Sepsis, defined as a systemic inflammatory response of the body to an infection, is frequently encountered in hospitals and is a major cause for mortality. While sepsis can originate from almost every organ or tissue, the gastrointestinal tract seems to play a central role in the pathophysiology of sepsis. During sepsis, systemic inflammation can induce the production of pro and anti-inflammatory mediators, therefore gastrointestinal inflammation. Gastrointestinal inflammation, hypoperfusion and ischemia-reperfusion injury can eventually result in gastrointestinal barrier disruption, with failure of the barrier function and as a result increased permeability of pathogenic molecules and microbiota. On the other hand, intra and extra luminal, gastrointestinal inflammatory processes can lead to intestinal barrier failure as well. The gastrointestinal tract can, as a result, also be the source of sepsis.
As the gastrointestinal barrier seems to play a central role, we hypothesized that future therapies should also focus on modulating this leaky barrier during sepsis. Identified potential targets for modulation are intestinal alkaline phosphatase and protease activity. This project therefore aims to investigate the role of intestinal barrier function, intestinal alkaline phosphatase (IAP) and protease activity in a translational model for sepsis. As our laboratory experiments occur in a well-controlled environment, we aim to collect human samples to cross-validate our laboratory set-up with the clinical setting of sepsis.
To achieve these goals, this project consist of 2 stages. During the first stage of the project, the effect of exogenously administered IAP will be determined in a caecal ligation and puncture (CLP) mouse model for sepsis. For this, a CLP-procedure will be performed. In this procedure, 50% of the caecum is ligated after which it is punctured with a 21 G needle to induce a reproducible, polymicrobial abdominal sepsis. 1 IU/g IAP is administered twice a day for 2 consecutive days and mice are monitored clinically. On the second day, the abdomen is reopened, the distal ileum is ligated and 4kDa-FITC Dextran is injected directly to the lumen. Mice are kept sedated for one hour, after which they are sacrificed, blood is collected and abdominal tissue samples are collected. Blood is analyzed fluorospectrophotometrically to assess the migration of 4kDa FITC over the gastrointestinal barrier as a measure for intestinal permeability. Tissue samples are used to determine gene expression of cytokines and tight junction proteins, microscopy and to determine expression on the protein level. In collaboration with the department of Medical Microbiology, we will determine bacterial translocation as a result of the induced sepsis. For this, hemocultures and mesenteric lymph nodes will be cultured and identified with MALDI-TOF after enrichment. Similarly with the investigations for the effect of IAP, the effect of protease inhibitors (Nafamostat Mesylate, SPIx) will be determined in our CLP-model.
In the second stage of this project, human samples of patients with intra-abdominal sepsis will be collected. These samples will be used to determine expression of inflammatory cytokines and tight junction proteins in healthy and septic intestinal tissue. Results of our murine model will next be matched with the clinical results to validate our experimental model.
Results are statistically analyzed with t-tests, ANOVA and linear regression models when appropriate.