Correspondence: Tetsuya Imamura Ph.D., Department of Lower Urinary Tract Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Email: firstname.lastname@example.org
Cold stress as a result of whole-body cooling at low environmental temperatures exacerbates lower urinary tract symptoms, such as urinary urgency, nocturia and residual urine. We established a model system using healthy conscious rats to explore the mechanisms of cold stress-induced detrusor overactivity. In this review, we summarize the basic findings shown by this model. Rats that were quickly transferred from room temperature (27 ± 2°C) to low temperature (4 ± 2°C) showed detrusor overactivity including increased basal pressure and decreased voiding interval, micturition volume, and bladder capacity. The cold stress-induced detrusor overactivity is mediated through a resiniferatoxin-sensitve C-fiber sensory nerve pathway involving α1-adrenergic receptors. Transient receptor potential melastatin 8 channels, which are sensitive to thermal changes below 25–28°C, also play an important role in mediating the cold stress responses. Additionally, the sympathetic nervous system is associated with transient hypertension and decreases of skin surface temperature that are closely correlated with the detrusor overactivity. With this cold stress model, we showed that α1-adrenergic receptor antagonists have the potential to treat cold stress-exacerbated lower urinary tract symptoms. In addition, we showed that traditional Japanese herbal mixtures composed of Hachimijiogan act, in part, by increasing skin temperature and reducing the number of cold sensitive transient receptor potential melastatin channels in the skin. The effects of herbal mixtures have the potential to treat and/or prevent the exacerbation of lower urinary tract symptoms by providing resistance to the cold stress responses. Our model provides new opportunities for utilizing animal disease models with altered lower urinary tract functions to explore the effects of novel therapeutic drugs.
Cold stress produced by sudden change or continuous exposure to low environmental temperature seriously affects urinary tract functions, along with other physiological responses. For instance, whole-body cooling increases the heart rate and/or blood pressure.[2-4] Sudden whole-body cooling elicits urinary sensations and frequent urination in healthy people, even in the absence of LUTS. For patients having LUTS, it especially exacerbates symptoms such as urinary urgency, nocturia and residual urine. Thus, the response to cold stress potentially provides insightful understanding into the mechanism(s) of LUTS.[5-8] For this reason, we have established an animal model to investigate cold stress-exacerbated LUTS.
Rats stimulated with cold stress show remarkable detrusor overactivity that causes an increase in basal pressure, and decreases in voiding interval, micturition volume and bladder capacity. By using the cold stress model, we have found important aspects of the mechanism(s) that elicit cold stress-induced detrusor overactivity (Fig. 1). The present review shows that cold stress-induced detrusor overactivity is mediated, at least in part, through a RTX-sensitive C-fiber sensory nerve pathway. The response is closely associated with the sympathetic nervous system that mediates transient hypertension and sensory input from the lowered temperature of the skin. Other components of the response to cold stress include TRPM8 channels within the skin cells and/or sensory neurons.[10, 11] In addition, α1-AR antagonists have the potential to treat cold stress-exacerbated LUTS.[9, 12] Finally, traditional Japanese herbal mixtures composed of Hachimijiogan have the potential to provide resistance to the cold stress responses.
Cold stress-induced detrusor overactivity model
We produced environmental cold stress by quickly and smoothly transferring rats from RT (27 ± 2°C) to LT (4 ± 2°C) environments. Two days before cystometric investigations, the bladders are prepared by cannulation. The micturition patterns of conscious rats were monitored for 20 min before transfer to the LT environment. Immediately after transfer to the cold room, the rats showed violent shivering, pilomotor responses and vigorous movements in the cages.
Changes in micturition patterns occurred in two phases during the 40 min of exposure to LT (Fig. 2a). During the first 20 min after transfer, phase I, the rats showed detrusor overactivity patterns, such as increased micturition frequency (Fig. 2a,b); for example, less than 5-min voiding interval; decreased micturition volume and less than 1-mL bladder capacity (Fig. 2c). Basal pressure significantly increased, and micturition pressure tended to increase, though the change is not significant. Also, residual volume does not change. During the second 20 min of LT exposure, phase II, the detrusor overactivity patterns slowly mitigated and nearly disappeared (Fig. 2a). The micturition patterns then approximated those at RT. The basal and micturition pressure, and residual volume did not undergo significant further changes. However, voiding interval (Fig. 2b), micturition volume and bladder capacity (Fig. 2c) gradually increased, achieving levels that were significantly higher than in phase I and similar, but somewhat lower to those at RT. After 40 min of LT exposure, the rats were returned to RT. The detrusor overactivity patterns quickly and completely disappeared (Fig. 2a), and all of the measured variables recovered to the baseline RT values.
