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.