Clinical evidence suggests that psychological stress is linked to bladder dysfunction.  This is supported by experimental data using rodent models with recent work reporting that repeated exposure to environmental (water avoidance) stress results in increased urinary frequency in female mice and enhanced bladder contractile responses. Mirabegron, a beta3-adrenoceptor agonist, and solifenacin, a competitive muscarinic (M3) antagonist, are commonly used clinically to manage the symptoms of bladder overactivity; however, there is a lack of evidence to support their effectiveness for bladder dysfunction caused by psychological stress. Therefore, the aim of this study was to determine if mirabegron and solifenacin are effective in managing the changes in voiding behaviour and detrusor contractility induced by water avoidance stress in female mice.

Female mice (12-14 weeks) were randomly allocated to control (Unstressed, n=6), water avoidance stress (Stressed, n=6), mirabegron treated (Stress + Mira, n=6) and solifenacin treated (Stress + Soli) experimental groups. Treated mice were administered mirabegron and solifenacin in their drinking water during the full 10-day stress protocol. Stressed mice and drug treated stressed mice were placed on a central pedestal surrounded by room temperature water for 1hr/day for 10 days. Controls were age-matched and housed normally without environmental stress exposure. Voiding behaviour was assessed prior to (Day 0) and on days 1, 3, 5 and 10 of the stress protocols using voiding pattern analysis. Mice were euthanised 24 hours after the final stress exposure. At this time, a blood sample was also taken to measure plasma corticosterone levels. Bladders were removed, catheterised and intravesical pressure responses recorded during distension, in response to pharmacological stimulation and following electrical field stimulation (EFS).  Intraluminal and serosal fluid samples were collected following bladder distension and levels of ATP and acetylcholine quantified.

A significant increase in urinary frequency was observed following stress exposure, with a 6-fold change evident by day-10 (p<0.001) (Figure 1A).  Mice treated with mirabegron or solifenacin during the stress exposure displayed significantly fewer voiding events compared to the Stressed group (p<0.001), and voiding frequency in drug treated animals was similar to unstressed controls (Figure 1A). The increased frequency observed with stress was associated with a significant decrease in average void size and increase in number of small voids (0.2cm2) compared to the mirabegron-treated, solifenacin-treated and control groups, with no difference in total voided volume or bladder compliance between the groups. 
There was a significant increase in plasma corticosterone levels in the Stressed group, 107± 21.9 µg/mL, compared to the unstressed group, 44.8 ± 8.89 µg/mL (p = 0.034).  Solifenacin significantly decreased plasma corticosterone levels, 37.0 ± 10.5 µg/mL (p = 0.026), and, although not significantly different to the stressed group, mirabegron treatment also decreased plasma corticosterone levels, 45.8 ± 17.0 µg/mL (p = 0.059).  
Isolated whole bladder contractile responses to cholinergic stimulation, with cumulative carbachol concentrations, were similar between the groups, with no significant difference in the pEC50 values.  However, the maximal contractile response to carbachol was significantly enhanced following 10-days stress exposure (43.5 ± 1.21 mmHg Unstressed vs 57.5 ± 3.35 mmHg Stressed (p<0.001)). Maximal contractile responses in the solifenacin and mirabegron treated groups were however not significantly different from the stressed or control groups. When responses to carbachol were expressed as a percentage of the KCl response, no significant differences in the maximal responses were observed between the groups.  The phasic component of the carbachol-induced (1µM) contractile response was also quantified, and while amplitude was unchanged, the frequency of contractions was significantly enhanced in the Stressed (p<0.001) and Stress + Mirabegron groups (p<0.01) compared to unstressed controls (Figure 1 B).  However, treatment with solifenacin significantly reduced the frequency of phasic contractions to control levels (Figure 1 B).  Following carbachol pre-contraction, β-adrenoceptor mediated relaxation was investigated using isoprenaline which produced a concentration-dependent decrease in intravesical pressure in isolated bladder from all experimental groups.  While there was a tendency towards a reduction in the maximal relaxation response in bladders from the Stress and Stress + Mirabegron group, this change was not statistically significant. Contractile bladder responses to purinergic stimulation with αβmATP or EFS were not changed by stress exposure or drug treatment, nor were levels of ATP or acetylcholine released into the intraluminal or serosal fluid.

Mirabegron and solifenacin treatment prevented increased voiding behaviour in animals exposed to psychological stress.  With no differences in urinary volume detected, the changes observed were in voiding phenotype rather than urine production, with drug treatment returning voiding behaviour to unstressed control levels, despite continued stress exposure.  Overactive bladder symptoms correlate with increased anxiety and depression in patients.  While there is some evidence that both mirabegron and solifenacin can reduce the associated symptoms of anxiety and depression in clinical studies (Kelleher et al., 2005; Kinjo et al., 2019), here we observed that oral treatment with solifenacin and mirabegron can reduce the hormonal stress response associated with environmental stress. Central and peripheral muscarinic and adrenergic receptors have previously been linked to regulation of corticosterone release (Rhodes et al, 2005; Walker et al, 1991) and the reduction in the hormonal stress response may contribute to the effectiveness of these drugs in managing the voiding changes associated with water avoidance stress exposure. While voiding frequency was reduced by mirabegron and solifenacin treatment, the increase in bladder contractility to stimulation with carbachol observed following stress was not altered in either of the drug treated animal groups.  This would suggest that the increase in contractility does not play a causal role in the voiding dysfunction observed with stress and may instead be a local compensatory mechanism in response to stress.

This study is the first pre-clinical study to our knowledge to show that management of bladder dysfunction caused by psychological stress can benefit from the use of an antimuscarinic such as solifenacin or the β3-adrenoceptor agonist, mirabegron.

Kinjo, M., Yamaguchi, T., Tambo, M., Okegawa, T., & Fukuhara, H. (2019). Effects of Mirabegron on Anxiety and Depression in Female Patients with Overactive Bladder. Urol Int, 102(3), 331-335.
Rhodes, M. E., Billings, T. E., Czambel, R. K., & Rubin, R. T. (2005). Pituitary-adrenal responses to cholinergic stimulation and acute mild stress are differentially elevated in male and female M-2 muscarinic receptor knockout mice. Journal of Neuroendocrinology, 17(12), 817-826.