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.

Repeated dermal application of the common preservative methylisothiazolinone triggers local inflammation, T cell influx, and prolonged mast cell-dependent tactile sensitivity in mice

“…Many of these products contain the biocide preservative MI. We previously reported that vaginal exposure to MI provokes short-term provoked genital pain in ND4 mice lasting up to 21 days after 10 intravaginal exposures to 0.5% MI in sensitized mice [ 3 ].…”

“…Here we investigated the inflammatory and nociceptive effects of dermal applications of MI which are a much more likely route of pervasive exposures to this toxic chemical that are linked to rising allergic sensitivity [ 3 ] and tissue injury [ 4 – 9 ]. We found that 10 dermal exposures to 0.5% MI induced pain responses that lasted much longer–up to 70 days after the last challenge.…”

“…The average of these measurements per mouse was calculated for each timepoint and compared to baseline measurements as percent decrease from baseline. Based on published criteria [ 3 , 19 , 21 , 25 ], mice were deemed to be hyperalgesic when they exhibited a 33% or greater reduction in experimental vulvar withdrawal thresholds compared to baseline. Any increase in sensitivity below the 33% decrease from baseline threshold is considered to be a negligible increase in tactile sensitivity.…”

Generating primary murine vaginal fibroblast cell lines

“…We have developed a murine model of allergy-driven genital pain that recapitulates clinical characteristics of the chronic pain condition localized provoked vulvodynia, including mast cell accumulation in vaginal tissue and long-lasting tactile sensitivity to stimulation of the anogenital ridge, using repeated exposures to the allergenic cosmetic preservative methylisothiazolinone (MI) [8 , [11] , [12] . Primary fibroblast cell lines derived from vulvar biopsies of vulvodynia patients have been shown to exhibit a heightened cytokine response when treated with yeast and bacterial antigens [5 , 6] .…”

“…Here, we describe a novel method for the establishment of murine vaginal fibroblast cell lines, which provide a cell type-specific in vitro experimental system for investigating fibroblast behavior in pre-clinical rodent models of disease. We validate this methodology in two ways: 1) we evaluate surface marker expression and visually examine morphology of cells and 2) we demonstrate that primary fibroblasts derived from vaginal canals of previously sensitized ND4 mice repeatedly exposed to an allergic irritant in our model of chronic allergy-provoked genital pain replicate the heightened cytokine response to bacterial and yeast antigens observed in fibroblast cell lines derived from vulvar biopsies taken from vulvodynia patients [5] , [6] , 8] .…”