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. 2014 Jul;155(7):1280-1287.
doi: 10.1016/j.pain.2014.03.023. Epub 2014 Apr 2.

TRPA1 mediates bladder hyperalgesia in a mouse model of cystitis

Jennifer J DeBerry  1 , Erica S Schwartz, Brian M Davis
Affiliations

TRPA1 mediates bladder hyperalgesia in a mouse model of cystitis

Jennifer J DeBerry et al. Pain. 2014 Jul.

Abstract

Urinary bladder pain is a primary symptom associated with interstitial cystitis/painful bladder syndrome. We used systemic injections of cyclophosphamide (CYP), an alkylating antineoplastic agent, to induce cystitis and examine the roles of 2 channels previously demonstrated to be required for inflammatory visceral hyperalgesia: transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1). Injection of CYP (100 mg/kg, i.p.) every other day for 5 days was accompanied by bladder edema and urothelial ulceration, but without significant plasma extravasation or infiltration of neutrophils. Toluidine blue staining showed a significant increase in the number of degranulated bladder mast cells after CYP treatment. Despite this mild pathology, CYP-treated mice exhibited bladder hyperalgesia 1 day after the final injection that persisted 7 days later. Although many previous studies of visceral hyperalgesia have reported changes in dorsal root ganglion neuron TRPV1 expression and/or function, we found no change in bladder afferent TRPV1 expression or sensitivity on the basis of the percentage of bladder afferents responsive to capsaicin, including at submaximal concentrations. In contrast, the percentage of bladder afferents expressing functional TRPA1 protein (i.e., those responsive to mustard oil) increased ∼2.5-fold 1 day after CYP treatment, and remained significantly elevated 7 days later. Moreover, bladder hyperalgesia was reversed by acute treatment with the TRPA1 antagonist HC-030031 (300 mg/kg, i.p.). Our results indicate that CYP-induced bladder hyperalgesia can be induced without robust inflammation or changes in primary afferent TRPV1. However, significant changes were observed in TRPA1 expression, and blockade of TRPA1 alleviated CYP-induced bladder hyperalgesia.

Keywords: Bladder; Calcium imaging; Cystitis; Hyperlagesia; TRPA1; TRPV1; Visceromotor reflex.

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Conflict of interest statement

None of the contributing authors have any conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1
CYP treatment (100 mg/kg) every other day for five days alters urinary bladder morphology. A–C: Hematoxylin and eosin-stained sections of urinary bladder were taken one day following 3rd vehicle injection (Control; A) or CYP injection (1d post-CYP; B), or seven days later (7d post-CYP; C). Epithelial ulceration (arrowheads) and edema (arrows) between the submucosa and transitional epithelium were observed 1d post-CYP mice. By 7d post-CYP, edema was resolved and ulcerations were absent. D–F: PGP9.5 staining of bladder nerves in control mice (D), 1d post-CYP (E) and 7d post-CYP (F). Despite significant changes in the edema seen 1d post-CYP, there are not obvious differences in the number or density of large nerve bundles (arrows) or small clusters of nerve fibers (arrow heads). Transitional epithelium (epi) is not seen in E because the extent of edema increased the bladder wall thickness to the point that it could not be included at this magnification. Epithelium (D&F) exhibited non-specific, edge artifact staining. Calibration bar = 400 μm in panels A–C; 200 μm in panels D–F.
Fig. 2
Fig. 2
CYP treatment (100 mg/kg, i.p.) every other day for five days induced mild bladder inflammation. After the final CYP injection, bladder weight was significantly increased one day and seven days later compared to controls (A), but there was no change in extravasation of Evans blue (B) or neutrophil-based myeloperoxidase activity (C). There was a greater number of degranulated mast cells in bladder tissue from CYP-treated mice (D), and a trend toward an increase in the total number of bladder mast cells (D, inset). n=3–4 mice/group; * indicates p<0.05; NS indicates not significant.
Fig. 3
Fig. 3
CYP treatment (100 mg/kg) changes the kinetics of depolarization-evoked Ca2+ transients in lumbosacral (L6-S1) bladder afferents. Retrogradely labeled, healthy, dissociated urinary bladder afferents were identified using bright field (A) and fluorescence (B) microscopy. Despite no change in baseline intracellular Ca2+ ([Ca2+]i) (C) at either 1 day (1d post-CYP) or seven days (7d post-CYP) following three injections of CYP, the change in intracellular Ca2+ (Δ[Ca2+]i) in response to application of 50 mM K+ was significantly greater in both 1d post-CYP and 7d post-CYP treatment groups compared to control (D). The decay of evoked Ca2+ transients (T50) was significantly longer in the 7d post-CYP group (E). n=8–12 mice/group; * indicates p<0.05 versus control; NS indicates not significant. Calibration bar = 20 μm.
Fig. 4
Fig. 4
CYP treatment (100 mg/kg) increases the percentage of lumbosacral (L6-S1) bladder afferents expressing functional TRPA1, but not TRPV1. The number of TRPV1- and TRPA1-expressing bladder afferents was determined using Ca2+ imaging. Expression of TRPV1, as determined by percentage of cells exhibiting Ca2+ transients in response to application of capsaicin (Cap; 1 μM; n=4–7 mice/group), did not change either one or seven days following CYP treatment (A). Dose-response experiments (B) further indicated no change in sensitivity to Cap (500, 200, or 100 nM; n=1–4 mice/group). In contrast, there was a significant increase in the percentage of bladder afferents responsive to application of the TRPA1 agonist, mustard oil (MO; 100 μM; n=4–8 mice/group) at both post-treatment time points (C); this increase was confirmed via an additional TRPA1 agonist, cinnemaldehyde (Cinn; 100 μM; n=2 mice/group). The magnitude and decay of MO-evoked Ca2+ transients did not significantly change. * p<0.05 indicates 1d post-CYP versus control; # indicates p<0.05 7d post-CYP versus control; NS indicates not significant.
Fig. 5
Fig. 5
CYP treatment (100 mg/kg) induced robust urinary bladder hyperalgesia. The visceromotor reflex (VMR), quantified by abdominal muscle electromyography (EMG), was used to assess nociceptive responses to bladder distention. Compared to control mice, CYP-treated mice exhibited augmented EMG responses when tested one day (1d post-CYP) and seven days (7d post-CYP) following the 3rd CYP injection. Bladder hyperalgesia was completely blocked by pre-treatment with the TRPA1 antagonist, HC-030031 (300 mg/kg, i.p.), one hr prior to testing. n=6–8 mice/group; * indicates p<0.05 1d post-CYP versus control; # indicates p<0.05 7d post-CYP versus control; + indicates p<0.05 versus 1d post-CYP + vehicle.
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