Chronic psychological stress is linked to the development and worsening of overactive bladder (OAB) characterized by urinary symptoms, e.g. frequency, urgency, incontinence, and nocturia [1]. 
Water avoidance stress (WAS) is a well-accepted stress-inducing model employed to induce abnormal bladder symptoms resulting from psychological stress [2]. Animals subjected to 10 days of WAS exhibited bladder dysfunction, e.g. increased urinary frequency, decreased urine volume, and histopathological alterations [3]. Studies in rats showed that exposure to WAS for one day was considered acute stress, while ten days represented chronic stress [3].
However, it remains elusive whether prolonged exposure to WAS longer than 10 days could alter the voiding pattern and bladder contractile properties. Therefore, this study aimed to investigate the voiding pattern profile of mice exposed to acute WAS (1 day) to prolonged WAS (28 days). Bladder contractile properties and tonic response to muscarinic agonist carbachol (CCh) were determined in mice exposed to acute WAS and prolonged WAS.

Adult male C57BL/6NJcl mice (8-10 weeks old) were randomly divided into four groups; control 1-day (C1, n=8), stress 1-day (S1, n=9), control 28-day (C28, n=9), and stress 28-day (S28, n=9) groups. Mice in the stressed groups were subjected to WAS protocol by being placed on a platform in the middle of a polypropylene box (43 cm length, 29 cm width, and 20 cm height) filled with water at room temperature for 1 hour from 10 a.m. to 11 a.m. for either 1 or 28 consecutive days. Mice in the control groups were housed in standard cages. 
Voiding spot analysis was monitored on days 1, 7, 10, 14, 21, and 28. A standard cage was lined with filter paper (15 x 26.5 cm2) on the bottom of the cage. Mice were individually placed in the cage and had free access to food but water deprivation for 4 hours from 1 p.m. to 5 p.m. After 24 hours, urine-stained filter paper was captured under the UV light and analyzed with Image J Software to determine the number of urine spots and voided area.
After 1-day or 28-day exposure to WAS, mice were euthanized, and the bladders were collected and trimmed into a rectangle shape to conduct Ex vivo organ bath experiment. Bladder contractile properties were stimulated with KCl (80 mM). After washing with Krebs solution, cumulative carbachol (CCh) concentrations at 0.1, 0.3, 1.0, 3.0, 10, and 30 μM were added into the tissue chamber. Tonic responses to CCh were analyzed in percentage change from their baseline contraction. 
All procedures were approved by the institutional committee for the ethical use of animals, Prince of Songkla University, Hat Yai, Songkhla, Thailand. Data were expressed as mean standard error of the mean (S.E.M.). Voiding spot analysis was compared using unpaired t-test. Multiple comparison analysis was conducted using One-way ANOVA followed by Dunnett's test to compare the contractile response to KCl and CCh with the baseline. Statistical significance was determined at P<0.05 using GraphPad Prism 9.0 software.

There was no significant difference in the total voided area between the S1 and C1 groups (C1 at day1; 37.25 ± 4.44 cm2 vs. S1 at day1; 43.77 ± 8.20 cm2) and the total number of urine spots (C1 at day1; 2.38 ± 0.57 vs. S1 at day1; 2.25 ± 0.37). Mice in the S28 group showed a significant decrease in total void area after exposed to WAS for 10 days compared to the control group (C28 at day10; 52.88 ± 7.88 cm2 vs. S28 at day10; 21.80 ± 5.90 cm2, **P<0.01, unpaired t-test) (figure 1). However, we did not observe a significant change in urine spot number after 10 days of WAS (C28 at day 10; 2.60 ± 0.81 vs. S28 at day 10; 2.57 ± 1.11). Interestingly, after 28 days of WAS exposure, there was no significant difference in the total voided area (C28 at day28; 49.57 ± 12.34 cm2 vs. S28 at day28; 42.94 ± 7.33 cm2) and urine spot number (C28 at day28; 2.50 ± 0.85 vs. S28 at day28; 2.88 ± 0.91) between the control (C28) and the stress (S28) groups.
The bladder strips of the S1 group showed a significant increase in tonic contractile response to CCh at concentrations of 1.0, 3.0, 10, and 30 μM compared to the baseline (*P<0.05, ****P<0.0001, One-way ANOVA followed by Dunnett's test), whereas the C1 group was significantly increased at 3.0 and 10 μM of CCh (*P<0.05, **P<0.01, One-way ANOVA followed by Dunnett's test).
In prolonged 28-day WAS exposure, the tonic contractile response of the S28 group was significantly increased in response to CCh at concentrations of 3.0 and 10 μM (*P<0.05, One-way ANOVA followed by Dunnett's test), which showed a similar tonic response in C28 group (*P<0.05, One-way ANOVA followed by Dunnett's test).

In voiding spot analysis, mice subjected to acute WAS for one day did not show a significant alteration of total urine spot number and voided area. However, the bladder contractile properties exhibited increased sensitivity to a muscarinic agonist. 
Although a decrease in urine volume and a higher proportion of small urine spots was detected after 10 days of WAS, mice exposed to WAS for 28 days displayed an equivalent tonic contractile response to CCh compared to the control 28-day group, which is correlated with no significant alteration in voiding pattern.  
These findings imply that acute stress exposure to WAS for one day could affect muscarinic signaling in the bladder tissue without significant changes in voiding patterns. Prolonged exposure to 28-day WAS did not exhibit abnormal voiding patterns but reversed abnormal voiding patterns after 10 days of stress exposure and reduced the heightened response to muscarinic stimulation in the urinary bladder.

Acute exposure to 1-day WAS enhanced tonic contractile response to muscarinic agonist in the urinary bladder without a significant change in voiding pattern. Notably, chronic exposure to 28 days of WAS could reverse impaired voiding patterns after 10 days of WAS exposure. Monitoring both acute and prolonged chronic stress of the WAS model in mice can lead to an extended understanding of the effect of psychological stress exposure on urinary bladder functions and changes in bladder contractile properties in animal models. These findings could be essential information in employing WAS as a model to study stress-related urinary bladder dysfunction. Further investigation is essential to examine the related mechanisms in adaptive responses of the urinary bladder function in long-term exposure to water avoidance stress model.

Reynolds, W. S., McKernan, L. C., Dmochowski, R. R., & Bruehl, S. (2023). The biopsychosocial impacts of anxiety on overactive bladder in women. Neurourology and urodynamics, 42(4), 778–784. https://doi.org/10.1002/nau.25152
West, E. G., Sellers, D. J., Chess-Williams, R., & McDermott, C. (2021). Bladder overactivity induced by   psychological stress in female mice is associated with enhanced bladder contractility. Life  sciences, 265, 118735.
Smith, A. L., Leung, J., Kun, S., et al. (2011). The Effects of Acute and Chronic Psychological Stress on   Bladder  Function  in  a  doi:10.1016/j.urology.2011.06.0   Rodent  Model.  Urology,  78(4),  967.e1–967.e7.

Continence 12S (2024) 101433
DOI: 10.1016/j.cont.2024.101433