Abstract

Introduction: Recent literature implicates gut epithelia mucosa and intestinal microbiota as important players in post-stroke morbidity and mortality. As most studies have focused on the acute effects of stroke on gut dysbiosis, our study objective was to measure chronic, longitudinal changes in the gut microbiota and intestinal pathology following ischemic stroke.

Hypothesis: We hypothesized that mice with experimental ischemic stroke would exhibit chronic gut dysbiosis and intestinal pathology at up to 35 days post-stroke compared to sham controls.

Methods: Male C57BL/6J mice were subjected to 60 minutes of transient middle cerebral artery occlusion (tMCAO) or sham-surgery followed by reperfusion and euthanasia at day 35. To determine the long-term effects of tMCAO on gut dysbiosis, fecal boli were collected pre- and post-tMCAO on days 0, 3, 14, and 28. Following DNA extraction, amplification of the V3-V4 region of the 16s rRNA gene was performed via PCR, then library preparation and high-throughput sequencing on the HiSeq Ilumina platform. Longitudinal changes between tMCAO and sham-injured mice were analyzed with the DESeq2 pipeline and MicrobiomeAnalyst. Pathological indices of gut dysbiosis were assessed via Periodic acid-Schiff stain and immunolabeling of tight junction and mucin proteins.

Results: Bioinformatics analysis revealed significant differences in abundance among taxa at the phylum, family, and species levels between baseline samples and post-injury fecal microbiome samples in tMCAO mice compared to sham mice that persisted up to 28 days post-stroke. The most persistent changes in post-stroke microbial abundance were decreases in beneficial bacteria families, Bifidobacteriaceae and Lactobacillaceae, and significant increases in the harmful bacteria families, Helicobacteraceae and Enterobacteriaceae. Intestinal histopathology at day 35 showed evidence of chronic intestinal inflammation that included marked increases in immune cell infiltration with mild-moderate epithelial hyperplasia and villous blunting.

Conclusion: These results demonstrate that acute, post-stroke disruption of the gut-brain-microbiota axis progresses to chronic gut dysbiosis and intestinal inflammation.