Diabetes mellitus (DM) causes urine storage dysfunction such as overactive bladder and urinary incontinence as well as voiding dysfunction associated with underactive bladder. The present study using genetically mutated type 2 DM mice was conducted to examine the mechanisms that cause the lower urinary tract dysfunction, especially manifestations during urine storage. Since DM induces cognitive impairment [1], we examined by using dual analysis of voluntary voiding behavior and reflex micturition [2] if the forebrain is involved in the lower urinary tract dysfunction caused by DM. Furthermore, since DM affects the autonomic nervous system such as the cardiovascular function via sympathetic nerves [3], we assessed the intravesical pressure-volume relationship during cystometry and examined the sensitivity of bladder smooth muscle strip to β-adrenergic stimulation for relaxing detrusor in the DM mice.

Female type 2 DM mice (+Leprdb /+Leprdb = db/db) (8–10-week-old, BKS.Cg-Dock7m +/+ Leprdb/J strain) and their age-matched wild-type (WT) mice (m+/+/m+/+ = m+/m+) were used. To evaluate voluntary voiding behavior, conscious mice were individually placed in metabolic cages for 3 consecutive days, and data of voided urine and water-intake were continuously recorded into a computer. Urine volume per voiding (μl) and voiding frequency (times/day) were evaluated. To examine reflex micturition during cystometry, mice were decerebrated under sevoflurane anesthesia. Intravesical pressure was recorded via a PE-50 tube inserted into the bladder dome. Cystometric recordings were performed in the animals at supine position under unanesthetized conditions by infusing saline (10 μl/min) into the bladder at room temperature. Pressure-volume relationship (μl/mmHg), voided volume (VV, μl), postvoid residual volume (RV, μl), and volume threshold for inducing a voiding contraction (VT, μl) were evaluated. Quantitative real-time PCR assays were performed on β3-adrenoceptor expressed in the mouse bladder muscle. To assess a relaxation of in vitro bladder muscle strip, body of bladder was excised and then bladder strips approximately 5 to 6 mm wide and 7 to 10 mm long were prepared without mucosa. The bladder strips were suspended in a 15-ml organ bath filled with Krebs solution composed of 119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 15 mM NaHCO3, 1.2 mM NaH2PO4, and 5 mM glucose at 37 °C and gassed with 95% O2/5% CO2. Changes in strip tension were measured with an isometric force transducer and recorded with Power-Lab acquisition system. Prior to testing each drug concentration, the strips were stimulated by 3 µM carbachol for evoking its contraction. When contraction became stable, a selective β3-adrenergic agonist, BRL 37344 (3 µM and 10 µM) was applied. Relaxing responses in strips were normalized to the weight of each strip. Maximal relaxation of the tension was used for data analysis, which were expressed as a percent inhibition of the carbachol-induced tension of the bladder strip. Values are expressed as mean ± S.E.M. Group comparisons were made using two-way repeated measures ANOVA or unpaired t-test, and p < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Evaluation of voluntary voiding behavior in metabolic cages revealed that mean urine volume per voiding and voiding frequency in DM and WT mice were 219.1 ± 15.9 μL and 145 ± 11.1 μL (**p=0.002) and 35.4 ± 3.6 times/day and 9.7 ± 0.7 times/day (***p<0.0001), respectively. Cystometric evaluation showed that voided volumes of DM mice and WT mice were 233.6 ± 28.7 μL and 84.4 ± 4.9 μL (***p=0.0002), respectively. In addition, postvoid residual volumes of DM mice and WT mice were 20.9 ± 3.4 μL and 16.0 ± 4.2 μL, respectively, showing no difference between the two groups (p=0.38). Pressure-volume relationships of DM mice and WT mice were as shown in Figure 1, indicating that DM mice utilized higher bladder compliance during urine storage than WT mice did. Quantitative real-time PCR assays of bladder smooth muscle revealed that mRNA expressions (per GAPDH) of DM and WT mice were 14.5 ± 1.2 and 22.5 ± 1.9 (**p=0.0034), respectively. The examination on relaxation of in vitro detrusor strip revealed that BRL 3 μM decreased tensions of bladder muscle strip of DM and WT mice in a same fashion (by 16.3 ± 1.4 % and 14.5 ±1.0 %, respectively; p=0.13), whereas BRL 10 μM decreased those of DM and WT mice by 38.4 ± 2.2 % and by 27.8 ± 2.1 %, respectively, showing a significant difference (*p=0.03).

Generally, in WT mice, expelled urine volume in voluntary voiding behavior is greater than voided volume in reflex micturition [2]. As shown in Figure 2, in WT (m+/m+) mice the urine volume per voiding in voluntary voiding behavior was much greater than the voided volume in reflex micturition, whereas in DM (db/db) mice there was no difference in voided volumes between voluntary voiding behavior and reflex micturition. The result suggests that the forebrain of conscious DM mice failed to exert its inhibitory control to the brainstem to hold urine storage. The in vitro experiments using bladder muscle strip revealed that DM detrusor is more sensitive to β3-adrenergic stimulation than WT detrusor, while quantitative PCR analysis showed that mRNA expression in DM mice was significantly fewer than that in WT mice.

The comparison between ‘voluntary voiding’ (in conscious behavior) and ‘reflex micturition’ (under decerebrate, unanesthetized conditions) suggests the possibility that the conscious DM mouse lacks the robust inhibitory modulation from the forebrain to the brainstem that is important in the control of micturition. The combination of the in vivo cystometry evaluation on the pressure-volume relationship and of the in vitro experiment examining detrusor relaxation shows that the bladder of DM mice is more sensitive to β3-adrenergic stimulation than that of WT mice, suggesting that DM enhances the sympathetic innervation and facilitates the detrusor relaxation during urine storage. This is the first study to demonstrate the alteration of conscious control in the forebrain affected by DM in voiding behavior, and the sympathetic modulation enhanced by DM in bladder muscle relaxation.

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Continence 7S1 (2023) 100859
DOI: 10.1016/j.cont.2023.100859