A behavioral study of the development of vaginal hyperalgesia and an immunohistochemical study of the development of cyst innervation after transplant surgery.

<p>Note that sensory and sympathetic fibers appear within the cysts two weeks before significant hyperalgesia develops. <i>A</i>. Hyperalgesic severity (post-transplant AUC minus baseline AUC) at different times after transplant surgery in the same rats. Error bars are ± SEM (<i>n</i> = 18). *, differs from two weeks; #, differs from six weeks, <i>p</i><0.05. For the 4 week time point, <i>*t</i> = 0.058. <i>B</i>. Photomicrographs of sensory (CGRP-positive and sympathetic (VMAT2-positive) fibers in the cysts labeled at two, four, six, and ten weeks after transplant surgery (n = 3–5 rats/survival time). Calibration bar is 50 µm for all images.</p

Evans Blue dye extravasation in intact and denervated cysts harvested at different times after transplant surgery.

<p>Note that the cysts' sensory innervation becomes functional (i.e., contributes to EB dye extravasation) three weeks after transplant surgery, which is one week before the hyperalgesia becomes significant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031758#pone-0031758-g001" target="_blank">Fig. 1A</a>). *, differs from intact at the same time period, <i>p</i>≤0.05. Within the Intact group: #, differs from the one-week time period, (<i>p</i>≤0.05). Error bars are ± SEM (<i>n</i> = 8–12/group).</p

Effect of cyst removal or sham-cyst removal performed before the cysts acquire mature innervation on the development of vaginal hyperalgesia.

<p><b><i>A</i></b>. This diagram depicts the time-course of experimental testing (baseline, post-transplant) and surgical procedures (1, 2 or 4 weeks after transplant). <b>B</b>–<b>D.</b> The graphs show percent escape response as a function of vaginal distention volume before (baseline, solid line) and 5–10 weeks after transplant surgery (dashed line). Error bars are ± SEM. The inset bar graphs depict the severity of hyperalgesia individually for each rat in that group (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031758#pone-0031758-g001" target="_blank">Fig. 1</a>). Solid bars indicate cyst removal or sham-cyst removal at 1 or 2 weeks post-transplant; the hatched bars indicate cyst removal or sham-cyst removal at 4 weeks post-transplant. <b>B.</b> Pre-innervation cyst removal (1 or 2 weeks post-transplant) prevents the development of hyperalgesia (n = 5). <b>C.</b> When cyst removal is performed just after innervation has become active, but before innervation is mature; i.e., 4 weeks post-transplant, the development of hyperalgesia is prevented in two of three rats. <b>D.</b> In contrast, after sham-cyst removal, regardless of when the surgery is performed relative to the innervation (either 2 or 4 weeks after transplant), hyperalgesia develops in all rats (n = 4).</p