Inflammatory Renin-Angiotensin System Disruption Attenuates Sensory Hyperinnervation and Mechanical Hypersensitivity in a Rat Model of Provoked Vestibulodynia

Abstract

Vestibulodynia is characterized by perivaginal mechanical hypersensitivity, hyperinnervation, and abundant inflammatory cells expressing renin-angiotensin system proteins. We developed a tractable rat model of vestibulodynia to further assess the contributions of the renin-angiotensin system. Complete Freund's adjuvant injected into the posterior vestibule induced marked vestibular hypersensitivity throughout a 7-day test period. Numbers of axons immunoreactive for PGP9.5, calcitonin gene-related peptide, and GFRα2 were increased. Numbers of macrophages and T cells were also increased whereas B cells were not. Renin-angiotensin-associated proteins were abundant, with T cells as well as macrophages contributing to increased renin and angiotensinogen. Media conditioned with inflamed vestibular tissue promoted neurite sprouting by rat dorsal root ganglion neurons in vitro, and this was blocked by the angiotensin II receptor type 2 receptor antagonist PD123319 or by an angiotensin II function blocking antibody. Sensory axon sprouting induced by inflamed tissue was dependent on activity of angiotensin-converting enzyme or chymase, but not cathepsin G. Thus, vestibular Complete Freund's adjuvant injection substantially recapitulates changes seen in patients with provoked vestibulodynia, and shows that manipulation of the local inflammatory renin-angiotensin system may be a useful therapeutic strategy.

Perspective: This study provides evidence that inflammation of the rat vestibule induces a phenotype recapitulating behavioral and cytological features of human vestibulodynia. The model confirms a crucial role of the local inflammatory renin-angiotensin system in hypersensitivity and hyperinnervation. Targeting this system holds promise for developing new nonopioid analgesic treatment strategies.

Keywords: Allodynia; angiotensin II receptor type 2; axon sprouting; growth factors; hormones.

Figure 1

Mechanical sensitivity of posterior vestibule of rats receiving injections of saline or complete Freund’s adjuvant (CFA) assessed by response to von Frey filament testing. Rats were tested 1 day prior to (−1) and 1, 3, and 6 days after vestibular injection and implantation of minipumps containing water vehicle alone or with the AT2 antagonist PD123319. * p<0.05 vs Day -1. ** p<0.001vs Day -1 and Saline for each respective Day. α p=0.002 vs Day 1 CFA. # p<0.002 vs Day -1 and CFA for each respective Day. @ p≤0.001 vs Day 1 and 3 CFA and Day 1 and 3 CFA+PD. N=6 for each group, 2-way repeated measures anova with Holm-Sidak post-hoc testing.

Figure 2

Innervation of the rat vestibule. Posterior vestibule was injected with saline (Sal) or complete Freund’s adjuvant (CFA), and rats received infusions of water vehicle (Saline and CFA) or PD123319 (Saline+PD and CFA+PD). Vestibular tissue sections were immunostained for the pan-neuronal marker PGP9.5 (A, E, H, K), the peptidergic axon marker CGRP (B, F, I, L) and the nonpeptidergic axon marker GFRα2 (C, G, J, M). Quantitative analysis (D) compares axon density (total area in μm² of epidermal immunoreactive axons per mm of epidermis). Bar in M = 100μm for all micrographs. 1: p≤0.01 vs Saline; 2: p<0.05 vs CFA. N=6 for all groups and markers, 1-way anova with Student-Newman-Keuls post-hoc comparisons.

Figure 3

Renin-expressing macrophages in the rat vestibule. Cells were immunostained for the macrophage marker CD68 in combination with renin (REN). Vestibular tissue sections 7d after injection showed cells immunoreactive for renin alone (thin arrow), CD68 alone (fat arrow) or both proteins (arrowhead) after saline injection (A). CFA-injected tissue showed greater numbers immunoreactive cells (B, C) and in percentages of CD68-ir cells expressing REN, and REN-ir cells expressing CD68 (D). CD68- and REN-ir cells in rats receiving saline injection plus infusion of PD123319 (PD, E) and CFA with PD123319 (F). Scale bar in F = 100μm for all micrographs. 1: p<0.05 vs Saline; 2: p<0.05 vs CFA; 3: p<0.05 vs Saline+PD. n=6 for all groups and determinations, 1-way anova with Student-Newman-Keuls correction for multiple comparisons.

