Abstract| Volume 36, ISSUE 2, P186, April 2023

30. Vaginal Epithelial Disruption with Bleomycin Instillation: A New Murine Model for Vaginal Fibrosis

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      Adolescents who undergo vaginal reconstruction are often left with debilitating vaginal fibrosis. There are limited prevention methods (poorly fitting stents, estrogen therapy) and treatment options (vaginal dilators, estrogen or hyaluronan (HA) treatment), both of which have poor patient compliance. Our goal is to create a model for vaginal fibrosis, to aid in the development of new prevention and treatment options. We have previously shown a surgical murine model that heals regeneratively, and we hypothesize that by combining epithelial disruption and bleomycin instillation, similar to models of laryngotracheal stenosis, we can establish vaginal fibrosis.


      Six to seven week old C57BL/6J mice underwent general anesthesia, and control mice had normal saline instilled intravaginally (NS). For experimental animals, a wire brush was rotated 6 times intravaginally followed by 2.5U/kg bleomycin instillation (ED/B). This was repeated 5 times over ten days. Vaginal tissue was harvested 1 day, 3 weeks, or 6 weeks after the last instillation. Tissue was analyzed using trichrome staining, immunohistochemistry (IHC), gene array, qPCR, and a hydroxyproline assay. Average size of HA and size distribution was also determined. Statistical significance was determined by t-test between NS and ED/B at each time point.


      We found increased pro-fibrotic markers and decreased anti-fibrotic markers 3 weeks after the last ED/B exposure compared to NS controls. This was confirmed by qPCR, which showed increased expression of Acta2, Col1a1, and Col3 3 weeks after ED/B exposure compared to NS, with no differences observed at 1 day or 6 weeks. Trichrome staining also showed no difference in total collagen between NS and ED/B at 1 day or 6 weeks, however at 3 weeks, there was increased collagen in ED/B compared to NS. Hydroxyproline showed similar trends. Using IHC, we found a decrease in total number of fibroblasts and macrophages 3 weeks after ED/B compared to NS, with no differences found at 1 day or 6 weeks between groups. Lastly, we found an increase in average size of HA and the size distribution of HA 3 weeks after ED/B compared to NS, again with no differences found at 1 day and 6 weeks after exposure.


      Using epithelial disruption combined with bleomycin instillation, we established vaginal fibrosis, shown by increased collagen content, 3 weeks after the last exposure. However, at 1 day post exposure, fibrosis was not yet established; and by 6 weeks, the vaginal tissue had returned to baseline. We can utilize this model and these time points to not only study the mechanisms of fibrosis but also prevention and treatment options.