Pathophysiological Role of Transient Receptor Potential Ankyrin 1 in a Mouse Long-Lasting Cystitis Model Induced by an Intravesical Injection of Hydrogen Peroxide

Chronic inflammatory bladder disorders, such as interstitial cystitis/bladder pain syndrome, are associated with poor quality of life. The exact pathological processes remain unclear, but accumulating evidence suggests that reactive oxidative species (ROS) are involved in urinary bladder disorders. Transient receptor potential ankyrin 1 (TRPA1), the most sensitive TRP channel to ROS, was shown to be responsible for urinary bladder abnormalities and hyperalgesia in an acute cystitis model. However, the roles of TRPA1 in chronic inflammatory bladder are not fully understood. We previously established a novel mouse cystitis model induced by intravesical injection of hydrogen peroxide (H2O2), resulting in long-lasting frequent urination, bladder inflammation, pain-related behavior, and histopathological changes. In the present study, we investigated the pathophysiological role of TRPA1 in the H2O2-induced long-lasting cystitis mouse model. Under anesthesia, 1.5% H2O2 solution was introduced transurethrally into the bladder of female wild-type (WT) and TRPA1-knockout mice and maintained for 30 min. This increased the number of voids in WT mice at 1 and 7 days after injection, but reduced the number in TRPA1-knockout mice at 1 day but not 7 days after injection. Spontaneous locomotor activities (increase in freezing time and decrease in distance moved) were reduced at 3 h after injection in WT mice, whereas the spontaneous visceral pain-related behaviors were attenuated in TRPA1-knockout mice. Furthermore, upregulation of c-fos mRNA in the spinal cord at 1 day after injection was observed in WT but not TRPA1-knockout mice. However, there was no difference in histopathological changes in the urinary bladder, such as edematous thickening in the submucosa, between WT and TRPA1-knockout mice at 1 or 7 days after injection. Finally, Trpa1 mRNA levels in the L5-S1 dorsal root ganglion were not altered, but levels in the urinary bladder were drastically increased at 1 and 7 days after injection. Taken together, these results suggest that TRPA1 contributes to acute bladder hyperactivity such as frequent urination and bladder pain, but does not appear to play a major role in the pathological processes of long-lasting cystitis.


INTRODUCTION
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 nonselective 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 cyclophosphamideinduced 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 (H 2 O 2 ) (Homan et al., 2013). The H 2 O 2 -induced longlasting 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 H 2 O 2 -induced long-lasting cystitis model using TRPA1-knockout (KO) mice.

Animals
All experiments were performed according to the ethical guidelines recommended by the Kyoto University Animal Research Committee. The protocol was approved by the Kyoto University Animal Research Committee (permit number: 2015-40, 2016-40). Trpa1 +/+ (wild-type; WT) and Trpa1 −/− (TRPA1-KO) mice lines were bred from heterozygous mice with a C57BL/6 × 129 S1 background that were obtained from Jackson Laboratory (Bar Harbor, ME). Mouse lines were backcrossed to C57BL/6 J mice for at least 10 generations, and genotyped by genomic PCR using primers 5 ′ -tcatctgggcaacaatgtcacctgct-3 ′ and 5 ′ -tcctgcaagggtgattgcgttgtcta-3 ′ . Female WT and TRPA1-KO mice aged between 5 and 6 weeks old were used, while female C57BL/6 J mice of the same age were purchased from Japan SLC (Shizuoka, Japan) and used in some experiments. All mice were housed under constant ambient temperature (24 ± 1 • C) and humidity (55 ± 20%), with an alternating 12 h light/dark cycle (lights came on automatically at 8:00 a.m.). Food and water were freely available.

H 2 O 2 -Induced Cystitis Model
The H 2 O 2 -induced cystitis model was generated as previously reported (Homan et al., 2013). Briefly, under 2-3% isoflurane (Pfizer, NY) anesthesia, a polyethylene tube (PE-10; Clay-Adams, Parsippany, NJ) was introduced into the bladder transurethrally and the lower abdomen was pressed gently to withdraw urine. Next, 50 µL of 1.5% H 2 O 2 solution (Wako Pure Chemical Industries, Osaka, Japan) in sterile saline was introduced into the bladder through the catheter. The H 2 O 2 solution was drained from the bladder after 30 min by pressing the lower abdomen.

Measurement of the Number of Voids and Spontaneous Locomotor Activities
Mice were kept in an individual plastic cage (10 × 20 × 30 cm: width × length × height) lined with filter paper (Advantec Chromatography Paper No. 50; Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and allowed to acclimate for 30 min before experiments. After replacing the filter paper, the mouse was videotaped for 15 min, and the number of voids was quantified from the videotape by counting urine spots on the filter paper. Subsequently, freezing time and move distance were analyzed using the ANY-maze video tracking system (Stoelting Co., Wood Dale, IL).

Statistical Analysis
Data are expressed as means ± S.E.M. Statistical analysis was performed with the GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Unpaired t-tests or Mann-Whitney U-tests were used to determine mRNA expression levels. The number of voids, freezing time, and distance moved were analyzed with two-way ANOVA, followed by the Tukey posthoc test. In all cases, statistical significance was defined by a p-value < 0.05.

