Medizinische Universität Graz - Research portal
Selected Publication:
Obermueller, B.
Investigation of the intestinal ultrastructure, apoptosis and status of the tight junctions in the early phase of murine sepsis.
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Medical University of Graz; 2020. pp. 91
[OPEN ACCESS]
FullText
- Authors Med Uni Graz:
- Advisor:
- Castellani Christoph
- Singer Georg
- Sperl Daniela Ingrid
- Altmetrics:
- Abstract:
- Introduction: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. It is responsible for a large proportion of deaths on non-cardiologic intensive care units and is thus referred to as “silent killer”. Among many other organs, sepsis may cause alterations of the gut barrier, one of the most important barriers of the human body, leading to increased bowel wall permeability. This hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. It leads to passage of bacteria or bacterial toxins to the body, further fueling the inflammatory process and aggravating the patients´ conditions. This project aims to elucidate the underlying mechanisms for intestinal hyperpermeability in the early phase of sepsis. Methods: Eighteen male C57Bl/6 wild type mice were split to two groups. All mice received one single gavage of fluorescein isothiocyanate-dextran (FITC) 30min before intervention. One group (n=10) underwent cecal ligation and puncture (CLP) to induce sepsis. The other group (n=8) was sham operated. Animals were euthanized 8 hours after the induction of sepsis. A clinical sepsis score was applied to all mice. Serum FITC Dextran levels were determined by photometry. Serum pro-inflammatory cytokines were examined by Luminex® ELISA. Bowel wall inflammation was examined histologically. Electron microscopy was performed to assess the surface of ileum and colon (scanning electron microscopy, SEM) and the intercellular junctional complex (transmission electron microscopy, TEM). Additional information of tight junctions (TJ) was gained by PCR and ELISA for TJ components (claudin 2, claudin 4, occludin and tight junction protein 1). Intestinal epithelial cell (IEC) apoptosis was addressed by HOECHST staining and apoptosis marker PCR (Bax, Bak, Bad, Caspase-3, Lamin-B, Bcl-2) in ileum samples. The cellular ultrastructure was examined with Small Angle X-Ray Scattering (SAXS). Results: Septic animals exhibited significantly increased permeability for FITC 8h post-operatively. The clinical sepsis score and significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin 2 and decreased expressions of claudin 4, tight junction protein 1 and occludin as signs for increased TJ porosity. Light microscopy showed significant dilatation of intercellular spaces at the basal sections of IECs in septic animals confirmed by increased intercellular spaces at the level of TJs and adherens junctions in electron microscopy (TEM). HOECHST staining and PCR of ileum samples for apoptosis markers proofed no differences in intestinal epithelial cell apoptosis between the groups. The bowel surface showed no significant differences between the groups in SEM surface analysis or conventional histology. In small angle X-ray scattering no increase in number or size of nano-pores could be shown in the bowel wall. Conclusions: Intestinal hyper-permeability in septic animals is most likely caused by alterations of intercellular contacts and TJ porosity and not by apoptosis or altered ultrastructure of intestinal epithelial cells in this murine model of early sepsis.