Artemin Immunotherapy Is Effective in Preventing and Reversing Cystitis-Induced Bladder Hyperalgesia via TRPA1 Regulation

Abstract

Injury- or disease-induced artemin (ARTN) signaling can sensitize primary afferents and contribute to persistent pain. We demonstrate that administration of an ARTN neutralizing antibody, anti-artemin (α-ARTN), can block the development of, and reverse already established, bladder hyperalgesia associated with cyclophosphamide-induced cystitis in mice. We further demonstrate that α-ARTN therapy blocks upregulation of TRPA1, an ion channel contributing to persistent bladder pain during cyclophosphamide-induced cystitis, and decreases phospho-ERK1/2 immunoreactivity in regions of the spinal cord receiving bladder afferent input. Thus, α-ARTN is a promising novel therapeutic approach for treatment of bladder hyperalgesia that may be associated with interstitial cystitis/painful bladder syndrome, as well as cystitis associated with antitumor or immunosuppressive cyclophosphamide therapy.

Perspective: α-ARTN therapy effectively prevented and reversed ongoing bladder hyperalgesia in an animal model of cystitis, indicating its potential as an efficacious treatment strategy for ongoing bladder pain associated with interstitial cystitis/painful bladder syndrome.

Keywords: Bladder; TRPA1; artemin; cystitis; growth factor; pain; visceral.

Figure 1

The experimental time line was as follows: cystitis was induced by injection of cyclophosphamide (CYP; 100 mg/kg, i.p.) on Days 1, 3, and 5; an ARTN neutralizing antibody (α-ARTN; 10 mg/kg, i.p.) was administered to one group of mice 30 min prior to CYP on day 1, and to another group of mice 30 min following CYP on day 5. Experimental endpoints were collected from these groups on Days 6 and 13, respectively.

Figure 2

CYP upregulates bladder-derived ARTN mRNA concurrent with upregulation and sensitization of bladder afferent TRPA1. (A) Bladder-derived ARTN and NGF mRNAs were significantly upregulated 1d post-CYP. Expression peaked at >5-fold and 2.5-fold above control levels, respectively. Expression at 7d post-CYP did not differ from control. GDNF mRNA expression was unchanged. (B) Bladder afferent TRPA1 mRNA expression was significantly increased at 1d post-CYP and remained increased at 7d post-CYP. In contrast, CYP had no effect on TRPV1 mRNA expression at either time point. (C) Isolated bladder afferents from naive mice exhibited robust Ca²⁺ influx to application of 50 mM K⁺ (black dash). Approximately 15% of these responded to 100 μM MO (gray dash). 75% of MO-responsive afferents exhibited profound desensitization (tachyphylaxis) of TRPA1 channel with repeated MO application (10 min insterstimulus interval). Rescue from tachyphylaxis by ARTN was observed in 28.5% of these neurons (no example shown). (D) ARTN exposure (gray bar; 7 min) was efficacious in recruiting de novo responses to MO in 45.8% of bladder afferents that were initially insensitive to MO. (E) As predicted by afferent TRPA1 mRNA expression, the percentage of bladder afferents exhibiting MO-evoked Ca²⁺ transients was significantly increased 1d post-CYP relative to controls when mice were pre-treated with IgG (the control for α-ARTN). This CYP-induced increase in MO-responsive afferents was prevented in mice pre-treated with α-ARTN. * indicates p<0.05 versus control, ^# indicates p<0.05 α-ARTN versus IgG.

Figure 3

Treatment with α-ARTN before or after CYP injections prevents and reverses, respectively, CYP-induced bladder hyperalgesia. (A) Relative to controls (open circles), mice treated with CYP exhibited augmented abdominal EMG responses to noxious distension pressure at 1d post-CYP (black circles) that persisted at 7d post-CYP (gray circles). * p<0.05, 1d post-CYP versus control; ^# p<0.05, 7d post-CYP versus control. (B) At 1d post-CYP, mice pre-treated with IgG (black triangles) exhibited abdominal EMG responses like those of mice treated with CYP alone (panel A). Pre-treatment with α-ARTN prevented the development of hyperalgesia at 1d post-CYP (black squares). Post-treatment with α-ARTN after CYP, when hyperalgesia was already established, reversed CYP-induced hyperalgesia at 7d post-CYP (gray squares). ⁺ p<0.05, 1d post-CYP + IgG pre-tx versus control.

Figure 4

α-ARTN treatment normalizes spinal pERK expression after bladder stimulation and prevents CYP-induced upregulation of MO-responsive bladder afferents and. (A) Spinal neuronal pERK expression (white arrows) in response to noxious bladder distension was significantly increased 1d post-CYP in regions of the dorsal spinal cord receiving lumbosacral bladder afferent input. Pre-treatment with α-ARTN prior to CYP prevented upregulation of distension-evoked spinal pERK. SDH: superficial dorsal horn; DCM: dorsal commissure; CC: central canal; SPN: sacral parasympathetic nucleus. * p<0.05, 1d post-CYP versus naive; ⁺ p<0.05, α-ARTN versus 1d post-CYP.