Enteric Glial Mechanotransduction and Mechanical Stress Induced Postoperative Ileus - Despite the implementation of enhanced recovery protocols for gastrointestinal (GI) surgeries (ERAS), 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. A better understanding of the pathogenic mechanism(s) of POI is required to develop better therapeutic strategies. General Hypothesis: Our recent work, strong pilot, feasibility and preliminary data (Figs 1-18) and publications support the novel hypothesis that the glial Piezo-1“mechanosensor” modulates motility, and the mechanical stress’’ on the bowel during GI surgery results in amplification in the Piezo-1/Cx43-ATP signaling pathway to cause disruption of motility, POI and POGD in the context of intestinal inflammation. Targeting a glial mechanosensor (Piezo-1) is a paradigm shift in our thinking about the pathogenesis of POI, and it seems reasonable, since gut physical manipulation (“mechanical stress”) is sufficient to cause POI. We assembled a dream team of investigators and plan to utilize an impressive array of novel innovative models and techniques for mouse or human studies on mechanogated channels. These include glial specific cKO mice for Piezo-1, Cx43 HCs and mitochondrial complex-1, RiboTag–gene reporter mice for glia or neurons, human models to study glia, isolated neural networks of ganglia and Early/Late human specimens to study impact of surgical stress on mechanogated channels, bulk-RNAseq in RiboTag, Ca2+wave studies, and a ‘first in man’ patch clamp technique for whole cell recordings. The “mechanotransduction hypothesis” is tested in 3 specific aims in mice and humans. Aim 1 will determine the role of Piezo-1 and Cx43 HCs in glial mechanotransduction and intestinal motility, using Ca2+reporter mice, pharmacologic agents or glial cKO mice for deletion of Piezo-1 and Cx43. Aim 2 will unravel the pathogenic role of Piezo 1 channels and Cx43 HCs in POI using a surgical model of “mechanical stress” induced POI. Studies will (1) Investigate the protective effect of deleting Piezo1 and Cx43 in glia on inflammation and GI dysmotility (hallmarks of the disorder) in mouse POI. (2) Bulk-RNAseq analysis in glial or neuronal RiboTag-Piezo1cKO or - Cx43cKO mice (and controls) will evaluate whether deletion of specific channels can protect against mechanical stress induced POI. (3) We will investigate Ca2+waves in glial conditional knockouts for Piezo-1 and Cx43 HCs. (4) Cell-stretch (FlexCELL) is used to test if “mechanical stress” can cause a reactive glial phenotype by activating Piezo-1 channels. (5) And we will evaluate mitochondrial dysregulation in enteric gliosis. AIM 3 will test whether findings from animals are translatable to humans on Piezo-1 and Cx43 – mechanotransduction and evaluate impact of “surgical stress” in Early/Late samples of ileum, from 65 right colectomy patients. OVERALL IMPACT: Studies will address a critical gap in knowledge, identify the “glial Piezo mechanosensor” operating in motility and the pathogenic mechanism of POI induced by mechanical stress during GI surgery, perhaps offering a possible new way to protect the gut.