The transfers to and from the cold room were handled very gently and smoothly so as to avoid unnecessary stress in the rats. We confirmed that these transfers did not affect their behavior. Throughout the cystometric investigations, we infused an isotonic sodium chloride solution (saline) maintained at RT into the bladder at a rate of 10 mL/h. We confirmed that the temperature of the infused saline did not change during the LT exposure. Additionally, we confirmed that diuresis was not promoted under the LT condition. Some studies from other groups directly stimulated the urinary bladder with an infusion of ice-cold water;[14-18] however, aside from our studies, there are no others that investigated the onset of urinary sensations and frequent urination induced by sudden whole-body cooling. Our cold stress model accurately captured a common human experience, and the ensuing response of the rats was very similar to the human physiological response to similar conditions.
Relationship between cold stress-induced detrusor overactivity and C-fiber sensory nerve pathway
The activation of C-fiber sensory nerves is widely accepted as one of the mechanisms of LUTS exacerbation. We focused on RTX-sensitive C-fiber sensory nerve pathways to determine if they mediate the cold stress-induced detrusor overactivity. One day before the cystometric investigations, we subcutaneously injected 0.3 mg RTX/kg bodyweight into healthy rats. That RTX dose decreases the S100- and CGRP-positive nerve fibers that are components of the C-fiber sensory nerves within the urinary bladder. Cold stress cystometric investigations were then carried out as described earlier. Although there were no significant differences between non-RTX-treated and RTX-treated rats at RT, the cold stress micturition patterns of the RTX-treated rats were different. The voiding interval (Fig. 2b), micturition volume and bladder capacity (Fig. 2c) during LT phase I of the RTX-treated rats were significantly greater than those of untreated rats. During LT phase II, the micturition patterns and values of the RTX-treated rats were similar to those of LT phase I. Furthermore, as a result of the mitigation of responses that occurred in untreated rats during phase II (Fig. 2c), there were no significant differences between any of the variables in the non-treated and the RTX-treated rats during the final 20 min of LT exposure. When the rats were returned to RT, the micturition patterns of the RTX-treated rats recovered to the RT patterns, which were similar to the untreated rats returned to RT. The RTX-dependent inhibition of detrusor overactivity induced during LT Phase I suggests that the C-fiber sensory nerve pathway mediates a portion of the cold stress-induced detrusor overactivity (Fig. 1).
Cold stress-induced detrusor overactivity and blood pressure
A universally common experience is that whole-body cooling as a result of exposure to low environmental temperature increases blood pressure,[2, 4] as well as urinary sensations and urinary frequency. By 10 min after transfer to LT, the blood pressure of the rats significantly increased compared with that at RT, and it was maintained through the LT exposure period (Fig. 2d). Thus, activity of the sympathetic nervous system associated with transient hypertension partially correlated with the cold stress-induced detrusor overactivity (Fig. 1).
Cold stress-induced detrusor overactivity and skin surface temperature
As measured with a digital thermography camera, within 5–10 min after transfer to LT, the hind leg skin surface temperature of the rats decreased in correlation with the cold stress-induced detrusor overactivity (Fig. 2d). By the end of LT phase I (20 min) and through phase II (40 min), the skin temperature was relatively stable (Fig. 2d). Thus, while skin temperature had become stable by 20 min of LT and was maintained for the duration of exposure, the cold stress-induced detrusor overactivity gradually disappeared. The time-dependent reductions of LT stimulated-responses represent an adaptive response that is universal in normal healthy humans. Thus, a momentary cold stimulus can act as a trigger for the urinary responses, and the present results suggest that the cold stress-induced detrusor overactivity is associated with the resulting sudden decrease of skin temperature. After regaining homeostasis of skin temperature, even at levels significantly lower than they are at RT, the cold responses diminish. Therefore, the decrease of skin temperature is closely associated with the cold stress-induced detrusor overactivity (Fig. 1).
We focused on the mammalian TRP channel expression in the skin because the cold stress-induced detrusor overactivity is correlated with skin surface temperature. The mammalian TRP channel family consists of 28 channels that are subdivided into five different classes: TRPV (vanilloid), TRPC (canonical), TRPM (melastatin), TRPML (mucolipin) and TRPA (ankyrin). TRP channels can be activated by physical (voltage, mechanical, heat or cold) stress or chemical (pH, osmolality) stimuli.[20-24] TRPM8 can be activated by both menthol and LT stimuli (less than 25–28°C).[25-28] The cell bodies of temperature-sensitive neurons, consisting mostly of C- and Aδ-fibers, are located in both the trigeminal ganglia and DRG. TRPM8 channel protein and mRNA are found in approximately 10–15% of the trigeminal ganglia and 5–10% of the DRG.[29-31] TRPM8 channel knockout mice, which are not different from wild-type mice in overall appearance, general behavior, viability, core body temperature or anatomy of the sensory ganglia, have significantly less cold sensitivity and almost no menthol sensitivity.[32, 33] In addition, the TRPM8-expressing sensory neurons with dichotomizing axons within DRG might have a role in urinary urgency evoked by cold sensation. Therefore, we focused on correlations between cold stress-induced detrusor overactivity and TRPM8 channels.
