinal dysfunction (POGD) associated with prolonged hospitalizations, increased morbidity and health care costs into the billions. Novel therapies in the pipeline are limited. A better understanding of the pathogenic mechanism(s) of POI is required to develop better therapeutic strategies. Emerging evidence indicates that enteric glia play an important role in health and disease of the gastrointestinal tract. Endothelin (ET-1) signaling'` in `reactive enteric glia' in the gut is a novel target of investigation for us in the pathogenesis of POI and POGD (Gulbrensan & Christofi, Gastro, 2018). Our recent work, strong pilot, feasibility and preliminary data (Figs 1-16), abstracts and publications support the novel hypothesis that glial ET-1 / ETBR signaling in enteric glia disrupts motility and it contributes to the pathogenesis of POI and POGD in the context of intestinal inflammation. Pathogenic mechanism(s) involving endothelins include glial hypersensitivity to ET-1/ETBR signaling, induction of a reactive glial phenotype, enteric gliosis, enteric neuropathy, and alterations in neural-motor pathways. Our study will also investigate the therapeutic potential of targeting ETBR in POI and POGD. We will test our general hypothesis in 3 specific aims in mice and humans: Aim 1 will focus on the Mechanism of ET-1/ETBR inhibition of motility. A multi-tier approach is used to study ETBR on motility in mouse and human, including muscle contraction, peristaltic waves, pellet propulsion, ascending and descending reflexes and GI transit. ETBR signaling in glia, neurons and motility is studied in cell-specific Ca2+ reporter mice for glia, neurons, excitatory and inhibitory neuron pathways, transgenic models of glial conditional ETBR ablation or ET-1 up regulation, and ETBR-EGFP reporter mice. Translatability of findings from mice to humans is tested on Ca2+waves in human EGC networks, `first in human' patch clamp recordings in networks of myenteric ganglia', and ETBR studies of ascending and descending reflexes. Aim 2 will focus on the pathogenic mechanism of ETBR signaling in enteric glia in POI – Pathogenic mechanisms include hypersensitivity in ET-1/ETBR signaling, induction of a reactive glial phenotype analyzed by RNAseq in RiboTag mice, and mice generated by crossing RiboTag with glial-specific ETBR knockouts. Translatability of findings to humans is tested in hEGCs or networks of ganglia with Ca2+or patch clamp recordings. Impact on enteric neuropathy is tested in mice with glial ablation of ETBR or ET-1 up regulation in Ca2+reporter mice for neurons. Aim 3 will focus on glial ET-1 and ETBR as a therapeutic target in POI using a gut surgical manipulation model of POI and colectomy patients with POI, and pharmacological antagonists in mice, or testing in mice with glial ablation of ETBR or up regulation of ET-1. Overall, a novel glial ET-1 / ETBR pathogenic mechanism of postoperative ileus may be identified as a relevant therapeutic target for prophylactic treatment.