Veterinary Biosciences - Theses

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Type: Masters Research thesis × Start date: 2020 × End date: 2023 × Subject: colitis, goblet cells, mucus, mucin, immunohistochemistry, Ki-67, dogs ×

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    Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis
    Lim, Chelsea Xiaoyun ( 2023-08)
    Background: Intestinal mucus barrier disruption occurs with chronic inflammatory enteropathies. The lack of studies evaluating changes in mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced expression of goblet cells and/or mucin 2 (MUC2), which are key players in mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is also unknown if Ki-67 expression, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to severity of colonic inflammation in dogs with GC and LPC. Objectives: Study objectives included assessing if Ki-67 expression is upregulated in dogs with GC compared to LPC and dogs without colitis; comparing GBC and MUC2 expression among GC, LPC and non-inflamed colon; and exploring the use of RNAscope in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Methods: Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs between January 2015 and March 2022. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colonic signs (NC) (n=10) group based on the final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per slide were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using computer image analysis. Pearson correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic (WH) score and measured variables. Linear regression models were used to compare the relationships between WH scores with KI67%, GBC%, and MUC2%; and between GBC% and MUC2% across groups. Results: Median WH scores were highest in dogs with GC. Median KI67% was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation in all groups. Median MUC2% normalised to WH score (MUC2%*) was highest in the NC group (10.02%; range, 3.05-39.09%), which was two and three-fold higher compared to the GC and LPC groups respectively. Mucin 2 expression had a strongly positive correlation with GBC% in the NC group (5.71% increase in MUC2%; 95%-CI, 0.503–0.987, p =.12), while no significant trends were observed in the GC and LPC groups. With increased severity of colonic inflammation, MUC2 expression sharply declined in the LPC group (-8.90%; 95%-CI, -17.98-0.19, p =.05) but remained static in the GC group. Conclusion and relevance: Dogs with GC had the most severe histologic colonic inflammation. Dogs without colitis had the highest Ki-67 expression. Goblet cell expression did not correlate with histologic severity of colonic inflammation overall. Dogs with GC and LPC likely have differences in pathways regulating MUC2 biosynthesis and secretion. Changes in MUC2 expression appear influenced by pathways regulating GBC activity rather than quantity in GC and LPC. Development of therapeutic strategies aimed at modifying GBC function and MUC2 expression may help improve mucus barrier integrity and mucosal healing in dogs with chronic colitis.