Effect of TRPA1 Deletion on the Number of Voids in H 2 O 2 -Induced Cystitis Mice
The number of voids was measured in WT and TRPA1-KO mice at 1 and 7 days after intravesical injection of saline (controls) or H 2 O 2 . Consistent with our previous report (Homan et al., 2013), an intravesical injection of 1.5% H 2 O 2 significantly increased the number of voids 1 day after injection [F (1, 69) = 71.82, p < 0.001]. This increase was significantly suppressed in TRPA1-KO mice [F (1, 69) = 12.30, p < 0.001]. Both H 2 O 2injected WT and TRPA1-KO groups exhibited a significant increase in the number of voids compared with saline-injected WT and TRPA1-KO groups, respectively, and the number of voids in H 2 O 2 -injected TRPA1-KO mice was significantly lower than in H 2 O 2 -injected WT mice (Figure 1A). At 7 days after injection, the number of voids was significantly increased in H 2 O 2 -injected groups [F (1, 67) = 16.31, p < 0.001], with significant increases observed in both WT and TRPA1-KO mice. However, there was no significant difference between WT and TRPA1-KO H 2 O 2 -injected groups [F (1, 67) = 0.1099, p = 0.7413; Figure 1B].

Effect of TRPA1 Deletion on Visceral Pain-Related Behaviors in H 2 O 2 -Induced Cystitis Mice
Reduced spontaneous locomotor activity in rodents is considered evidence of visceral pain-related behavior, as previously reported in a cyclophosphamide-induced cystitis mouse model (Miki et al., 2011). We previously reported a decrease in spontaneous locomotor behavior at only 3 h after intravesical H 2 O 2 injection (Dogishi et al., 2015). In the present study, to examine the effect of TRPA1-KO on visceral pain-related behavior, spontaneous locomotor activities including freezing time and distance moved were analyzed over a 15 min period in freely moving WT and TRPA1-KO mice at 3 h after H 2 O 2 injection. Intravesical H 2 O 2 injection significantly increased freezing time [F (1, 39) = 8.323, p < 0.01] and reduced the distance moved [F (1, 39) = 4.717, p < 0.05] in WT but not in TRPA1-KO mice. TRPA1 deficiency significantly reduced freezing time [F (1, 39) = 11.23, p < 0.01]. The freezing time in WT mice was significantly increased in the H 2 O 2 -injected group compared with the saline-injected control group, but a significant increase was not observed in H 2 O 2injected TRPA1-KO mice. Furthermore, the freezing time in H 2 O 2 -injected TRPA1-KO mice was significantly shorter than that in H 2 O 2 -injected WT mice (Figure 2A).
Similarly, H 2 O 2 -injected WT mice displayed a significant decrease in the distance moved compared with saline-injected WT mice. In TRPA1-KO mice, no significant difference was observed between saline-and H 2 O 2 -injected groups, and moving distance in the H 2 O 2 -injected group was increased compared with H 2 O 2 -injected WT mice, but not significantly ( Figure 2B).

Effect of TRPA1 Deletion on Upregulation of c-fos mRNA in the Spinal Cord of H 2 O 2 -Induced Cystitis Mice
Activation of bladder sensory neurons responsible for bladder hyperactivity and pain-related behaviors is correlated with the induction of c-fos mRNA expression, an immediate early gene, in the spinal cord (Avelino et al., 1999;Dinis et al., 2004). To determine whether TRPA1 deletion affects neuronal activity in the spinal cord caused by H 2 O 2 -induced cystitis, c-fos mRNA levels in the L5-S1 spinal cord, the area of termination of most bladder afferents (Nadelhaft and Booth, 1984), were examined 1 day after intravesical saline or H 2 O 2 injection. In WT mice, H 2 O 2 injection caused a significant upregulation in the relative expression of c-fos mRNA compared with the saline-injected control group. By contrast, in TRPA1-KO mice, there was no significant difference between saline-and H 2 O 2 -injected groups (Figure 3).

Effect of TRPA1 Deletion on Histopathological Changes in the Bladder of H 2 O 2 -Induced Cystitis Mice
Cystitis induced by intravesical H 2 O 2 injection was histopathologically examined by HE staining of the bladder of WT and TRPA1-KO mice. In H 2 O 2 -injected mice, severe edematous thickening in the submucosa was observed compared with the saline-injected control group at 1 day after injection, which was partially alleviated by 7 days after injection in both WT and TRPA1-KO mice. There was no difference in histopathological changes between WT and TRPA1-KO mice (Figure 4).

Expression of Trpa1 mRNA in the Bladder and DRG of H 2 O 2 -Induced Cystitis Mice
The effects of H 2 O 2 injection on Trpa1 mRNA levels in the urinary bladder and L5-S1 DRG were examined. Intravesical H 2 O 2 injection drastically elevated the relative expression levels of Trpa1 mRNA in the bladder on day 1 and 7. By contrast, there were no differences in the expression levels of Trpa1 mRNA in the L5-S1 DRG between saline-and H 2 O 2 -injected groups at 1 and 7 days after injection (Figure 5).

DISCUSSION
In the present study, using an intravesical H 2 O 2 -induced longlasting 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 H 2 O 2 injection, but levels were not altered in the DRG.
We confirmed that an intravesical injection of H 2 O 2 produced long-lasting frequent urination, visceral pain-related behaviors, and bladder inflammation, as previously reported (Homan et al., 2013;Dogishi et al., 2015). Since H 2 O 2 can activate TRPA1 Sawada et al., 2008), it is conceivable that H 2 O 2 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 H 2 O 2 . Thus, direct stimulation of bladder TRPA1 by exogenous H 2 O 2 appears not to play a major role in the induction of cystitis. Since the H 2 O 2 solution was immediately drained from the bladder at 30 min after injection, and because H 2 O 2 remaining in the bladder was rapidly degraded, H 2 O 2induced 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 wellknown 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 H 2 O 2injected TRPA1-KO mice, this suggests that activation of Cfibers 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 H 2 O 2 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, H 2 O 2 -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 H 2 O 2 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 longlasting 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 H 2 O 2 -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.