A total of 44 eight-week-old male C57BL/6 mice (weight, 20–25 g; Orient Bio, Inc., Seongnam, Korea) were used in this study. Animal experiments were performed after obtaining approval from the Institutional Animal Care and Use Subcommittee of our university (approval number: 190813-661). Animal were monitored daily for health and behavior, and animals were fed sterilized commercial standard laboratory food and water. Mice were maintained at room temperature (23°C±2°C), 40%–60% relative humidity, under a 12-hour light/dark cycle and specific pathogen-free conditions. Animals were euthanized by 100% CO2 gas exchange in a closed container at a CO2 exchange rate of 10%–30% of container volume/min. Cardiac and respiratory cessation were confirmed before harvesting tissues [15]. Diabetes was induced in 8-week-old C57BL/6J mice by injecting STZ intraperitoneally (i.p.) (STZ, 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) in saline for 5 consecutive days. Compared with a single high-dose STZ (200 mg/kg) induction, this low-dose STZ-induced minimal toxicity and significantly reduced mortality in the induced diabetic mice (DM), as described previously [16,17]. Twelve weeks after completing STZ injections (after induction), mice with a tail vein blood glucose level >300 mg/dL and exhibiting a significant body weight reduction were considered to have DM. Fasting and postprandial blood glucose levels were determined using an Accu-Check blood glucose meter (Roche Diagnostics, Mannheim, Germany) [18].

Twelve weeks after induction, mice were anesthetized with ketamine (100 mg/kg, Yuhan Corp., Seoul, Korea) and xylazine (5 mg/kg, Bayer Korea, Seoul, Korea) i.p. and placed supine on a thermoregulated surgical table. Bladders were exposed by a lower abdominal midline incision, and then domes were punctured to insert a PolyE 120 Tubing, (ID 0.015 inches [0.38 mm], OD 0.042 inches [1.09 mm]; Smiths Medical International Ltd., Minneapolis, MN, USA). Catheters were secured with sutures, tunneled subcutaneously (to leave an exposed end through an incision in the back of the neck), and heat-sealed [19].

Conscious cystometry was performed in metabolic cages 5 days after catheter implantation [19]. The exposed end of the catheter was opened and connected to a pressure transducer (Biopac Systems, Goleta, CA, USA), and saline (154 mmol/L NaCl) was infused into the bladder using a pump 0.6 mL/hr for one hour [19]. Maximum pressure (highest pressure during voiding), threshold pressure (pressure before voiding contraction), basal pressure (lowest pressure between voids), voiding pressure (average calculated pressure between voids), and systolic interval (time between voids) were recorded.

Whole urinary bladders were harvested from age-matched treatment-naïve mice 5 days after bladder function evaluation, as described previously [10]. Tissues were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS) for 24 hours at 4°C. After washing 3 times with PBS, samples were embedded in optimal cutting temperature compound, placed in a cryostat at -20°C for 20 minutes, and thick sectioned (12 µm) for full-thickness bladder staining.

After washing several times with PBS, samples were immersed in 3% bovine serum albumin for 1 hour at room temperature to minimize nonspecific antibody binding, and incubated with antibodies to PECAM-1 (an endothelial cell marker; Millipore, Temecula, CA, USA; 1:50; Cat# MAB1398Z), NG2 chondroitin sulfate proteoglycan (a pericyte marker; Millipore; 1:50; Cat# ab5320), collagen I (Abcam, Cambridge, UK; 1:50; Cat# ab34710), E-cadherin (Abcam; 1:50; Cat# ab11512), tyrosine hydroxylase (TH, Abcam; 1:50; Cat# ab112), choline acetyltransferase (ChAT, Abcam; 1:50; Cat# ab18736), calcitoningene related peptide (CGRP, Abcam; 1:50; Cat# ab36001), and βIII-tubulin (a neuronal marker; Abcam; 1:100; Cat# ab107216) for 48 hours at 4°C. After washing several times with PBS, the samples were incubated with Donkey anti-Rabbit IgG H&L (DyLight 550) (Abcam; 1:50; Cat# ab98489), FITC Affinipure goat anti-Armenian hamster IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.; 1:50; Cat# 127-095-160), goat antirabbit IgG H&L (Alexa Fluor 488) (Abcam; 1:50; Cat# ab150081), rhodamine (TRITC) AffiniPure donkey anti-rat IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.; 1:50; Cat# 712-025-150), donkey anti-Sheep IgG (H+L) FITC (Thermofisher; 1:50; Cat# A16042), rhodamine (TRITC) AffiniPure donkey anti-Goat IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.; 1:50; Cat# 705-025-147), and Alexa Fluor 488-donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch Laboratories, Inc.; 1:50; Cat# 703-545-155). After washing several times with 1×PBST, the samples were mounted in media containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc.; Burlingame, California, US; Cat# H-1500) to stain nuclei. Sections were visualized, and images were obtained using a confocal microscope (K1-Fluo, Nanoscope Systems, Inc., Daejeon, Korea), and quantitative analysis was performed using an image analyzer system (Image J 1.34, National Institutes of Health, Bethesda, MD, USA; http://rsbweb.nih.gov/ij/).

