An Avant-Garde Model of Injury-Induced Regenerative Vaginal Wound Healing

Abstract

Objective: To design and validate a novel murine model of full-thickness (FT) vaginal wound healing that mirrors postinjury tissue repair and underscores the impact of estrogen signaling-driven healing kinetics, inflammation, and neovascularization. Approach: Five-week-old female CD1 mice were subjected to two 1-mm FT wounds. To assess wound healing kinetics, vaginas were harvested at 6, 12, 18, 24, 48, and 72 h and 7 days postinjury. Wounds from all time points were analyzed by hematoxylin and eosin and trichrome to, respectively, assess the rate of wound closure and tissue deposition. Inflammatory leukocyte (CD45), neutrophil (Ly6G), and macrophage (F480 and CD206) infiltration was examined by immunohistochemistry (IHC) and the resulting anti-inflammatory M2 (CD206)/total (F480) macrophage ratio quantified. Neovascularization (CD31) and estrogen receptor-α (ERα) expression levels were similarly determined by IHC. Results: We observed rapid healing with resolution of mucosal integrity by 48 h (p < 0.05), and overall neutrophils and polarized type 2 macrophages (M2) apexed at 12 h and reduced to near control levels by day 7 postinjury. Tissue repair was virtually indistinguishable from the surrounding vagina. CD31⁺ vessels increased between 12 h and day 7 and ERα trended to decrease at 12 h postinjury and rebound at day 7 to uninjured levels. Innovation: A proof-of-concept murine model to study vaginal wound healing kinetics and postinjury regenerative repair in the vagina was developed and verified. Conclusion: We surmise that murine vaginal mucosal repair is accelerated and potentially regulated by estrogen signaling through the ERα, thus providing a cellular and molecular foundation to understand vaginal healing responses to injury.

Keywords: estrogen receptor; novel murine injury model; regenerative wound healing; vaginal injury; wound kinetics.

Julie C.-E. Hakim, MD, FRCSC, FACOG

Figure 1.

Murine vaginal wound model. (A) Minimally invasive intravaginal injury method. (B) H&E staining of a vaginal cross section demonstrates full-thickness injury (circle) where (i) represents the vaginal lumen, (ii) indicates the vaginal epithelial layer, and (iii) depicts vaginal stroma and smooth muscle layers. H&E, hematoxylin and eosin.

Figure 2.

Murine vaginal wound healing. (A) Representative H&E and trichrome images of murine vaginal wound healing kinetics. (B) Epithelial gap (in μm) of murine vaginal wound healing. (C) Total collagen (% positive area) in injured murine vaginal tissue healing. Data represent mean ± SEM (NI group n = 2, 6–72-h group n = 4, and 7-day group n = 3), 4 images/mouse. *Indicates p < 0.05 compared with uninjured controls, as determined by ANOVA and Dunnett's post hoc test. Scale bar represents 100 μm. ANOVA, analysis of variance; NI, no injury.

Figure 3.

Inflammatory and angiogenic changes over time in murine vaginal wound resolution. (A) Representative images of wild-type uninjured female and injured mice at 12 h and 7days postinjury. (B) Quantification of CD31⁺ vessel numbers per area of the submucosa. (C) Quantification of total CD45⁺ leukocytes, (D) Ly6G⁺ neutrophils, (E) F4/80⁺ macrophages, and (F) anti-inflammatory CD206⁺ M2 macrophages per area of submucosa. (G) Anti-inflammatory M2 macrophage burden represented by the CD206⁺/F480⁺ ratio. Collective quantification data represent mean ± SEM (NI group n = 2, 12-h group n = 4, and 7-day group n = 3), 4 images per mouse. *Indicates p < 0.05 compared to uninjured controls as determined by ANOVA and Dunnett's post hoc test. Scale bar represents 100 μm. ANOVA, analysis of variance; NI, no injury.

Figure 4.

Estrogen plays a role in murine vaginal wound healing. ERα density measuring vaginal wound resolution between uninjured mice and mice at 12 h and 7 days postinjury, respectively, illustrated by IHC images (A) and quantification per area of submucosa (B) of ERα. Data represent mean ± SEM (NI group n = 2, 12-h group n = 4, and 7-day group n = 4). ERα, estrogen receptor alpha; IHC, immunohistochemistry.