Figure 4

Angiotensinogen-expressing macrophages in the rat vestibule. Cells were immunostained for the macrophage with marker CD68 coexpressing angiotensinogen (AGT). Vestibular tissue sections 7d after injection showed cells immunoreactive for AGT alone (thin arrow), CD68 alone (fat arrow) or coexpressing both proteins (arrowhead) after saline injection (A). CFA-injected tissue showed greater numbers immunoreactive cells (B, C) but not in percentages of CD68-ir cells expressing AGT, and AGT-ir cells expressing CD68 (D). CD68- and AGT-ir cells in rats receiving saline injection plus infusion of PD123319 (PD, E) and CFA with PD (F) showed reduced antigen expression relative to CFA. Scale bar in F = 100μm for all micrographs. 1: p<0.025 vs Saline; 2: p<0.05 vs CFA; 3: p<0.05 vs Saline+PD. n=6 for all groups and determinations except CFA+PD (5), 1-way anova with Student-Newman- Keuls or Dunn’s correction for multiple comparisons.

Figure 5

Renin-expressing T cells in the rat vestibule. Cells were immunostained for the T cell marker TCRα/β (TCR) in conjunction with renin (REN). Vestibular tissue sections 7d after injection showed cells immunoreactive for renin alone (thin arrow), TCR alone (fat arrow) or coexpressing both proteins (arrowhead) after saline injection (A). CFA-injected tissue showed increased numbers immunoreactive cells (B, C) and in percentages of TCR-ir cells expressing renin (D). TCR- and renin-ir cells in rats receiving saline injection plus infusion of PD (E) are comparable to saline-injected controls (C,D), and CFA with PD (F) show little change in numbers of immunoreactive cells (C) but reduced colocalization (D) relative to CFA alone. Scale bar in F =100μm for all micrographs. 1: p<0.050 vs Saline; 2: p<0.050 vs CFA; 3: p<0.050 vs Saline+PD. n=6 for all groups and determinations, 1-way anova with Student-Newman- Keuls test for multiple comparisons.

Figure 6

Angiotensinogen-expressing T cells in the rat vestibule. Cells were immunostained for the T cell marker TCRα/β (TCR) and angiotensinogen (AGT). Vestibular tissue sections 7d after injection showed cells immunoreactive for AGT alone (thin arrow), CD68 alone (fat arrow) or coexpressing both proteins (arrowhead) after saline injection (A). CFA-injected tissue showed greater numbers immunoreactive cells (B, C) and in percentages of TCR-ir cells expressing AGT, and AGT-ir cells expressing TCR (D). TCR- and AGT-ir cells in rats receiving saline injection plus infusion of PD123319 (PD, E) and CFA with PD123319 (F) are shown. Scale bar in F = 100μm- for all micrographs. 1: p<0.050 vs Saline; 2: p<0.050 vs CFA; 3: p<0.050 vs Saline+PD. n=6 for all groups and determinations, 1-way anova with Student-Newman- Keuls correction for multiple comparisons.

Figure 7

B cells and mast cells in the rat vestibule. Tissue sections from vestibules of rats injected with saline at 7d post-injection showed cells immunoreactive for the B cell marker CD79 (A). Injection of CFA did not affect numbers of CD79-positive cells (B). Mast cells stained with Giemsa were observed in saline injected tissue in either quiescent (arrow) or degranulating (arrowhead) states (C) in roughly equal numbers (D). Following CFA injection, mast cells were more abundant, and most were in the process of degranulation (D, E). Scale bar = 50μm. 1: p<0.050 vs Saline; 2: p<0.005 vs CFA, n=6 for all groups, 1-way anova with Student-Newman- Keuls correction for multiple comparisons.

Figure 8

Conditioned medium from rat vestibular tissue promotes ANGII/AT2-enhanced neurite outgrowth from dissociated rat dorsal root ganglion neurons. Media conditioned with tissue injected with saline (A, Sal) promotes typical neurite formation by dissociated neonatal small-diameter neurons detected by peripherin-ir. Tissue injected with CFA promotes much more aggressive neurite outgrowth (B, C). Addition of antibody directed to angiotensin II (ANGII Ab) did not affect outgrowth in saline injection-conditioned medium (D), but prevented the increase by CFA-injected tissue (E). Addition of the AT2 antagonist PD123319 did not alter neurite formation in saline injected tissue-conditioned medium (F) but normalized outgrowth in CFA-injected tissue-conditioned medium (G). Scale bar in G= 100 μm. 1: p<0.001 vs Saline; 2: p<0.001 vs CFA. n=6 for all groups, Kruskal-Wallis 1-way anova on ranks.

Figure 9

Effects of peptidase inhibitors on neurite outgrowth. Neonatal rat dorsal root ganglion neurons were dissociated and grown in control medium (Con) or in medium conditioned by CFA-injected vestibular tissue (+CFA). Conditioned medium contained vehicle (Water) or solutions containing the ACE inhibitor enalapril maleate (EM), Cathepsin G inhibitor (Cath), or the mast cell chymase inhibitor chymostatin (Chymo). 1: p<0.05 vs Con; 2: p<0.05 vs CFA; 3: p<0.05 vs EM; 4: p<0.05 vs Cath. Kruskal-Wallis 1-way anova on ranks.