Lower urinary tract symptoms, such as urinary frequency, urgency, nocturia, and abdominal visceral pain, lead to impaired quality of life. These symptoms are features of chronic inflammatory bladder disorders including interstitial cystitis/bladder pain syndrome (IC/BPS). Many hypotheses about the pathogenesis of chronic cystitis have been proposed, such as urothelial dysfunction, inflammation, neural hyperactivity, an autoimmune response, and toxic urinary agents (Homma et al., 2016). However, the exact pathological processes involved remain unclear.

Accumulating evidence suggests that reactive oxidative species (ROS) contribute to bladder disorders. Excess ROS are a feature of various bladder pathological conditions including bladder outlet obstruction (Lin et al., 2005), bladder ischemia/reperfusion (Yu et al., 2004), and bladder inflammation models (Chien et al., 2003). Furthermore, ROS are abundantly produced by inflammatory cells such as macrophages, neutrophils, and mast cells when they infiltrate an inflamed bladder (Brooks et al., 1999; Winterbourn, 2002; Chien et al., 2003; Ndengele et al., 2005). ROS induce bladder hyperactivity by activating capsaicin-sensitive C-fiber afferent pathways (Masuda et al., 2008; Nicholas et al., 2017). In cyclophosphamide- or ifosfamide-induced acute cystitis animal models, the metabolite acrolein enters the urothelium and causes bladder inflammation, which is prevented by ROS scavengers or antioxidants (Yildirim et al., 2004; Batista et al., 2007; Song et al., 2014). In addition, a human study demonstrate that the serum total antioxidant capacity in IC/BPS patients is lower than that in healthy controls (Ener et al., 2015). Thus, it is likely that ROS play a critical role in the etiology and/or pathology of chronic cystitis.

Transient receptor potential ankyrin 1 (TRPA1), a non-selective cation channel, is highly expressed in a subset of nociceptive C-fibers where it acts as a polymodal nociceptor (Wu et al., 2010). TRPA1 is activated by various irritants and oxidative stimuli including ROS, and contributes to nociceptive and inflammatory pain generation (Jordt et al., 2004; Bautista et al., 2006; Andersson et al., 2008; Sawada et al., 2008). In the lower urinary tract, TRPA1 is expressed in the urothelium or detrusor of the urinary bladder in addition to the C-fibers (Du et al., 2008; Streng et al., 2008). This is because intravenous administration of a TRPA1 antagonist does not alter the voiding function, while intravesical infusion of a TRPA1 agonist increases the micturition frequency (Streng et al., 2008; Minagawa et al., 2014), indicating that TRPA1 does not play a major role in bladder function under physiological conditions. By contrast, in a cyclophosphamide-induced cystitis model, bladder hyperalgesia, and voiding frequency are caused by activation of TRPA1 (Meotti et al., 2013; DeBerry et al., 2014). Moreover, human studies reveal that Trpa1 mRNA levels in the urinary bladder are markedly elevated in patients with IC/BPS (Homma et al., 2013) and bladder outlet obstruction (Du et al., 2008). Thus, it is likely that ROS-sensitive TRPA1 may play a key role in the pathogenesis or pathology of chronic cystitis, although this is not fully understood at present.

We previously established a novel long-lasting cystitis mouse model by intravesical injection of hydrogen peroxide (H₂O₂) (Homan et al., 2013). The H₂O₂-induced long-lasting cystitis model is characterized by long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes (Homan et al., 2013; Dogishi et al., 2015). In the present study, we investigated the pathophysiological roles of TRPA1 in the H₂O₂-induced long-lasting cystitis model using TRPA1-knockout (KO) mice.

