Development of the standard mouse model for human bacterial vaginosis induced by Gardnerella vaginalis

Abstract

Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a diminished number of protective bacteria in the vaginal flora. Instead, it is accompanied by a significant increase in facultative and strict anaerobes, including Gardnerella vaginalis (G. vaginalis). BV is one of the most common gynecological problems experienced by reproductive age-women. Because an ideal and standard animal model for human BV induced by G. vaginalis is still underdeveloped, the main objective of this study was to develop a mouse model for human BV induced by G. vaginalis to demonstrate the clinical attributes observed in BV patients. A total of 80 female ICR mice were randomly assigned to 4 groups and intravaginally inoculated with different doses of G. vaginalis: NC (uninfected negative control), PC1 (inoculated with 1 × 10⁵ CFU of G. vaginalis), PC2 (inoculated with 1 × 10⁶ CFU of G. vaginalis) and PC3 (inoculated with 1 × 10⁷ CFU of G. vaginalis). The myeloperoxidase (MPO) activity and serum concentrations of cytokines (IL-1β, IL-10) in mice administered with G. vaginalis were significantly higher than those of the control group. Gross lesion and histopathological analysis of reproductive tract of mice inoculated with G. vaginalis showed inflammation and higher epithelial cell exfoliation compared to the control group. In addition, vaginal swabs from the mice inoculated with G. vaginalis showed the presence of clue cells, which are a characteristic feature of human BV. Altogether, our results suggested that G. vaginalis is sufficient to generate comparable clinical attributes seen in patients with BV.

Keywords: Gardnerella vaginalis; bacterial vaginosis; cytokine; epithelial exfoliation; mouse model.

Figure 1

Study design and Gardnerella vaginalis inoculation. All mice were intraperitoneally injected with β-estradiol-3-benzoate (1 mg/kg) for 3 days, then PC1, PC2 and PC3 were intravaginally inoculated with 1 × 10⁵ CFU, 1 × 10⁶ CFU and 1 × 10⁷ CFU of G. vaginalis, respectively. NC was intravaginally inoculated with PBS instead of G. vaginalis.

Figure 2

Gram staining of vaginal smears (A,B), assessment of MPO activity in vaginal tissue (C), and serum concentration of IL-1β and IL-10 (D). (A) Gram-stained smears of vaginal fluid (before G. vaginalis inoculation). No bacterium were detected in the epithelial cells of the mouse vagina prior to G. vaginalis inoculation (1000× magnification). (B) Gram-stained smears of vaginal fluid (4 days after G. vaginalis inoculation). The presence of clue cells, characterized by vaginal epithelial cells covered in bacteria, was confirmed in the vaginal smear of infected mice. The red arrows indicate Gram-variable-staining G. vaginalis (1000× magnification). (C) Assessment of MPO activity in vaginal tissue. (D) Serum concentration of IL-1β and IL-10. The error bars in the graph represent the calculated standard deviation. The significance level of p < 0.05 was denoted as “*”, indicating a statistically significant difference. The symbol “+” indicates no statistically significant difference but rather a tendency. The value from each individual animal was measured and used for the statistical analysis (20 mice/group). Significant differences in values between groups was determined based on ANOVA. Dunnett’s multiple comparisons test was used for the statistical evaluation of differences between the control (NC) and the infection groups (PC1, PC2, PC3). NC, negative control; PC1, positive control inoculated with 1 × 10⁵ CFU of G. vaginalis; PC2, positive control inoculated with 1 × 10⁶ CFU of G. vaginalis; PC3, positive control inoculated with 1 × 10⁷ CFU of G. vaginalis.

Figure 3

Gross lesion and histopathological examination of vagina. (A) The gross lesion of the excised reproductive tract (vagina and uterine horns) of mice. (B) The quantitative analysis of the reproductive tract length. For the quantitative analysis of the reproductive tract length, the distance from the ovary to the cervix was measured using ImageJ software (National Institutes of Health, MD, United States), and subsequently utilized for the statistical analysis. (C) Quantitative analysis of the thickness of keratinized stratified squamous epithelium. For the quantitative analysis of the thickness of keratinized stratified squamous epithelium, three different areas of the keratinized stratified squamous epithelium were randomly measured in each mouse vagina using the ZEN3.4 software (Carl Zeiss AG, Oberkochen, Germany). (D) Images of the hematoxylin and eosin stained (H&E) stained vaginal tissue sections. Scale bars located in the lower right-hand corner of each figure. The red arrows indicate the exfoliated epithelial cells. NC, negative control; PC1, positive control inoculated with 1 × 10⁵ CFU of G. vaginalis; PC2, positive control inoculated with 1 × 10⁶ CFU of G. vaginalis; PC3, positive control inoculated with 1 × 10⁷ CFU of G. vaginalis.