Functional roles of TRPM8 channels expressed within the skin in cold stress-induced detrusor overactivity
The TRPM8 channel agonist menthol elicits detrusor overactivity in healthy, conscious rats (Fig. 1).[10, 11] Spraying a liquid stream of 90% menthol solution once every 5 min for 20 min at RT induced detrusor overactivity patterns that significantly decreased voiding interval (Fig. 3a), micturition volume and bladder capacity (Fig. 3b). The micturition patterns are very similar to the patterns of cold stress-induced detrusor overactivity that occur immediately after LT exposure. The basal pressures of menthol-sprayed rats tend to increase, though the increases are not statistically significant. In contrast, the basal pressures of the cold stress-exposed rats significantly increased. The difference in response might be a result of the nature of local stimulation by menthol spray and whole-body stimulation by LT.[10, 11]
To investigate the roles of TRPM8 channels in the menthol spray- and cold stress-induced detrusor overactivity mechanism(s), we used a TRPM8 channel antagonist, BCTC. In rats intravenously injected with 0.1 μmol/kg BCTC, the menthol spray-induced detrusor overactivity was completely blocked. After menthol spraying, voiding interval (Fig. 3a) and bladder capacity (Fig. 3b) of BCTC-treated rats did not decrease. In the BCTC-treated rats, the cold stress-induced detrusor overactivity that immediately occurs after transfer to LT was also partially abolished. Additionally, the decreases of voiding interval (Fig. 3c), micturition volume and bladder capacity (Fig. 3d) were inhibited in a dose-dependent manner.
Another cold-sensitive TRP channel, TRPA1, is important in the transduction of cooling sensations that are activated by noxiously cold temperatures of less than 17°C.[35-39] However, the TRPA1 channel agonist, cinnamon, does not elicit detrusor overactivity that is similar to the menthol-induced detrusor overactivity. Although BCTC has a blocking effect for another thermosensitive channel, TRPV1 channels, the channels expressed in the skin are not likely to be functionally associated with the cold stress-related detrusor overactivity. TRPV1 is present in the skin, but it is responsive only to temperatures that are greater than 43°C. The present results suggest that both menthol spray- and cold stress-responses are, in part, mediated through the neurological pathway involving TRPM8 channels (Fig. 1).[10, 11] Consequently, the evidence suggests that TRPM8 channels expressed in the skin act as triggers for cold stress-induced detrusor overactivity (Fig. 1).
Functional roles of α1-AR in cold stress-induced detrusor overactivity
In addition to the RTX-sensitive C-fiber sensory nerve pathway, the cold stress-induced detrusor overactivity is partially mediated through the sympathetic nervous system that is also associated with transient hypertension. Thus, we have focused on α1-AR that are activated by epinephrine and increase vascular tone, and consequently, blood pressure. α1-AR are present at parasympathetic nerve terminals in the urinary bladder,[40, 41] as well as in the bladder urothelium where they mediate afferent bladder activity.[42-45] Therefore, we investigated the role of α1-AR in cold stress-induced detrusor overactivity by using the α1-AR antagonists naftopidil (high affinity for α1D-AR), silodosin (high affinity for α1A-AR), tamsulosin (affinity for α1A/1D-AR) and prazosin (non-selective for α1-AR subtypes).
All of the α1-AR antagonists partially mitigate cold stress-induced detrusor overactivity. For instance, reductions in voiding interval (Fig. 4a) and bladder capacity (Fig. 4b) after transfer to LT conditions (phase I) are inhibited in a dose-dependent manner by the antagonists. Except for prazosin, the antagonists prevent the increase in blood pressure that routinely occurs on exposure to the LT environment. Therefore, cold stress-induced detrusor overactivity that occurs simultaneously with increasing blood pressure is mediated, at least in part, by stimulation of the α1-AR subtypes α1D-AR and α1A-AR.[9, 12] The present results also suggest that administration of α1-AR antagonists have the potential to treat bladder storage symptoms, such as urinary frequency and nocturia, that are exacerbated by cold stress (Fig. 1).
