Repeated vulvovaginal fungal infections cause persistent pain in a mouse model of vulvodynia

Abstract

Provoked vestibulodynia, the most common form of vulvodynia (unexplained pain of the vulva), is a prevalent, idiopathic pain disorder associated with a history of recurrent candidiasis (yeast infections). It is characterized by vulvar allodynia (painful hypersensitivity to touch) and hyperinnervation. We tested whether repeated, localized exposure of the vulva to a common fungal pathogen can lead to the development of chronic pain. A subset of female mice subjected to recurrent Candida albicans infection developed mechanical allodynia localized to the vulva. The mice with allodynia also exhibited hyperinnervation with peptidergic nociceptor and sympathetic fibers (as indicated by increased protein gene product 9.5, calcitonin gene-related peptide, and vesicular monoamine transporter 2 immunoreactivity in the vaginal epithelium). Long-lasting behavioral allodynia in a subset of mice was also observed after a single, extended Candida infection, as well as after repeated vulvar (but not hind paw) inflammation induced with zymosan, a mixture of fungal antigens. The hypersensitivity and hyperinnervation were both present at least 3 weeks after the resolution of infection and inflammation. Our data show that infection can cause persistent pain long after its resolution and that recurrent yeast infection replicates important features of human provoked vulvodynia in the mouse.

Fig. 1

Development of vulvar mechanical allodynia in a subset of mice after multiple rounds of vulvovaginal candidiasis with C. albicans strain SC5314. (A) The experimental timeline illustrates the experimental procedures across three rounds of vulvovaginal infections with SC5314. Inoculations of 5 × 10⁴ cells were given on day 0 for each of three infections (for second and third inoculations, SC5314 was administered no more than 1 week after the previous vulvar and hind paw sensitivity testing). Behavioral measurements of vulvar mechanical sensitivity [von Frey (VF)] were taken at baseline and at three points during each infection: days 4, 11, and 32. Note that for each day 32 measurement, yeast was absent from the vaginal cavity for 3 weeks before testing. (B and C) Frequency histograms showing the number of subjects displaying 50% withdrawal thresholds (jumping up with all four paws; see Materials and Methods) in five arbitrarily defined bins (0: 0 to 0.99 g; 1: 1.0 to 1.99 g; 2: 2.0 to 2.99 g; 3: 3.0 to 3.99 g; 4+: >4.0 g) at each testing session. Infection status is indicated by shading. Mice were inoculated vaginally with saline (B) or 5 × 10⁴ SC5314 cells (C) on day 0 of each infection round (n = 15 to 18 per group); all mice received fluconazole (FLU; 15 mg/kg, orally, once daily) from day 4 to day 11. *P < 0.05 by χ² analysis compared to within-group baseline. (D) Mice displaying ≥33% decreases in withdrawal threshold compared to their own baseline at each testing session. *P < 0.05 compared to Saline + FLU group by one-tailed Fisher's exact test; ^•P < 0.10 compared to Saline + FLU group.

Fig. 2

Allodynic mice previously infected with multiple rounds of vulvovaginal candidiasis have increased expression of total, peptidergic, and sympathetic fibers compared to nonallodynic mice and controls (n = 4 to 6 mice per group). (A, E, and I) Saline + FLU group. (B, F, and J) Non-allodynic Candida + FLU subgroup. (C, G, and K) Allodynic Candida + FLU subgroup. (D, H, and L) Bars represent mean ± SEM fiber length (μm) per unit area (μm²). Total nerve fiber density (top row; PGP 9.5–IR) is significantly increased in the allodynic Candida + FLU subgroup, with long fibers lining the lamina propria beneath the epithelium (C and D). Peptidergic nerve fibers immunoreactive for CGRP, which normally consist of fine processes throughout the lamina propria that occasionally penetrate the basal cell layer of the epithelium, are significantly increased in the allodynic Candida + FLU group (G and H) and represent about half of the total fiber population (compare y axes of D and H). Sympathetic nerve fibers immunoreactive for VMAT2 sparsely innervate the upper lamina propria in normal and nonallodynic mice, whereas a significant increase in innervation density is observed in the allodynic Candida + FLU group (K and L). *P < 0.05; **P < 0.01 compared to all other groups by one-way ANOVA followed by Tukey's post hoc test. Scale bars, 50 μm [(C), (G), and (K)]. Arrows point to fibers.

Fig. 3

Development of vulvar mechanical allodynia in a subset of mice after a single, extended SC5314 infection. (A) Experimental time-line illustrating the experimental procedures. An inoculation of 5 × 10⁴ SC5314 cells was given on day 0; fluconazole (FLU; 15 mg/kg, orally, once daily) treatment occurred from day 14 to day 21. Behavioral measurements of vulvar mechanical sensitivity [von Frey (VF)] were taken at baseline (day −7) and at 14, 21, 42, and 70 days after inoculation. (B and C) Frequency histograms showing the number of subjects (n = 10 to 15 per group) in the Saline + FLU (B) and Candida + FLU (C) groups displaying 50% withdrawal thresholds in five arbitrarily defined bins (0: 0 to 0.99 g; 1: 1.0 to 1.99 g; 2: 2.0 to 2.99 g; 3: 3.0 to 3.99 g; 4+: >4.0 g) at each testing session. Infection status is indicated by shading. *P < 0.05 by χ² analysis compared to within-group baseline.

Fig. 4

Development of vulvar mechanical allodynia in a subset of mice after repeated vulvar injections of zymosan. (A) Frequency histogram showing the proportion of female mice displaying chronic allodynia after one to six weekly (or less) injections of vulvar saline (left; n = 10), vulvar zymosan (middle; n = 19), or hind paw zymosan (right; n = 6). “No” indicates that chronic allodynia was never observed, even after six injections. (B) Representative patterns of vulvar mechanical sensitivity over time, using data from the first six mice to be tested (#1 to #6). Vulvar zymosan was injected into mice so indicated 4 hours before the data points highlighted in gray.