Neurourology and Urodynamics 27:348–352 (2008)
Cold Environmental Stress Induces Detrusor Overactivity Via Resiniferatoxin-Sensitive Nerves in Conscious Rats Tetsuya Imamura,* Osamu Ishizuka, Naoki Aizawa, Chen Zhong, Teruyuki Ogawa, Tsuyoshi Nakayama, Tomoaki Tanabe, and Osamu Nishizawa Department of Urology, Shinshu University School of Medicine, Matsumoto Nagano, Japan Aims: We determined if cold environmental stress induced detrusor overactivity in conscious rats. We then examined the role of resiniferatoxin (RTX)-sensitive nerves in this response. Methods: Three days prior to cystometric investigation, the urinary bladders of 12 female rats were cannulated. Six of the rats were treated with RTX 24 hr prior to cystometric investigation. The rats were exposed to three ambient temperature conditions: room temperature (RT, 27 C) for 20 min, low temperature (LT, 4 C) for 40 min, and RT again for 20 min. During each exposure, cystometric patterns of the rats were recorded. Additionally, neuronal structures of urinary bladders were visualized by immunohistochemistry. Results: When the conscious rats were suddenly transferred from RT to LT, the cooled rats exhibited micturition patterns of detrusor overactivity. After 20 min at LT, the response slowly improved. After returning to RT, the overactive detrusor response disappeared, reverting to patterns similar to those before transfer to LT. When the RTX-treated rats were exposed with cold stress, they also exhibited detrusor overactivity. However, it was significantly mitigated compared to the non-RTX-treated normal rats. The normal rats had distinct neuronal structures labeled with S100 and calcitonin gene-related peptide antibodies in the urinary bladders, but the RTX-treated rats had few. Conclusion: Detrusor overactivity of the conscious rats was induced by cold environmental stress. A portion of the cold-stress detrusor overactivity might be mediated by RTX-sensitive neurological pathway. The cold-stress model would be useful to investigate lower urinary tract functions. Neurourol. Urodynam. 27:348–352, 2008. ß 2007 Wiley-Liss, Inc. Key words: cold; overactive detrusor; rat; resiniferatoxin; stress; urinary bladder INTRODUCTION
Bladder Cannulation
Changes of environmental temperatures induce various physiological responses. For instance, cold environmental stress elicits urinary sensations and frequent urination along with increasing heart rate and blood pressure.1 – 3 Seasonal or continuous cold environmental stress can aggravate existing lower urinary tract dysfunctions such as urinary urgency, frequent urination, or cystitis.4 – 6 The mechanisms of urinary bladder sensation have been vigorously investigated by instillation of ice-cold water into the bladders of patients7 – 12 or experimental animals13 maintained at normal environmental temperature. The cold stimulus applied in this fashion activates afferent C fibers in the urinary bladder.14 – 16 To our knowledge, there are no studies that investigate the onset of urinary sensations and frequent urination induced by sudden whole body cooling. In this study, we determined if quick and large changes of environmental temperature affect micturition patterns in conscious rats. We found that the cold environmental stress induced drastic changes, and these changes were mediated, at least in part, through a resiniferatoxin (RTX)-sensitive neurological pathway.
Three days prior to cystometric investigation, 12 female rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (75 mg/kg body weight, Sankyo Eeru Medicine Co., Tokyo, Japan) and xylazine (15 mg/kg body weight, Bayer, Leverkusen, Germany). The urinary bladders were exposed and incised at the top. A polyethylene catheter (PE50, Nippon Becton Dickinson, Tokyo, Japan) was inserted through the incision and fixed at that site with a 5-0 silk thread. The free end of the catheter was tunneled subcutaneously and exteriorized at the back of the neck. All the rats were maintained individually in cages under a 12-hr alternating light–dark cycle, with freely available food and water.
MATERIALS AND METHODS Animals
Eighteen Female Sprague-Dawley (SD) rats at postnatal week 10 (Japan SLC Inc., Shizuoka, Japan) were used for the experiments. Animals were treated in accordance with National Institutes of Health Animal Care Guidelines and the guidelines approved by the Animal Ethics Committee of Shinshu University School of Medicine.
ß 2007 Wiley-Liss, Inc.