In the present study, using an intravesical H₂O₂-induced long-lasting cystitis mouse model (Homan et al., 2013), we showed that TRPA1 is involved in initial bladder hyperactivity, but apparently not in the pathological processes involved in long-lasting cystitis, since (1) TRPA1 deletion reduced the initial increase in the number of voids and the decrease in spontaneous locomotor behaviors, which were accompanied by a reduction in c-fos mRNA upregulation in the spinal cord; (2) TRPA1 deletion had no effect on the delayed frequent urination; (3) TRPA1 deletion had no effect on histopathological changes in the urinary bladder at 1 or 7 days after injection. Furthermore, we found that Trpa1 mRNA levels in the urinary bladder were drastically increased at 1 and 7 days after H₂O₂ injection, but levels were not altered in the DRG.

We confirmed that an intravesical injection of H₂O₂ produced long-lasting frequent urination, visceral pain-related behaviors, and bladder inflammation, as previously reported (Homan et al., 2013; Dogishi et al., 2015). Since H₂O₂ can activate TRPA1 (Andersson et al., 2008; Sawada et al., 2008), it is conceivable that H₂O₂ injected intravesically could directly stimulate TRPA1 in the bladder, leading to the generation of long-lasting cystitis. However, the present findings showed no apparent differences in histopathological changes in the urinary bladder between WT and TRPA1-KO mice injected with H₂O₂. Thus, direct stimulation of bladder TRPA1 by exogenous H₂O₂ appears not to play a major role in the induction of cystitis. Since the H₂O₂ solution was immediately drained from the bladder at 30 min after injection, and because H₂O₂ remaining in the bladder was rapidly degraded, H₂O₂-induced cystitis appears to be caused by non-selective insults to the bladder wall, probably by lipid peroxidation, protein oxidation, and DNA damage, as we discussed previously (Homan et al., 2013). In addition, we confirmed that TRPA1 deletion had no effects on the mRNA expression levels of antioxidant enzymes, GPx1, and catalase (Supplementary Figure 1).

The present behavioral experiments revealed that initial bladder hyperactivity, including frequent urination and visceral pain-related behaviors, was mediated, at least in part, through TRPA1 activation. In the lower urinary tract, sensations in the urinary bladder are conveyed to the spinal cord through primary sensory afferent neurons consisting of two types of fibers; myelinated (Aδ) and unmyelinated (C). It is well-known that C-fibers respond to noxious stimuli, while Aδ-fibers respond to bladder filling under physiological conditions (Fowler et al., 2008). Several pieces of evidence suggest that intravesical resiniferatoxin- or capsaicin-induced desensitization of C-fibers results in an increased bladder capacity and reduced bladder pain perception through inactivation of spinal cord neurons in an animal model of acute cystitis (Dinis et al., 2004; Saitoh et al., 2009). Taken together with the present results showing the loss of c-fos mRNA upregulation in the spinal cord of H₂O₂-injected TRPA1-KO mice, this suggests that activation of C-fibers through TRPA1 stimulation enhances the activity of spinal cord neurons, resulting in frequent urination and abdominal visceral pain during the initial phase of long-lasting cystitis. Acute damage to bladder urothelial cells induced by exogenous H₂O₂ injection causes hyperpermeability of the urothelial barrier (Homan et al., 2013). Thus, the submucosa is exposed to irritants in the urine, which may activate TRPA1 expression on the bladder terminal of C-fibers. Alternatively, H₂O₂-induced acute inflammation of the bladder may be accompanied by bladder vascular hyperpermeability and infiltration of abundant inflammatory cells, including neutrophils, into the submucosa (Homan et al., 2013; Dogishi et al., 2017). Several lines of evidence suggest that ROS produced from infiltrated inflammatory cells contribute to bladder hyperactivity (Chien et al., 2003; Masuda et al., 2008). Consequently, it is conceivable that excessive ROS produced from infiltrated inflammatory cells in the submucosa may activate TRPA1. Under severe initial bladder inflammation, it is possible that TRPA1 may be sensitized to ROS by various inflammatory mediators (Gouin et al., 2017).