Resistance to the cold stress responses provided by traditional Japanese herbal mixtures
To treat for cold stress-exacerbated LUTS, we were interested in Japanese herbal mixtures. HARNCARE (THC-002; TAIHO Pharmaceutical, Tokushima, Japan), TJ-107 (Goshajinkigan; Tsumura, Tokyo, Japan) are produced from the traditional Japanese herbal mixture, Hachimijiogan. THC-002 is a galenical solution extracted from Hachimijiogan and refined by removal of the starch component. After 7 days of administering THC-002 by stomach tube, the skin temperature of the treated rats was significantly increased compared with the THC-002-free control rats at RT. Although the skin temperature of THC-002-treated rats tended to be higher than that of the control rats during LT exposure, THC-002 did not completely prevent the decrease in skin temperature. Compared with control rats (Fig. 5a), THC-002 partially inhibited the cold stress-induced detrusor overactivity associated with increased micturition frequency and decreased micturition volume (Fig. 5b). Additionally, TRPM8 channel protein (Fig. 5c,d) and mRNA expression levels within the skin of THC-002-treated rats were significantly lower than that of control rats. THC-002 and Goshajinkigan both mitigated the C-fiber sensory nerve pathway-related detrusor overactivity by decreasing the tachykinin neurotransmitters and/or receptors related to C-fiber activation in the urinary bladder[46-49] and urethra. In future studies, we will investigate the mechanism(s) of increased skin temperature and decreased TRPM8 expression levels within the skin by administration of THC-002. At present, we simply report the empirical observation that some traditional Japanese herbal mixtures effectively mitigate detrusor overactivity elicited by cold stress (Fig. 1).
Some people without clinically evident cardiovascular disease experience cool hands and/or legs, even in a warm environment. These sensations are an indication of cold sensitivity, which is an important concept in oriental medicine. People having cold sensitivity show high LUTS storage symptoms compared with people who are not sensitive to cold. In addition, some cold-sensitive patients tend to complain about a change for the worse of their symptoms during cold periods. Some galenicals produced from traditional Japanese herbal mixtures can reduce the sensitivity to cold,[51, 52] possibly by improving blood circulation. Although we did not investigate changes of blood circulation or protein factors associated with improved circulation, our clinical studies show that treatment by THC-002 can reduce cold sensitivity and mitigate LUTS storage symptoms. Therefore, the traditional Japanese herbal mixtures, mainly composed of Hachimijiogan, have the potential to provide resistance to cold stress-exacerbated LUTS.
Advantages and disadvantages in cold stress-induced detrusor overactivity models
To investigate LUTS, many researchers have strived to develop disease models, utilizing chemical irritants or physical destruction of tissues and/or pharmacological techniques. However, these models do not necessarily reflect normal disease processes. Our cold stress model seriously affects physical changes in basal metabolism, blood circulation and/or peripheral nerve systems, and induces detrusor overactivity in healthy rats without disease or injury. In addition, the model imitates micturition patterns that simulate human physiological responses as a result of sudden drops to low temperatures.
Many disease animal models are used to investigate LUTS, such as spontaneously hypertensive, diabetic or ovariectomized rats. We often find that the lower urinary tract dysfunctions of these animals vary greatly among individuals at RT. However, our cold stress model mitigates the variability that is a result of intrinsic and/or potential lower urinary tract dysfunctions. Recently, we showed that the ovariectomized rats exhibit detrusor overactivity that is greater than that in sham-operated rats when exposed to LT, whereas the differences between ovariectomized and sham-operated rats at RT are minimal. Therefore, the advantages of our model are useful to investigate the mechanisms of pathogenesis, aggravation and/or treatment of LUTS (Fig. 1).
Despite these advantages, our model has two disadvantages. One is that it reflects systematic reactions as a result of “whole-body” cooling. Thus, the analysis of partial reactions and changes in various receptors, neurotransmitters and/or enzymes is limited (Fig. 1). Another is that our model provides only temporary and instantaneous stimulation, but not repeated and/or extended periods of cold stimulation. For patients having LUTS, repeated and/or continued cold environmental stress is the most serious problem. To overcome these disadvantages, we need more precise and detailed investigations.
The present review supports the validity of using conscious healthy rats as a model for investigating cold stress-exacerbated LUTS that is associated with detrusor overactivity (Fig. 1). RTX-sensitive C-fiber sensory nerve pathways involving α1-AR and TRPM8 channels in skin cells and/or sensory neurons mediate the cold stress-induced detrusor overactivity. In addition, decreases of skin temperature correlate with the detrusor overactivity. α1-AR antagonists and traditional Japanese herbal mixtures composed of Hachimijiogan have the potential to treat or prevent cold stress responses that can exacerbate LUTS. Therefore, our cold stress-detrusor overactivity model will be useful in the investigation of the mechanism(s) of pathogenesis, treatment and/or prevention for LUTS. Furthermore, our future studies of the cold stress response will explore changes in animal models that mimic the effects of human aging, metabolic disturbances, altered peripheral circulation and nervous system dysfunctions. Based on the outcome of these studies, we feel that novel combinations of drugs can be developed that will provide new insights into the onset and treatment of urinary cold stress responses.