Treatment With Resiniferatoxin (RTX)
This study focused on RTX-sensitive nerves to determine if the nerves mediated the effects of cold environmental stress. The nerves in six cannulated rats were desensitized with RTX (Sigma–Aldrich, Steinheim, Germany) 2 days after bladder cannulation and 24 hr prior to cystometric investigation. The RTX was given subcutaneously with 0.3 mg RTX/kg body No conflict of interest reported by the author(s). Karl-Erik Andersson led the review process. Abbreviations: RTX, resiniferatoxin; RT, room temperature; LT, low temperature; CGRP, calcitonin gene-related peptide; SMA, alpha smooth muscle actin; UP III, uroplakin III; DAPI, 4 0 , 6-diamidino-2-phenylindole dihydrochloride; BP, bladder pressure; MV, micturition volume; UT, urothelium; SM, smooth muscle layers. *Correspondence to: Tetsuya Imamura, Department of Urology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto Nagano 390-8621, Japan. E-mail:
[email protected] Received 9 March 2007; Accepted 3 July 2007 Published online 13 August 2007 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/nau.20497
Cold Stress Induces Detrusor Overactivity weight. above.
17
After injection, the rats were maintained again as
Cystometric Investigation
Three days after cannulation, the cystometric investigation was performed on unanesthetized and unrestricted rats (n ¼ 6; non-RTX-treated normal rats, n ¼ 6; RTX-treated rats). The bladder catheter was connected via a T-tube to a pressure transducer (P23 DC; Statham, Oxnard, CA) and a microinjection pump (Model 200; Muromachi-Kikai, Tokyo, Japan). The transducer was calibrated at 0 and 20 cmH2O. Each rat was placed in a metabolic cage, which enabled the measurement of micturition volume by means of a fluid collector connected to a force displacement transducer (Type 45196; NEC San-ei Instruments, Tokyo, Japan). Throughout the experiments, an isotonic sodium chloride solution maintained at room temperature (RT) was pumped into the bladder at a rate of 10 ml/hr with a syringe pump (Terumo Co., Tokyo, Japan). The flow rate was calibrated with a measuring cylinder. The rats were not given food and water during the experiment. The urinary bladder intravesicular pressure and micturition volume were recorded continuously on a pen oscillograph (Recti-Horiz-8K; NEC San-ei Instruments, 10 mm/min recording speed). The following cystometric variables were measured: basal pressure (cmH2O), micturition pressure (cmH2O), voiding interval (min), micturition volume (ml), and bladder capacity (ml). Bladder capacity was calculated by adding the micturition volume and the difference between the saline infusion volume and micturition volume. These values were averaged for each 20 min period described below. Environmental Temperature Conditions of Cystometric Investigation
The cystometric investigations were perfomed under the following environmental temperature conditions. The cannulated normal rats were placed singly in metabolic cages at RT, (27 2 C) for 20 min during which cystometric data were gathered. They were then quickly transferred in the metabolic cages to the cold room for low temperature exposure (LT, 4 2 C) for 40 min. The LT exposure was divided into two 20 min phases (Phase I and Phase II). During this time, the saline infusion was delivered through the polyethylene catheter to the urinary bladders within 1.5 min. For much of this time, it passed through the subcutaneous tissues where the temperature of the infusion was maintained. Afterwards, the rats were returned to the standard RT condition (re-RT) for 20 min. The transfers to and from the cold room were handled very gently and smoothly so as to avoid unnecessary stress in the rats. The cannulated RTX-treated rats were also treated as described above. Immunohistochemistry
Three normal rats and three rats that were treated with RTX 24 hr earlier, which were not cannulated, were sacrificed for immunohistochemistry. The urinary bladders were removed and rinsed with saline, and then fixed in 4% paraformaldehyde with 4% sucrose in 0.1 M phosphate buffer for 12 hr at 4 C. The tissues were embedded with OCT compound (Sakura Co., Tokyo, Japan) in dry-ice hexane. For immunostaining, the tissue sections (5 mm) were soaked in phosphate buffer saline (PBS) for 5 min at 4 C. Antigen retrieval was achieved by immersion of the sections in 10 mM sodium citrate and Neurourology and Urodynamics DOI 10.1002/nau
