Function and expression pattern of TRPM8 in bladder afferent neurons associated with bladder outlet obstruction in rats

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Abstract

We investigated the function and expression pattern of the transient receptor potential melastatin-8 (TRPM8) in urinary bladder afferent neurons from control and bladder outlet obstruction (BOO) rats. BOO was produced and, after six weeks, the effects of intravesical infusion of menthol, the agonist of TRPM8, were investigated using unanesthetized cystometry. The intravesical infusion of menthol produced an increase in the micturition pressure in both sham surgery and BOO rats. In BOO rats, increased basal and threshold pressure and a decreased micturition interval were observed. Next, the population of TRPM8-positive and the co-expression proportion of TRPM8 with neurochemical markers (NF200 or TRPV1) in the bladder afferent neurons were each compared between the control and BOO rats using retrograde tracing and immunohistochemistry. The population of TRPM8-immunoreactive bladder afferent neurons was larger in BOO rats (3.28 ± 0.43%) than in the control rats (1.33 ± 0.18%). However, there were no statistical differences between the control and BOO rats in the co-expression proportion of neither TRPM8-NF200 (84.1 ± 4.3% vs 79.7 ± 2.7%, p = 0.41) nor TRPM8-TRPV1 (33.3 ± 3.6% vs 40.8 ± 2.6%, p = 0.08) in the bladder afferent neurons. The present results suggest that the neuronal input through TRPM8-positive bladder afferent neurons are augmented after BOO, however, the neurochemical phenotype of the up-regulated TRPM8-positive bladder afferent neurons is not changed after BOO.

Introduction

Over active bladder (OAB), which is characterized by urgency or urge incontinence, is a bothersome symptom in the patients with benign prostatic hyperplasia (BPH) (Chapple and Roehrborn, 2006). BPH causes OAB following bladder outlet obstruction (BOO). The etiologies of detrusor overactivity caused by BOO have been demonstrated using an animal model. After partial urethral obstruction, histological and functional changes are observed in the detrusor (Speakman et al., 1987, Seki et al., 1992) and in the neuronal pathway of the micturition reflex (Steers and De Groat, 1988, Steers et al., 1991).

The evaluation of bladder cooling reflex (BCR), or the contraction of urinary bladder caused by the intravesical infusion of cold water, is an appropriate diagnostic tool for neurogenic detrusor overactivity (Bors and Blinn, 1957, Geirsson et al., 1999). Jiang et al. (2002) proposed that the BCR was carried via the C-fibers innervating the urinary bladder, because an intravesical infusion of small amount of cold water facilitated the action potential of C-fibers in the pelvic nerve in an anesthetized normal cat model. The cold-sensitive thermoreceptors that are linked to the C-fibers have been detected in the bladder wall (Fall et al., 1990, Mazieres et al., 1998). Additionally, the responsible afferents for the BCR are sensitized by the intravesical infusion of menthol (Lindström and Mazières, 1991, Geirsson, 1993).

As BCR is observed in some patients with BPH (Chai et al., 1998, Gotoh et al., 1999), it is therefore hypothesized that detrusor overactivity with BPH likely results from an increased excitability in C-fibers following BOO (Hirayama et al., 2003). In the rat BOO model, C-fiber dependent sacral micturition reflex enhancement has been demonstrated (Steers and De Groat, 1988) and capsaicin-sensitive C-fiber bladder afferents were found to be involved in functional alterations in bladder afferent pathways following BOO (Tanaka et al., 2003).

Transient receptor potential melastatin-8 (TRPM8) has been shown by two groups (McKemy et al., 2002, Peier et al., 2002) as a cold temperature- and menthol-sensitive cation channel. TRPM8 in the bladder wall was first identified by Stein et al. (2004) using immunohistochemistry and RT-PCR, therefore TRPM8 might be a candidate for inducing the BCR because intravesical infusion of menthol facilitated the micturition reflex in guinea pig (Tsukimi et al., 2005) and rat (Nomoto et al., 2008) models.

Several populations of C-fibers desensitized by capsaicin are considered to be responsible for the BCR in humans (Das et al., 1996) and guinea pigs (Tsukimi et al., 2005). These results suggest that TRPM8 may be expressed in capsaicin-sensitive, i.e. TRPV1-positive, bladder afferent neurons. However, in the rat, it is advocated that intravesically-infused menthol acts on the capsaicin-resistant afferents to facilitate the micturition reflex, because capsaicin pretreatment had no effect on this reflex (Nomoto et al., 2008). We recently described the population and neuronal phenotype of TRPM8-expressing rat bladder afferent neurons. The co-expressed proportion of TRPM8 and TRPV1 in the bladder afferent neurons was 36.1 ± 4.0% under normal conditions (Hayashi et al., 2009).

We hypothesized that the neuronal phenotype of TRPM8-expressed bladder afferents, which appears to induce the BCR, might alter following BOO. These post-BOO alternations might affect the micturition reflex induced by the intravesical infusion of menthol. In other cases of micturition disorders, the increase in TRPM8-immunoreactive nerve fibers in the bladder wall was correlated with the symptom score in the patients with painful bladder syndrome (Mukerji et al., 2006).

In the present study, we compared the micturition reflex induced by intravesical infusion of menthol between control and BOO rats using unanesthetized cystometry. We also compared the population and neuronal phenotype (A-fiber or C-fiber and/or capsaicin-sensitive or -insensitive) of TRPM8-expressing bladder afferent neurons between control and BOO rats using the retrograde tracing technique and immunohistochemistry.

Section snippets

Animals

In this study, we used 26 female Wistar rats (250–270 g). The experimental protocols were approved by the Animal Research Committee of the Kurume University School of Medicine.

Procedures of bladder outlet obstruction

Under anesthesia by sodium pentobarbital treatment (50 mg/kg, i.p.), the bladder and proximal urethra were exposed via median laparotomy. The proximal urethra was freed from the vaginal wall by careful dissection to avoid injury to the periurethral blood vessel. Three-zero black silk suture was placed around the proximal

Cystometry

Fig. 1 shows the typical cystometrical recordings of micturition cycles obtained in BOO and sham surgery rats. It is known that the BOO rat model shows an increase in bladder weight, bladder capacity, micturition pressure, and residual volume. After release of the obstruction, the micturition volume increases (Mattiasson and Uvelius, 1982, Steers and De Groat, 1988). The BOO rat experiments conducted in the present study also revealed increased MP (p < 0.01; Table 1) and MI (p < 0.01; Table 1)

Discussion

The present study demonstrated that intravesical infusion of menthol increased the BP and TP, and decreased the MI, in BOO rats. We speculate that these three parameters are involved in the TRPM8-expressing bladder afferent neuron, because the population of TRPM8-expressing bladder afferent neurons increased after BOO. Intravesical infusion of menthol might affect the bladder compliance and micturition reflex after BOO, which might be induced by the reinforcement of neuronal input originating

Acknowledgement

The authors thank Dr. S. Kobayashi and Dr. H. Hosokawa (Division of Biological Information, Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Kyoto, Japan) for kindly providing the anti-TRPM8 antibody.

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