Innervation Changes Induced by Inflammation in the Murine Vagina

Abstract

Vulvodynia is a prevalent chronic pain disorder associated with high medical costs and often ineffective treatments. The major pathological feature is proliferation of vaginal nerve fibers. This study aimed to develop a highly reproducible animal model to study neuroproliferation in the vagina and aid the identification of appropriately targeted treatments for conditions such as vulvodynia. Mild chronic inflammation was induced using microinjection of complete Freund's adjuvant in the distal vagina of C57Bl/6 mice. Control mice received saline. Inflammation and innervation density were assessed at 7 and 28 days after a single administration or 14 days following repeated administration of complete Freund's adjuvant or saline. Histochemistry and blinded-analysis of images were used to assess vaginal morphology (H & E) and abundance of macrophages (CD68-labeling), mast cells (toluidine blue staining, mast cell tryptase-immunoreactivity), blood vessels (αSMA-immunoreactivity) and nerve fibers immunoreactive for the pan-neuronal marker PGP9.5. Subpopulations of nerve fibers were identified using immunoreactivity for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY). Single administration of complete Freund's adjuvant resulted in vaginal swelling, macrophage infiltration, vascular proliferation and increased abundance of nerve fibers immunoreactive for CGRP, SP, VIP and/or PGP9.5 but not NPY, evident at seven days. Inflammation further increased following repeated administration of complete Freund's adjuvant but nerve fiber proliferation did not. Nerve fiber proliferation continued to be evident at 28 days. The inter-individual differences within each treatment group were small, indicating that this model may be useful to study mechanisms underlying vaginal nerve fiber proliferation associated with inflammation.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin‐II types 1 and 2 receptors in rat primary sensory neurons

“…This agrees with a previous report that Ang‐II enhanced neurite outgrowth in cultured DRG neurons, an effect that was prevented by treatment with an AT2R antagonist (Anand et al ). A similar role for this receptor was seen in CFA‐induced vestibulodynia where disruption of the RAS system and antagonism of AT2R attenuated neuritogenesis (Chakrabarty et al ; Sharma et al ). Note that CFA‐induced cutaneous inflammation triggers an elevation in the local levels of renin and angiotensinogen, which are released by T‐cells and macrophages, recruited to the site of inflammation (Chakrabarty et al ; Chakrabarty et al ).…”

“…One mechanism proposed to underlie this connection is that pro‐inflammatory mediators promote neuritogenesis of both peptidergic and non‐peptidergic nociceptors and possibly their sensitization (Anand et al ). This hyper‐innervation has been shown in skin [reviewed in (Hendrix and Peters )] and in a model of vestibulodynia (Chakrabarty et al ; Sharma et al ). Thus, we decided to examine the impact of Ang‐II on DRG neurite outgrowth, first in vitro and then in vivo .…”

Clodronate Treatment Prevents Vaginal Hypersensitivity in a Mouse Model of Vestibulodynia

“…Normal innervation of the vagina includes multiple populations of nerve fibres, most abundant in the lamina propria ( Barry et al., 2017 ) and hyperinnervation in our model involves multiple populations of neurons, consistent with clinical vestibulodynia ( Bohm-Starke et al., 1999 ). Increased CD68+ putative macrophages accompanies vaginal hyperinnervation in our model of vestibuldynia ( Sharma et al., 2018 ). However, the role of macrophages in this model, and in clinical vestibuldynia, is unclear.…”

“…To investigate vestibulodynia pathophysiology we developed a mouse model of hyperinnervation in response to mild chronic inflammation, induced by microinjection of complete Freund’s adjuvant (CFA) at the vaginal entrance ( Sharma et al., 2018 ). Normal innervation of the vagina includes multiple populations of nerve fibres, most abundant in the lamina propria ( Barry et al., 2017 ) and hyperinnervation in our model involves multiple populations of neurons, consistent with clinical vestibulodynia ( Bohm-Starke et al., 1999 ).…”

Emerging Evidence of Macrophage Contribution to Hyperinnervation and Nociceptor Sensitization in Vulvodynia

“…For many years lack of suitable models of vulvodynia was a major barrier to the development of treatments that specifically target the pathophysiology of the disease. Recently developed models of vaginal hyperinnervation in rats and mice are an important advancement (Farmer et al, 2011; Barry et al, 2018; Chakrabarty et al, 2018; Sharma et al, 2018). Increased abundance of macrophages has been observed in these models accompanying increased vaginal nerve fibers, consistent with signs in patient biopsies, but the extent to which macrophages contribute to hyperinnervation or nociceptor sensitivity in vulvodynia remains unclear.…”