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microwaving at 100 C for 5 min. The specimens were coated with 1.5% normal donkey serum (Chemicon International Inc., Temecula, CA) and 1.5% non-fat milk in PBS for 1 hr at 4 C. The sections were incubated with a mouse monoclonal antiS100 antibody (1:50, Abcam Ltd., Cambridge, UK), a marker of neuronal structures, or guinea pig polyclonal anti-calcitonin gene-related peptide (CGRP) antibody (1:800, Progen Biotechnik GmbH, Heidelberg, Germany), a marker of afferent nerves, for 12 hr at 4 C. Following a rinse with PBS, they were incubated with secondary antibody consisting of donkey antimouse or anti-guinea pig IgG conjugated with Alexa fluor 594 (1:250, Molecular Probes, Eugene, OR) for 1 hr at 4 C. Subsequently, following sufficient rinsing, the specimens were incubated with anti-alpha smooth muscle actin (SMA, 1:100, mouse monoclonal, Progen), a marker of smooth muscle, or anti-uroplakin III (UP III, 1:100, goat polyclonal, Santa Cruz Biotechnology Inc., Santa Cruz, CA), a marker for urothelium, for 12 hr at 4 C. Following rinsing, they were incubated with secondary antibody consisting of donkey antimouse or anti-goat IgG conjugated with Alexa fluor 488 (1:250, Molecular Probes) for 1 hr at 4 C. Finally, cell nuclei were counterstained with 5 mg/ml 4 0 , 6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes). The slides were coated with Fluorescent Mounting Medium (Dako Cytomation, Carpinteria, CA) and observed with a Leica DAS Microscopethe (Leica Microsystems GmbH, Wetzlar, Germany). Statistical Analysis
The results were expressed as means standard error of mean. Two-way repeated measures ANOVA followed by the Scheffe’s test were used to compare non-RTX-treated normal rats (n ¼ 6) and RTX-treated rats (n ¼ 6) in each temperature condition. Differences with P < 0.05 were considered significant. RESULTS Behavioral Responses to Changing Temperature Conditions
The rats were relatively inactive during the 20 min exposure to RT conditions. Prior to urination, they moved about in the metabolic cages and then became inactive again afterwards. Immediately after transfer to the cold room, they exhibited violent shivering, pilomotor responses, and vigorous movements in the cages. The reactions slowly disappeared with adaptation to the LT conditions. Upon returning to RT, the rats remained quiet, similar to their behavior before transfer to the cold room. Influence of Cold Environmental Stress on Micturition Patterns
Micturition patterns of conscious normal rats were affected by the changes in temperature (Fig. 1). During the first 20 min after the rats were transferred from RT to LT Phase I, the rats exhibited micturition patterns consistent with detrusor overactivity. Basal pressure increased to 13.93 0.91 cmH2O (P < 0.01); however, micturition pressure did not change significantly (Table I), though it tended to increase during this period. During LT Phase I, the voiding interval decreased to 2.40 0.26 min (P < 0.01). Additionally, micturition volume and bladder capacity each decreased (P < 0.01) in response to the LT stress (Table I). During the second 20 min of exposure, LT Phase II, the detrusor overactivity slowly mitigated (Fig. 1). Although basal pressure and micturition pressure did not change significantly,
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Fig. 1. Effect of temperature changes on micturition patterns of normal conscious rats. These results are representative of the six normal rats studied. In LT Phase I, detrusor overactivity was evident in the altered frequency of changes in BP and MV. In LT Phase II, the detrusor overactivity was slowly mitigated. In re-RT, the detrusor overactivity disappeared. RT: room temperature condition (27 2 C); LT: low teperature condition (4 2 C); re-RT: return to RT condition, 20 min in each condition. BP: bladder pressure, MV: micturition volume. Asterisks: real-time temperature transitions.
