Pre-clinical research investigating physiological consequences of the menopausal transition has employed the 4-vinylcyclohexene diepoxide (VCD) chemical model of ovarian failure (1,2). These studies, however, have relied on young adult rodents, even though clinical menopause occurs in midlife and may interact with age-related changes in tissue function. To address this, we aimed to adapt the VCD model by utilising ex-breeder female mice, which better reflect the reproductive ageing stage at which women enter perimenopause. We sought to characterise how VCD-induced cycle disruption affects bladder physiology, assessed by in vivo cystometry and ex vivo isolated whole-bladder functional studies.

Ex-breeder female C57Bl/6J mice (9-month-old) were randomly allocated to Control or Perimenopause groups (n=16 per group). The perimenopause group were treated for 10 days, from day 0, with daily VCD (i.p. 160 mg/kg/day), while control mice received daily vehicle injections. Vaginal cytology was performed to assess oestrus cycle length before and after treatment, along with voiding behaviour. On day 100 (12-month-old), cystometry was performed in urethane-anaesthetised mice (n=8 per group). In separate animals, whole bladders were removed, catheterised and intravesical pressure recorded to assess bladder responses to distension, pharmacological stimulation or electric field stimulation (EFS) (n=8 per group). Data (mean ± SEM) were compared using Student’s t-tests.

Prior to commencing treatment, 69.6% of aged mice had a normal oestrus cycle length (4 or 5 days), while 30.4% had prolonged cycles (>5 days). In the 14-day period prior to the experimental endpoint, all VCD-treated mice exhibited abnormal cycles, characterised by persistent days in oestrus. Voiding pattern analysis showed no alterations in voiding behaviour.  

Cystometric analysis revealed no significant differences in baseline intravesical pressure, intercontractile interval, voiding threshold, or peak voiding pressure between perimenopausal and control animals.  

In isolated whole bladder studies, there was no significant change in bladder weight or volume following a fill to 20 mmHg, while the stretch-induced relaxation response following filling was significantly enhanced in bladders from perimenopausal animals, with bladders relaxing faster and reaching a lower baseline pressure (Figure 1A). Bladder responses to KCl, ATP and the β-adrenoceptor agonist isoprenaline were similar between groups.  However, the maximum response to the muscarinic receptor agonist carbachol was significantly enhanced (Figure 1B), with increased potency (pEC50: 6.24±0.09 Control v 5.96±0.08 Perimenopause; p<0.05) following VCD pretreatment. Nerve-mediated responses were enhanced with perimenopause, although not significantly (Figure 1C). The cholinergic component of the response was significantly increased, whilst the purinergic component declined in perimenopausal animals (Figure 1D).

Using aged female mice, we successfully adapted the VCD model to induce perimenopausal-like cycle disruption in animals that more closely approximate the reproductive age at which women enter the menopausal transition. Of note, in aged animals, abnormal oestrus cycles were observed in some mice prior to commencing treatment.  While VCD treatment did not alter key cystometric parameters in vivo, ex vivo studies revealed clear physiological changes in bladder responses. Bladders from perimenopausal animals demonstrated enhanced stretch-induced relaxation response following filling, suggesting altered passive mechanical properties. Muscarinic receptor-mediated contractile responses were also significantly increased, indicating upregulated post-junctional signalling pathways, with acetylcholine playing a greater role in neurotransmission. These findings demonstrate that physiological alterations can emerge in the bladder during the perimenopausal stage, even in the absence of overt changes in voiding behaviour and may represent compensatory mechanisms to normalise micturition.

These findings support the hypothesis that lower urinary tract changes can arise during perimenopause, even in the absence of detectable differences in in vivo voiding parameters and before the onset of overt menopausal hormone deficiency. Whilst the adapted VCD aged model may be more age-relevant, caution is needed given the abnormal cycling of some mice prior to induction of perimenopause.

Lohff, J. C., Christian, P. J., Marion, S. L., & Hoyer, P. B. (2006). Effect of duration of dosing on onset of ovarian failure in a chemical-induced mouse model of perimenopause. Menopause, 13(3), 482-488.
Brooks, H. L., Pollow, D. P., & Hoyer, P. B. (2016). The VCD mouse model of menopause and perimenopause for the study of sex differences in cardiovascular disease and the metabolic syndrome. Physiology, 31(4), 250-257.