By contrast, TRPA1 appears not to play a major role during the latter stages of long-lasting cystitis. At 7 days after intravesical H₂O₂ injection, frequent urination was partially alleviated, although it still persisted, and the decrease in spontaneous locomotor behaviors ceased, as reported previously (Homan et al., 2013; Dogishi et al., 2015). The observed severe edematous thickening of the submucosa was partially alleviated by 7 days after injection. Furthermore, we previously reported that the urothelial damage and hyperpermeability are recovered within several days, while bladder inflammation, such as accumulation of inflammatory cells and increased expression of inflammatory cytokines, persisted (Homan et al., 2013). Under such long-lasting inflammatory bladder conditions, excessive ROS production and/or sensitization of TRPA1 to ROS in the bladder may be recovered. We previously reported that bladder tissue remodeling, such as hyperplasia of the urothelium, vascularization, and fibrosis, is induced in the late phase of long-lasting cystitis (Homan et al., 2013; Dogishi et al., 2017). In addition to hyperactivity of bladder sensory neurons induced by long-lasting inflammation, bladder structural changes may affect the micturition function, leading to frequent urination. However, it is difficult to perform cystometric analysis in the present mouse cystitis model, although we could measure intercontraction interval and intravesical pressure in intravesical H₂O₂-induced rat cystitis model (Dogishi et al., 2017). Such technical problems by using genetically-modified mice limit to analyze the urodynamics in the mouse cystitis model. Further detailed investigations including cystometry will be needed to elucidate the roles of TRPA1 in the long-lasting bladder hyperactivity.

Recent evidence suggests that activation of TRPA1 may cause and/or enhance neurogenic inflammation (Gouin et al., 2017). However, the present results suggest that TRPA1 is not responsible for the occurrence and maintenance of bladder inflammation. Consistently, a TRPA1 antagonist attenuates visceral nociception in an ifosfamide-induced cystitis model, although ifosfamide-induced bladder inflammation is not suppressed (Pereira et al., 2013).

In the lower urinary tract, TRPA1 is expressed in both C-fibers and the bladder epithelium (Streng et al., 2008; Wu et al., 2010). This raises the question of which sites expressing TRPA1 are associated with initial bladder hyperactivity. In the present study, we found that Trpa1 mRNA levels were drastically upregulated in the urinary bladder from the initial to the late phases, but not in the L5-S1 DRG. Consistent with these findings, upregulation of Trpa1 mRNA was reported in the urinary bladder of patients with bladder outlet obstruction or IC/BPS (Du et al., 2008; Homma et al., 2013), suggesting that upregulation of TRPA1 expression in the urinary bladder may be pathologically correlated with bladder disorders. Given these expression changes, it is possible that TRPA1 expressed in the urinary bladder, rather than in the DRG, may be responsible for initial bladder hyperactivity. However, this interpretation may be a hasty judgement because the involvement of TRPA1 was observed only during the initial phase, but not in the late phase, although upregulation of Trpa1 mRNA persisted until at least 7 days after injection. Under inflammatory conditions, the sensitivity of TRPA1 in the DRG is reportedly enhanced without changes in expression levels, and this allegedly contributes to hyperalgesia (Zhou et al., 2013). Thus, functional sensitization of TRPA1 expressed in the DRG may contribute to initial bladder hyperactivity, including frequent urination and visceral pain-related behaviors. Further investigation is therefore required to identify the sites of TRPA1 expression responsible for the pathology of long-lasting cystitis.

In conclusion, the present study revealed that TRPA1 contributes to initial bladder hyperactivity, affecting the frequency of urination and abdominal visceral pain, but it does not appear to play a major role in the pathology of long-lasting cystitis. Therapeutic strategies targeting TRPA1 may be effective for minimizing bladder hyperactivity in acute cystitis, but its usefulness for chronic cystitis may be limited.

SO, TN, and SK designed the project. SO, KD, MKo and MKa performed the experiments. SO, KD, MKo, HS, KN, and TN analyzed the data. SO and TN wrote the manuscript. SK supervised the experiments and finalized the manuscript.