Results are expressed as the means ±standard error of the means of at least 4 independent experiments. The unpaired t-test was used to compare groups. The analysis was conducted using GraphPad Prism version 8 (Graph Pad Software, Inc., San Diego, CA, USA), and statistical significance was accepted for P-values <0.05.

Twelve weeks after completing STZ injections (after induction), fasting and postprandial blood glucose concentrations in mice were significantly higher in DM than in controls. In addition, mean body weight was significantly lower in DM (Table 1).

Bladder function was assessed in age-matched controls and in DM 5 days after catheter implantation (performed 12 weeks after induction) by conscious cystometry to obtain urodynamic parameters, as previously described [19]. Bladder enlargement appeared to be part of DBD [20], however, the underlying mechanism of diabetes-related bladder enlargement is largely unknown. In the present study we also demonstrated that compared with age-matched controls, the bladders of DM were significantly enlarged (Fig. 1A). The urination pattern of normal mice is regular and stable. However, urination frequency was significantly reduced in DM (Fig. 1B), and thus, the intercontractile interval (time between voids) was significantly greater in DM. On the other hand, basal pressure (lowest average pressure between voids), maximal bladder pressure (peak pressure during micturition), and threshold pressure (pressure immediately before micturition) were not significantly changed (Fig. 1C–F). These results confirmed that bladder dysfunction was significantly induced in DM.

Bladder mucosa, especially the LP, possesses a rich vascular network, which plays a fundamental role in the exchange of molecules across the bladder wall [21]. Therefore, to determine whether the microvascular characteristics of the LP were altered in DM, we performed immunofluorescence staining for PECAM-1 (an endothelial cell marker) and NG2 (a pericyte marker) in the bladders of DM and age-matched controls. We found that bladder LPs were rich in blood vessels, which concurs with previous studies [21]. In addition, endothelial cell and pericyte contents in the LPs of DM were significantly lower than in age-matched controls (Fig. 2). These results suggested that the effects of STZ on bladder microvasculature are similar to the deficits observed in diabetic retinopathy, nephropathy, and peripheral neuropathy.

Urothelium rests upon LP, and extracellular matrix (ECM) provides nutritive and informative support for cells, and collagen is the main structural protein of bladder ECM [22,23]. Immunofluorescence staining for collagen I (suburothelial layer) and E-cadherin (a urothelial cell-adhesion marker) were performed in DM and control bladders to determine whether the urothelial layer in LP is altered under diabetic conditions. We found that collagen I expression was significantly diminished in the suburothelial layer of diabetic bladders (Fig. 3A, B). Although no significant change in urothelial E-cadherin content was observed between normal and diabetic urinary bladder, we observed the area (as indicated by the arrow and elliptical yellow dotted line) of disrupted E-cadherin-positive urothelial linings in DM (Fig. 3A, C), which contributes to the decreased barrier function.

Under normal conditions, the bladder stores and expels urine in a coordinated, controlled manner as regulated by the central and peripheral nervous systems [24]. Immunofluorescence staining for TH (a classical marker of presynaptic sympathetic innervation) and ChAT (a biochemical marker of cholinergic neurons) were used to evaluate whether efferent nerves (sympathetic and parasympathetic nerves) in the detrusor were altered under diabetic conditions. TH and ChAT expressions were significantly lower in diabetic bladder detrusor muscles (Fig. 4). In addition, immunofluorescence staining for CGRP (sensory afferent nerve) and βIII-tubulin were performed to investigate the effect of STZ-induced diabetes on afferent nerves. We found that CGRP and βIII-tubulin expressions were significantly reduced in the detrusor muscles of diabetic mouse bladders as compared with age-matched controls (Fig. 5).