Investigation of Layer-specific Responses to Colon Anastomosis and Disturbed Healing Pathways in Non-steroidal Anti-inflammatory Drug (NSAID)-induced Colon Anastomotic Leakage

Abstract

<strong>Introduction: </strong> Colon anastomotic leakage (CAL) is a postoperative complication originating from disturbed colon anastomotic healing (CAH). Wound healing involves several well-coordinated stages, which have not been comprehensively studied for CAH or CAL. The first aim of this study was to provide transcriptional profiles of different intestinal layers of anastomotic tissues throughout distinct healing stages and to identify CAL-related genes. Non-steroidal anti-inflammatory drugs (NSAIDs) prevent PGE2 production by inhibiting the COX pathway. Although postoperative NSAID treatment has been recognized as a risk factor for CAL, its molecular and cellular effects on the anastomotic tissue have not been investigated in detail. The second aim of this study was to identify the causative pathways behind NSAID-induced CAL while investigating the roles of PGE2 in CAH. <br /> <strong>Methods: </strong> To achieve the first aim of this project, proximal colon anastomosis was constructed with 8 interrupted sutures in mice. 6 h, 24 h, and 72 h after surgery, anastomotic complications were assessed. Transcriptional profiles of the inner (mucosa and submucosa) and outer (muscularis externa) layers of the anastomotic and naive control tissues were analyzed with 3’ bulk mRNA sequencing to identify the layer-specific healing and leakage pathways. For the second aim of this project, mice were subjected to postoperative diclofenac sodium (DCF) treatment. Transcriptional and cellular changes induced by DCF treatment in anastomotic tissues were analyzed at postoperative day (POD) 2 with 3’ bulk RNA-Seq and flow cytometry. Tissue level PGE2 concentration was determined with nano-DESI MS imaging and ELISA. For testing the beneficial effects of PGE2 on CAH, DCF-induced and insufficient suture CAL mouse models were treated with dmPGE2 and an inhibitor of PGE2-degrading enzyme named SW033291. <br /> <strong>Results: </strong> Our data regarding the first aim of the project indicate that the mucosa/submucosa and muscularis externa enter the inflammation stage at 6 h, the proliferation stage at 24 h, and the tissue remodelling stage at 72 h during CAH. We observed that transcription profiles of the mucosa/submucosa, but not the muscularis externa, differ between CAH and CAL. Particularly, genes related to extracellular remodelling and wound healing showed lower expression in the mucosa/submucosa of CAL tissue compared to CAH. Results regarding the second aim of the project showed an enrichment of the adaptive immune responses in the anastomosis of the DCF-treated mice. Confirming the detrimental effects of adaptive immune responses, we observed a significant reduction in ACS and enrichment of extracellular matrix-associated pathways in DCF-treated Rag1-/- mice. We identified PGE2 as the most abundant prostaglandin in the anastomotic tissues. While dmPGE2 supplementation did not prevent anastomotic healing deficiencies in the DCFinduced CAL model, 10 mg/kg SW033291 treatment significantly reduced ACS in insufficient suture CAL mouse model. <br /> <strong>Conclusions: </strong> Mucosa/submucosa and muscularis externa are mostly in synchronization during the inflammation, proliferation, and extracellular remodelling stages during CAH. Transcriptional profiles within the anastomotic mucosa/submucosa differ between CAH and CAL in genes related to extracellular modeling and wound healing, indicating that genes of these pathways may contribute to CAL. Exaggerated adaptive immune response is one of the causative factors for DCF-induced CAL. As the main type of prostaglandin in the anastomotic tissue, PGE2 promotes CAH, however, the lack of PGE2 signalling may not be the sole reason behind DCF-induced CAL. Future studies would be needed to further descipher the effect of PGE2 signalling on NSAID-(un)related CAL.