Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia - Fig 1

<p><b>Timeline of oxazolone sensitization, challenge, and post-challenge outcome measures</b> (A) To measure oxazolone-driven vulvar tactile sensitivity, mice were topically sensitized with 2% Ox on the shaved flank (day 1) and subsequently challenged on the shaved labiar skin (days 5–14) with 1% Ox or EtOH vehicle for a total of 10 challenges. Tactile sensitivity was assessed in the ano-genital ridge area 1, 21, and 42 days after challenge cessation. Labiar skin was harvested at these time points from a separate cohort of mice for assessing molecular and cellular changes in the tissue. (B) To characterize T cell infiltration in Ox-challenged skin, mice were topically sensitized on their shaved back with 2% Ox (day 1) and challenged on both shaved flanks with 1% Ox or EtOH (days 5–14). Flank skin was harvested from both sides 1 day after challenge cessation for flow cytometric analysis of T cell infiltration. (C) To assess the effects of local mast cell depletion on Ox-induced tactile sensitivity and hyperinnervation, mice were topically sensitized with 2% Ox on the shaved flank (day 1), challenged on the shaved labia with 1% Ox or EtOH (days 5–14) and treated with intralabiar injection of saline or c48/80 (days 5–8 after Ox challenge cessation). Tactile sensitivity, mast cell levels and innervation were assessed 9, 21, and 35 days after the final Ox challenge.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p

Ten oxazolone challenges provoke tactile sensitivity that persists for 21 days after cessation of challenges.

<p>Sensitized mice that received ten daily Ox challenges on the labiar skin had increased tactile sensitivity in their ano-genital ridge area compared to controls. Percent decrease in labiar withdrawal threshold for each treatment group is displayed as mean ± SEM. Significance at each time point was determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis based on comparisons to previously sensitized mice challenged with EtOH (Ox/EtOH; * = p<0.05, *** = p<0.001) and untreated controls (NT; ## = p<0.01, ### = p<0.001). n = 9–12 mice per treatment group; data represent two independent experiments. Raw withdrawal thresholds at baseline and post Ox-challenge cessation for each animal are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169672#pone.0169672.s001" target="_blank">S1 Fig</a> and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169672#pone.0169672.s004" target="_blank">S1 Table</a>.</p

CD4⁺CD25⁺FoxP3⁺ T cells and IFN-γ producing CD8⁺CD103⁺ memory T cells accumulate in oxazolone challenged skin.

<p>Flow cytometric analysis of collagenase-digested, gradient-separated flank skin cells from Ox- (A-B) or EtOH- (C-D) challenged mice 1 day after the cessation of 10 challenges. Cells in (A) and (C) are scatter-gated for lymphocytes, single cells, live, and CD45⁺. Data are pooled from 10 mice per treatment group. Relative abundance of <i>Ifn-γ</i> and <i>Tbx21</i> (E) and total IFN-<i>γ</i> protein content (F) in labiar skin of Ox- vs. EtOH challenged mice after 10 challenges displayed as mean ± SEM (n = 5-6/treatment group). (G-H) Flow cytometric analysis of collagenase-digested, gradient-separated flank skin cells collected 1 day after 10 challenges from Ox-challenged mice and cultured for 18 hours with or without PMA/ionomycin; cells in (G) are live, singlet-gated, scatter-gated for lymphocytes, and CD45⁺CD3⁺CD8⁺.</p