Repeated Vaginal Exposures to the Common Cosmetic and Household Preservative Methylisothiazolinone Induce Persistent, Mast Cell-Dependent Genital Pain in ND4 Mice

Abstract

A history of allergies doubles the risk of vulvodynia-a chronic pain condition of unknown etiology often accompanied by increases in numbers of vulvar mast cells. We previously established the biological plausibility of this relationship in mouse models where repeated exposures to the allergens oxazolone or dinitrofluorobenzene on the labiar skin or inside the vaginal canal of ND4 Swiss Webster outbred mice led to persistent tactile sensitivity and local increases in mast cells. In these models, depletion of mast cells alleviated pain. While exposure to cleaning chemicals has been connected to elevated vulvodynia risk, no single agent has been linked to adverse outcomes. We sensitized female mice to methylisothiazolinone (MI)-a biocide preservative ubiquitous in cosmetics and cleaners-dissolved in saline on their flanks, and subsequently challenged them with MI or saline for ten consecutive days in the vaginal canal. MI-challenged mice developed persistent tactile sensitivity, increased vaginal mast cells and eosinophils, and had higher serum Immunoglobulin E. Therapeutic and preventive intra-vaginal administration of Δ⁹-tetrahydrocannabinol reduced mast cell accumulation and tactile sensitivity. MI is known to cause skin and airway irritation in humans, and here we provide the first pre-clinical evidence that repeated MI exposures can also provoke allergy-driven genital pain.

Keywords: allergy; mast cells; methylisothiazolinone; vulvar pain; Δ-9 tetrahydrocannabinol.

Figure 1

Population level sensitization to methylisothiazolinone (MI) in Europe and North America. (A) Location of epidemiological studies conducted in Europe (left) and North America (right); all points plotted at the approximate latitude and longitude of the original study with random noise added for easier visualization. (B) Sample size reported by epidemiological studies in Europe and North America using MI patch tests. Point placement corresponds with the year each study ended. Sample size may include multi-year studies if yearly data were not available. Color gradient indicates the highest concentration of MI in parts per million (ppm) tested in each study and the size of the point represents the proportion of the population that tested sensitive to MI. Studies in which MI ppm were not reported are excluded from the plot. The size of each point represents the percent of participants that were found to be sensitized to MI (n = 151 studies).

Figure 2

Sensitization, challenge, and treatment timelines. Schedule of MI in saline flank sensitizations and challenges (A–C). (B) Therapeutic intra-vaginal Δ-9-tetrahydrocannabinol (THC) treatment timeline. (C) Preventative intra-vaginal THC treatments.

Figure 3

Increased mast cell density in the vaginal canal and elevated tactile ano-genital sensitivity after 10 intra-vaginal MI challenges in previously sensitized ND4 female mice. Representative confocal images of vaginal canal tissue from MI sensitized mice challenged with MI (A–C) or saline D–F) at 1, 7, and 21 days after the 10th MI challenge, respectively. Mast cells stained with FITC-conjugated avidin (green) and nuclei counterstained with DAPI (blue); 200× magnification. (G) Density of avidin⁺ mast cells in 12 μm vaginal canal cryo-sections from sensitized mice challenged with MI or saline. Results reported as fold change in avidin signal in MI- over saline-treated mice. Dotted line denotes no change. Data pooled from 5–6 mice. (H) Serum IgE content in mice treated with MI or saline in the vaginal canal 1 day after the last MI/saline challenge. NT bar denotes serum IgE levels in naïve age-matched, untreated mice. Significance with respect to vehicle control group * = p < 0.05; 4–6 mice/treatment group. (I) Tactile sensitivity in MI and saline challenged mice, reported as mean ± SEM of the percent decrease from baseline in the withdrawal threshold for each treatment group; n = 17–18 mice/treatment group. Red dotted line = 33% hyperalgesia threshold. Significance with respect to vehicle control group *** p < 0.001.

Figure 4

Inflammatory changes in the vaginal canal and spinal cord, and increased eosinophil activity after 10 intra-vaginal MI challenges in previously sensitized ND4 female mice. Relative transcript abundance of interferon-γ (IFN-γ) and interleukin (IL)-6 transcripts in the vaginal canal tissue (A) and IL-1β and IL-6 in spinal cord tissue (B) of MI challenged mice one and seven days after 10 challenges, normalized to β2-microglobulin mRNA levels; 5–6 mice/treatment group. Black dotted line denotes no change in the relative abundance of transcripts. (C) Tissue eosinophil peroxidase levels measured by optical density (OD)/g of wet tissue, in the vaginal canal of mice one day after the 3rd and 10th MI challenge; 3–5 mice/treatment group. Significance with respect to vehicle control group ** p < 0.01. EPO, eosinophil peroxidase.

Figure 5

Reduced vaginal mast cell density and ano-genital sensitivity following six intra-vaginal therapeutic THC treatments after 10 vaginal MI challenges in previously sensitized ND4 female mice. (A,C) Representative confocal images of vaginal canal tissue from mice that were sensitized and challenged with MI and subsequently treated with THC (A) or untreated, that is, NT (C), at one day after the last treatment, respectively. Mast cells stained with FITC-conjugated avidin (green) and nuclei counterstained with DAPI (blue); 200× magnification. (B) Mast cell density displayed as fold change in avidin signal in THC-treated over NT mice at 1, 7, and 10 days after the 6th THC treatment; 6–7 mice/treatment group. Black dotted line denotes no change in MC abundance. (D) Anogenital tactile sensitivity of MI challenged mice treated with NT (black) or therapeutic THC (red) 1, 7, and 10 days after the 6th THC treatment; n = 8–9 mice/treatment group. Results displayed as mean ± SEM. Significance with respect to control group * p < 0.05.

Figure 6

Reduced vaginal mast cell density and ano-genital sensitivity following intra-vaginal preventive THC treatments before and during 10 vaginal MI challenges in previously sensitized ND4 female mice. (A,B,D,E) Representative images of vaginal canal tissue from THC-treated (A,B) and NT (D,E) MI-challenged mice one and seven days after the 10th MI challenge. Mast cells stained with FITC-conjugated avidin (green) and nuclei counterstained with DAPI (blue); 200× magnification. (C) Mast cell density displayed as fold change in avidin⁺ signal intensity of the THC-treated over NT mice; n = 3–7 mice/treatment group. (F) Ano-genital tactile sensitivity of preventive THC-treated and NT mice at 1, 7, and 21 days after the 10th MI challenge; 8–9 mice/treatment group. Red dotted line denotes 33% hyperalgesia threshold. Results displayed as mean ± SEM. Significance with respect to control group * p < 0.05 and ** p < 0.01.