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Introduction: Intraabdominal adhesions are an inevitable consequence of surgery. Despite good surgical technique, unavoidable ischemic and hypoxic events can exacerbate adhesiogenesis. Our laboratory has shown that the NK-1R, its ligand substance P (SP) and decreased peritoneal tissue plasminogen activator (tPA) secretion are correlated with adhesiogenesis in vivo. However, the mechanism by which NK-1R expression is regulated during adhesiogenesis remains unclear. We hypothesized that surgically-induced hypoxia, as a result of tying or clamping off vessels or electrocautery, may be a critical event modulating NK-1R expression and adhesion formation. The aim of this study was to evaluate the effect of hypoxia on NK-1R signaling and tPA secretion in vitro. Methods: Primary RPMCs were isolated 24-hrs after our previously described adhesion-inducing ischemic button surgery. Confluent cells were incubated under hypoxic (1% O2) or normoxic (21% O2) conditions for 24-hrs and exposed to SP (10 and 100 nM), a NK-1R antagonist (NK-1RA; CJ-12,255, Pfizer, Inc.), or SP+NK-1RA. Cells were harvested to assess the upregulation of NK-1R signaling by Western blot for phosphorylated-ERK1/2 proteins, key downstream intracellular signaling molecules in the NK-1R pathway. Media was harvested to measure tPA secretion via ELISA. In addition, rat peritoneal tissue was collected to assess NK-1R expression by immunofluorescence and tissue hypoxia by pimonidazole staining. Results: Pimonidazole staining was increased in peritoneal tissue after ischemic button surgery compared to non-operative controls. Immunofluorescence showed upregulation of the NK-1R in vivo, primarily localized to the mesothelial cell layer on the surface of ischemic buttons within 6-hrs of surgery compared to non-operative controls. In vitro, phospho-ERK1 levels were increased in hypoxic RPMCs by 1.6 fold with both 10nM SP (1.0 vs. 1.66 +/- 0.12; p<0.05) and 100nM SP (1.0 vs. 1.61 +/- 0.43; p<0.05) compared to normoxic controls. Phospho-ERK2 levels were also increased in hypoxic RPMCs by 1.6 fold (1.0 vs. 1.64 +/- 0.22; p<0.05) with 10nM SP and 1.8 fold (1.0 vs. 1.86 +/- 0.24; p<0.05) with 100nM SP versus normoxic controls. NK-1RA attenuated this effect by reducing phospho-ERK1 levels by 54% (1.0 vs. 0.46 +/- 0.13; p<0.01) and phospho-ERK2 levels by 51% (1.0 vs. 0.49 +/- 0.16; p<0.01). SP-mediated tPA secretion was decreased by 45% (100 +/-19 vs. 55.1+/-19, p=0.017) under hypoxic conditions while no significant reduction was seen in normoxia compared to controls. Conclusions: Ischemia, or hypoxia, contributes to adhesion formation through the upregulation of NK-1R signaling suggesting that early hypoxia-induced signaling pathways may be novel targets for adhesion prevention.