Acyloxyacyl hydrolase modulates pelvic pain severity

Abstract

Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2_CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.

Keywords: AOAH; QTL; allodynia; pelvic pain; pseudorabies virus.

Fig. 1.

Quantitative trait locus (QTL) analysis for pelvic allodynia. A: genetic background affects pelvic allodynia. Pseudorabies virus (PRV)-infected BALB/c (C) mice exhibit severe allodynia, whereas F1_CxB mice exhibit C57BL/6 (B6)-like allodynia. B: allodynia was quantified for individual F2_CxB mice at postinfection day 4 (PID4). C: log-of-odds scores (LOD) for association with individual SNPs was determined using R/qtl; dashed line indicates threshold for significance. Only single nucleotide polymorphism (SNP) rs6314295 exceeded threshold. D: allodynia of F2 mice relative to genotype. E: allodynia induced by PRV was significantly greater in C mice and CXB-6 mice, relative to B6, at PID4 (***P < 0.001). F: map of home genome around SNP rs6314295 with nearby genes, as indicated by arrow, which were identified in databases (NCBI map coordinates).

Fig. 2.

Identification of the gene encoding acyloxyacyl hydrolase (Aoah) as the candidate gene for pelvic pain. A: Aoah expression, relative to L19, was undetectable by qRT-PCR in sham mice but was induced by postinfection day (PID) and day 4. Bartha’s pseudorabies virus (PRV)-induced allodynia in WT (B) and Aoah^−/− mice (C). D: PRV induced no significant change in tactile sensitivity (50% threshold) of the plantar region of paw at PID4. E: pelvic allodynia was significantly higher in PRV-infected Aoah^−/− than in WT mice in response to the finest von Frey filament (*P < 0.05).

Fig. 3.

Acyloxyacyl hydrolase (AOAH) expression along the bladder-brain axis. Paraffin sections were stained with anti-AOAH antibodies or neuronal marker NeuN and visualized by immunofluorescence. A and B: AOAH expression in the bladder of C57BL/6 (B6) or Aoah^−/− mice, respectively. Lamina propria staining along fibers (arrows) occasionally projecting to luminal surface (arrowhead). C and D: AOAH expression in the sacral spinal cord of B6 or Aoah^−/− mice, respectively. AOAH-positive cell bodies (C and inset) were evident in dorsal horn lamina I and II. E and F: AOAH expression in the Barrington’s nucleus of B6 or Aoah^−/− mice, respectively. AOAH-positive cell bodies were evident throughout the Barrington’s nucleus; in E, the boundary with the cerebellum is shown for orientation. G: AOAH staining (green) colocalized with the pan-neuronal marker NeuN (red) in double-labeled cells (arrow, yellow). H: AOAH immunoreactivity was observed in the cerebellum with Purkinje cell bodies (arrow) and Purkinje cell fibers (arrowhead). Scale bars, 100 µm.

Fig. 4.

Acyloxyacyl hydrolase (AOAH) modulates sacral spinal cord excitability. Spontaneous action potentials and evoked potentials were quantified in sacral spinal cords ex vivo at ventral roots S1–S3. A: short-term depression (STD) was quantified in sham and pseudorabies virus (PRV)-treated mice as compound action potentials and expressed as percent initial pulse (%P1). At 2× current intensity (top) and 5× current intensity (bottom), and PRV-treated mice showed reduced STD at P2–P4. B: STD was quantified as compound action potentials and expressed as %P1. At 1× current intensity, Aoah^−/−mice showed reduced STD at P2. C: no difference of spontaneous firing activity in sacral spinal cords between Aoah^−/− and WT mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Acyloxyacyl hydrolase (AOAH) modulates visceral pain. A: male mice were infected with E. coli strain Sϕ874 and assessed for allodynia at postinfection day (PID)21. Pelvic allodynia was significantly higher in infected Aoah^−/− mice than in WT B6 mice. *P < 0.02. B: visceromotor reflex (VMR) was quantified in female mice as Electromyography (EMG) activity in response to bladder distension under anesthesia. EMG was significantly elevated in Aoah^−/− mice relative to WT B6 mice. *P < 0.05.

Fig. 6.

Acyloxyacyl hydrolase (AOAH) modulates mast cell and urothelial lesions. A: hematoxylin-eosin (H&E)-stained sections of urinary bladders of WT and Aoah^−/− mice. Representative histology images of H&E-stained section from WT (left) and Aoah^−/− mice (right). Scale bar, 200 µm (top), 100 µm (bottom). B: mast cells were quantified in toluidine blue-stained sections. Significantly more mast cells were identified in bladders of Aoah^−/− mice relative to WT mice (*P < 0.05). C: bladder section from an Aoah^−/− mouse infected with pseudorabies virus (PRV), where lesions (yellow arrows) were identified as areas of urothelium with multiple, contiguous TUNEL-positive nuclei (green) lacking overlying uroplakin III (red). D: lesions were quantified in nonserial bladder sections. Significantly more lesions were detected in bladders of Aoah^−/− mice infected with Bartha’s PRV relative to WT mice (*P < 0.05).

Fig. 7.

Elevated vascular endothelial growth factor (VEGF) in acyloxyacyl hydrolase (AOAH)-deficient bladders. A: VEGF was quantified in bladder homogenates by ELISA. Bladders were harvested from B6 mice, uninfected or infected with pseudorabies virus (PRV) to induce neurogenic cystitis or from AOAH-deficient mice. Bladder VEGF was significantly elevated in PRV-treated and AOAH-deficient mice relative to untreated B6 mice (*P < 0.05). B: immunofluorescent imaging of wild-type C57BL/6 (B6; left) and AOAH-deficient bladder sections (right) stained with anti-VEGF antibodies. VEGF immunoreactivity appears more intense in urothelium of AOAH-deficient bladder sections (compare arrows in left and right). Images were captured at ×20 magnification.