voiding interval increased to 4.86 0.69 min (P < 0.01), and micturition volume and bladder capacity increased to 0.79 0.10 ml (P < 0.01) and 0.85 0.10 ml, respectively (P < 0.01, Table I). When the rats were returned to RT (re-RT), the detrusor overactivity disappeared (Fig. 1), and all of the measured variables returned to RT values (Table I). Micturition Patterns of RTX-Treated Rats
The micturition patterns of the RTX-treated rats were also affected by changes of temperature (Fig. 2). When the RTXtreated rats were transferred to LT Phase I, the micturition patterns were similar to those of normal rats (Table II). Basal pressure and micturition pressure increased to 17.13 2.22 cmH2O (P < 0.05) and 57.51 9.74 cmH2O, respectively. The voiding interval decreased to 5.29 0.93 min (P < 0.05) while micturition volume decreased to 0.84 0.15 ml (P < 0.05), and bladder capacity decreased to 0.85 0.15 ml (P < 0.01). However, voiding interval, micturition volume, and bladder capacity of the RTX-treated rats were significantly greater than that of normal rats (P < 0.05, Table II), although there were no significant differences between normal and RTX-treated rats in RT. Thus, the cold-stress detrusor overactivity of RTX-treated rats was mitigated compared with that of normal rats (Fig. 2). During the second 20 min of LT exposure (LT Phase II), the micturition patterns were similar to that of LT Phase I (Fig. 2). For the RTX-treated rats, none of the measured variables changed significantly during LT Phase II. Further, there were no significant differences between any of the variables in the normal and the RTX-treated rats. When the rats were returned to RT (re-RT), the micturition patterns of the RTX-treated rats returned to the RT values (Table II). The decrease in basal pressure and the increases in voiding interval, micturition volume, and bladder capacity
were all significant. There were also no differences between the normal and the RTX-treated rats during re-RT. Neuronal Structures in Urinary Bladder tissues
Neuronal structures within urinary bladder tissues of normal and RTX-treated rats were visualized by indirect immunohistochemistry with S100 and CGRP antibodies. The urinary bladder tissue of normal rats had distinct S100positive neuronal structures under the urothelium and within the smooth muscle layers (Fig. 3A,B). CGRP-positive afferent nerves were also present within the smooth muscle layers (Fig. 3C). In contrast, there were fewer S100-positive nerve structures under the urothelium or within the smooth muscle layers in the RTX-treated rats than in normal rats (Figs. 3D,E). The number of CGRP-positive afferent nerves in the RTXtreated rats also decreased (Fig. 3F).
DISCUSSION
Changes of environmental temperature clearly affected the micturition patterns of conscious rats. When conscious rats were transferred from RT to LT, the rats experienced a sudden cooling of whole body mass. Voiding interval, micturition volume, and bladder capacity of the cooled rats significantly decreased compared to values at RT. After 20 min at LT, these variables slowly increased as the rats adapted during LT Phase II to the conditions. When the rats were returned to RT, the micturition variables recovered to the original values. These physiological responses simulated the urinary sensations and frequent urination that many people experience when exposed to sudden decreases of temperature. Thus, the onset of cold environmental stress induces detrusor overactivity in conscious rats.
TABLE I. Cystometric Parameters of Normal Rats at Each Environmental Temperature
Conditions
Basal pressure (cmH2O)
Micturition pressure (cmH2O)
Voiding interval (min)
Micturition volume (ml)
Bladder capacity (ml)
RT LT Phase I LT Phase II re-RT
7.73 0.74 13.93 0.91yy 13.24 0.63 8.74 0.59§§
51.22 3.78 59.04 4.49 53.04 5.33 45.11 3.54§
6.11 0.84 2.40 0.26yy 4.86 0.69{{ 6.85 0.88§
1.08 0.12 0.39 0.07yy 0.79 0.10{{ 1.13 0.14§§
1.21 0.15 0.44 0.08yy 0.85 0.10{{ 1.16 0.14§§
Note: y P < 0.05,
yy
P < 0.01 (RT versus LT Phase I); {P < 0.05, {{P < 0.01 (LT Phase I versus LT Phase II); §P < 0.05, §§P < 0.01 (LT Phase II versus re-RT).
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Fig. 2. Effect of temperature changes on micturition patterns of RTX-treated conscious rats. These results are representative of the six RTX-treated rats studied. In LT Phase I, cold-stress detrusor overactivity of the RTXpretreated rats was mitigated compare with that of normal rats. In LT Phase II, the detrusor overactivity did not change as much as it did in normal rats. In re-RT, the detrusor overactivity disappeared.
