Mechanisms and therapeutic interventions of postoperative gastric ileus

术后肠梗阻（POI）是腹部外科手术的主要并发症之一。 一些部署在战区的退伍军人受伤需要AS。AS后，患者通常 出现恶心、呕吐、腹胀和腹痛，这是术后 不适肠梗阻的发生率在胃肠道（GI）手术中最高， 肠梗阻，在剖腹手术患者中可高达40%。健康负担和长期的成本 在美国，由于POI导致的住院治疗估计每年高达14.7亿美元，说明 巨大的社会经济影响。缺乏有效的治疗方法促使人们进行新的实验研究， 阐明潜在的机制。最近对AS诱导的POI的病理生理学的认识， 确定了处理肠道引发的肠道炎症是一种促进机制， 临床上是治疗的相关目标。在其他炎症性疾病中，有证据表明， 胃肠道肌层内的巨噬细胞有助于炎症的发生和消退 通过激活分别分泌促炎细胞因子和抗炎细胞因子的M1和M2表型。 最近的研究指出迷走神经控制胆碱能抗炎通路。我们之前 确定脑干中的促甲状腺激素释放激素（TRH）起着生理作用（包括在 头期）以刺激支配胃肠道的迷走神经。在上一个赠款期间，我们报告说， 脑池内（IC）注射TRH可防止AS 2小时内发生的神经源性（早期）POI。我们 在术后6小时获得的初步数据表明：1）AS增加M1但不增加M2巨噬细胞， 胃外肌层中性粒细胞浸润，沿着胃排空延迟（GE）; 2）中央 通过IC注射稳定的TRH激动剂RX 77368激活迷走神经可防止上述增加，并减少 假手术组在不改变基础GE的情况下，降低AS诱导的延迟GE。在最后一次授予期间，我们还 已经确定AS诱导促动力激素（ghrelin）的血浆水平急剧下降， 影响迷走神经活动，并在AS前用IC TRH阻止这种反应。此外，我们还获得了 初步数据显示，新的长效和脑渗透性生长激素释放肽激动剂，HM 01给予 口服激活脑干中的迷走神经节前运动神经元并防止AS诱导的延迟GE。 基于这些报告和令人兴奋的支持性初步数据，我们将测试3个假设：目标1。作为 通过改变M1或M2的激活状态诱导大鼠胃外肌层炎症 大鼠胃外肌层巨噬细胞。2.中枢迷走神经刺激预防AS诱导的延迟性 GE通过激活胆碱能抗炎通路，使M1失活和/或激活M2 巨噬细胞，导致抑制胃外肌层的炎症。3.生长激素释放肽激动剂， HM 01是一种有前途的候选药物，通过口服给药，通过其双重有效的促动力和抗- 通过激活迷走神经胆碱能通路的炎症作用。这些目标将在鼠 AS诱导的POI模型结合神经解剖学的最新技术（PALITY技术 结合靶向双重或三重标记，包括迷走神经纤维的顺行追踪和3D成像， 肠神经元和巨噬细胞），分子生物学（激光显微切割结合RT-PCR， RNAscope，RT-qPCR，microRNA靶向，MILLIPLEX® Multiplex Assay使用Bio-RAD Bio-Risk 3D 由Luminex xMAP技术提供动力的悬浮系统）和功能研究（肠道运动和化学 迷走神经活动的刺激）。这些具体目标的完成，将在概念上推进实现 通过M1失活和/或M2活化作为潜在的抗炎药， 策略，并提供第一个临床前数据，以验证HM 01作为POI新疗法的候选药物。

Postoperative ileus (POI) is one of the main complication associated with abdominal surgery (AS) procedures. A number of Veterans deployed in the war zones undergo injuries requiring AS. After AS, patients usually develop nausea, vomiting, bloating, and abdominal pain which are major contributing factors to postoperative discomfort. The incidence of ileus is highest in gastrointestinal (GI) surgery with 24% of patients developing ileus and can be as high as 40% in laparotomy patients. The health burden and the cost of prolonged hospitalization due to POI have been estimated to be as much as $1.47 billion annually in the USA, illustrating its large socioeconomic impact. The lack of effective treatments has prompted novel experimental studies to elucidate the underlying mechanisms. Recent insight in the pathophysiology of POI induced by AS have identified intestinal inflammation triggered by handling of the intestine as a contributing mechanism which is clinically a relevant target for treatment. In other inflammatory conditions, there is evidence that resident macrophages within the GI muscularis contribute to both the initiation and the resolution of inflammation through activations of M1 and M2 phenotypes secreting pro- and anti-inflammatory cytokines respectively. Recent studies point to the vagus nerve controlling a cholinergic anti-inflammatory pathway. We previously established that thyrotropin-releasing hormone (TRH) in the brainstem plays a physiological role (including in the cephalic phase) to stimulate the vagus innervating the GI tract. In the last granting period, we reported that intracisternal (ic) injection of TRH prevents the neurogenic (early phase) of POI occurring within 2-h of AS. Our preliminary data obtained at 6-h post-surgery indicate that 1) AS increases M1 but not M2 macrophages and the infiltration of neutrophils in the gastric muscularis externa along with delay gastric emptying (GE); 2) central vagal activation by ic injection of the stable TRH agonist, RX77368 prevents the above increases and reduces the delayed GE induced by AS without modifying basal GE in sham group. In the last granting period, we also established that AS induces a sharp reduction of plasma levels of the prokinetic hormone, ghrelin known to influence vagal activity and the response is prevented by ic TRH before AS. In addition, we obtained preliminary data showing that the novel long acting and brain penetrant ghrelin agonist, HM01 administered orally activates vagal preganglionic motor neurons in the brainstem and prevents AS-induced delayed GE. Based on these reports and exciting supportive preliminary data, we will test 3 HYPOTHESES: Aim 1. AS induces inflammation in the rat gastric muscularis externa through changes in the activation status of M1 or M2 macrophages in the rat gastric muscularis externa. 2. Central vagal stimulation prevents AS-induced delayed GE by activating cholinergic anti-inflammatory pathway with the deactivation of M1 and or the activation of M2 macrophage leading to inhibiting the inflammation in the gastric muscularis externa. 3. The ghrelin agonist, HM01 is a promising candidate via oral administration to reverse POI by its dual potent prokinetic and anti- inflammatory actions through activation of vagal cholinergic pathway. These aims will be achieved in the rat model of AS-induced POI combined with state-of-the art technologies in neuroanatomy (CLARITY technique combined with targeted double or triple labeling, including anterograde tracing and 3D imaging of vagal fibers, enteric neurons and macrophages), molecular biology (Laser microdissection combined with RT-PCR, RNAscope, RT-qPCR, microRNA targeting, MILLIPLEX® Multiplex Assays using Bio-RAD Bio-Plex 3D suspension system powered by Luminex xMAP Technology) and functional study (gut motility and chemical stimulation of vagal activity). The completion of these specific aims will make a conceptual advance to target muscularis macrophages by deactivation of M1 and/or activation of M2 as a potential anti-inflammatory strategy and provide the first preclinical data to validate HM01 as a candidate for new therapy for POI.