Background: Acute occlusive mesenteric ischemia is a life-threatening disease and remains to be incompletely understood in modern gastrointestinal surgery. Recently it has been shown, that specifically enteric glia cells (EGCs) are important to maintain epithelial homeostasis in the intestine. After EGC ablation, letal intestinal inflammation occurs. EGCs of various subtypes and locations interact with the gut microbiome, synthesize trophic factors (i.e. TGF-ß, GDNF) and potentially protect enteric neurons from I/R and oxidative stress. We hypothesize that EGC function may therefore be pivotal for regeneration of epithelial integrity, cell protection and recovery of intestinal function after I/R. EGC activation after intestinal ischemia and reperfusion (I/R) has been suggested, but the relevance of such a reactive “gliosis” in the intestine in analogy to an “astrocytosis” in the brain (i.e. after ischemic stroke) is not fully understood. We therefore aim to investigate the role of EGCs after I/R.

Methods: We studied a superior mesenteric artery (SMA)-occlusion model in C57Bl/6 mice as well as in GFAP-GFP and GFAP-Td-Tomato reporter-mice, using the glial fibrillary acidic protein GFAP as an EGC marker. Anesthesized mice underwent median laparotomy and the SMA was identified and occluded with a microvascular clamp for 45 minutes. Intestinal samples were procured after 3 hours and 24 hours of reperfusion. To assess I/R injury, Parks’ score was performed in H/E stained paraffin slides and a standardized segment of ileum was tested for FITC dextran diffusion in an in-vitro assay confirming epithelial barrier breakdown. Immunofluorescence staining in intestinal whole mounts was performed for EGC markers. Intestinal samples were also analyzed for EGC related gene expression in the tunica muscularis. For further mechanistic studies, primary EGC cultures were established and subjected to in-vitro ischemic conditions.

Results: Parks’ score showed a significant I/R injury in the intestines of SMA-clamped animals at both timepoints, concomitant to an epithelial barrier breakdown as tested in an in-vitro FITC dextran diffusion assay, compared to sham animals. Immunfluorescence staining of the EGC marker GFAP in intestinal tunica muscularis whole mounts showed a morphologic upregulation of GFAP filaments after 45 minutes of intestinal ischemia at 3h and 24h of reperfusion (mean fluorescence intensity MFI: clamp3h vs. sham3h and clamp24h vs. sham24; One Way Anova p<0.001). GFAP gene expression in the tunica muscularis of animals with SMA-clamping was significantly upregulated after 3h of reperfusion. Furthermore, in GFAP reporter mice (GFAP-GFP) gene expression of the well-established transcription factor and hypoxia marker HIF1-α was significantly upregulated after 3h and 24h of reperfusion (clamp3h vs. sham3h, unpaired t-test p<0.05; clamp24h vs. sham24h, unpaired t-test p<0.01), concomitantly to HIF1-α dependent genes such as TGFß1 and VEGF-α. It was also found, that several proinflammatory cytokines (IL-6, Il-1ß) and chemokines (MCP-1) were significantly upregulated after limited mesenteric ischemia both in the animal and in-vitro models.

Conclusion: Mesenteric ischemia and reperfusion activates GFAP-positive EGC potentially via HIF-1α dependent pathways and produces an inflammatory micro-environment. Further study of the interactions of EGC and the intestinal epithelium may provide new insights into the regenerative mechanisms of the bowel after limited mesenteric ischemia.