This study focused on RTX-sensitive nerves, which are components of unmyelinated C afferents, to investigate the cold-stress detrusor overactivity. When rats treated with systemic RTX were exposed to the cold stress, the voiding interval, micturition volume, and bladder capacity decreased, but they were significantly higher than those of non-RTXtreated normal rats. Our previous article indicated that voiding interval, micturition volume, and bladder capacity of 12-week-old rats were significantly increased with RTX.17 However in our current study, there were no significant differences between non-RTX normal and RTX-treated 10week-old rats at RT. These findings indicate that the coldstress detrusor overactivity of the RTX-treated rats was partially mitigated. In contrast, basal pressure of both normal and RTX-treated rats increased during the cold stress. Also, micturition pressure tended to increase, though it did not reach statistical significance. These responses might reflect cold-induced increases in muscle tonus consistent with the observed shivering and pilomotor responses. Thus, while the increases of basal pressure and micturition pressures were induced by the cold stress, RTX-sensitive neurological pathways might not have mediated both of these pressures. These results suggest that RTX-sensitive neurological pathway mediated a portion of the cold-stress detrusor overactivity. The RTX-sensitive afferent nerves located within urinary bladder tissues are clearly associated with detrusor overactivity.18 –20 Desensitization of the nerves with capsaicin or RTX is used to treat bladder overactivity induced by different neurological diseases.21 – 24 To our knowledge, there is no RTXsensitive afferent nerves-specific antibody. However, S100positive neuronal structures25 and CGRP-positive afferent nerves26 present in urinary bladder tissues have been reported. We indirectly verified the decrease of the RTX-sensitive afferent nerves in the RTX-treated rats by immunohistochemistry. The RTX-treated rats had fewer S100-positive neuronal structures than the non-RTX-treated rats because the CGRPpositive afferent nerves were decreased by the treatment of
RTX. The RTX-sensitive afferent nerves are included in the CGRP-positive afferent nerves. The immunohistochemical findings supported that the urinary bladders of the RTXtreated rats lost a majority of the RTX-sensitive afferent nerves. Therefore, the RTX-sensitive afferent nerves present in the urinary bladders and/or receptors present on the nerves, such as TRPM827 – 29 might be involved in the regulation of detrusor activity and mediate a part of the overactivity associated with cold stress. This model suggests that cold environmental stress might reflect changes in various receptors, neurotransmitters, and/or enzymes expressed in urinary bladder tissues. Many researchers have strived to make disease models, utilizing chemical irritants or physical destruction of tissues, to investigate the mechanisms of pathogenesis, aggravation, and/or treatment for lower urinary tract dysfunctions. However, these models do not necessarily reflect normal disease processes. Our coldstress model induces detrusor overactivity in normal rats without disease or injury. In addition, the model causes various micturition patterns that simulate human physiological responses due to sudden changes of temperature. When this model is applied to experimental animals having receptors and/or neurotransmitters that can be controlled by pharmacological or genetic techniques, this model will help to elucidate the mechanisms of cold-stress-induced detrusor overactivity. These advantages could be useful to investigate lower urinary tract functions.
CONCLUSION
This study indicates that quick and large decreases of temperature affect micturition patterns of conscious rats. Cold environmental stress induced by transferring conscious normal rats from RT to LT conditions induces detrusor overactivity. A portion of the response is mediated by RTXsensitive neurological pathway. The cold-stress detrusor
TABLE II. Cystometric Parameters of RTX-Treated Rats at Each Environmental Temperature
Conditions
Basal pressure (cmH2O)
Micturition pressure (cmH2O)
Voiding interval (min)
Micturition volume (ml)
Bladder capacity (ml)
RT LT Phase I LT Phase II re-RT
11.53 2.54 17.13 2.22y 18.58 2.57 13.53 2.36§§
44.11 2.73 57.51 9.74 63.62 12.74 51.29 6.44
7.60 0.51 5.29 0.93y* 5.09 0.63 7.80 0.75§§
1.24 0.09 0.84 0.15y* 0.82 0.09 1.25 0.10§§
1.29 0.10 0.85 0.15yy* 0.86 0.09 1.28 0.10§§
Note: yP < 0.05, yyP < 0.01 (RT versus LT Phase I); §P < 0.05, §§P < 0.01 (LT Phase II versus re-RT); *P < 0.05 compared between non-RTX-treated normal rats and RTX-treated rats in each condition.
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Fig. 3. Neuronal structures in urinary bladder of normal and RTX-treated rats. A: Normal rats had distinct S-100 positive neuronal structures (arrows) under the urothelium. B: The structures (arrows) were also detected within smooth muscle layers along with (C) CGRP-positive afferent nerves (arrows). D: There were fewer S-100 positive nerve structures (arrows) under the urothelium of RTX-treated rats than in non-RTX normal rats. E: These structures (arrows) were also decreased within the smooth muscle layers. F: The RTX-treated rats had few CGRP-positive afferent nerves within smooth muscle layers. UT: urothelium stained with anti-uroplakin III; SM: smooth muscle layers stained with anti-SMA. Blue: nuclei.
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