Diabetic bladder dysfunction (DBD) is the most common diabetic complication, affecting >50% of patients, and it typically manifests as bladder overactivity or underactivity. Overactivity has long been postulated to be driven by increased C-fibers in the bladder and this increase has been found in both the male and female Akita diabetic mice (Type 1), although the mechanisms underlying this change is not known. To better understand this change we have developed a novel assay to visualize and quantitate the 3D architecture of C-fibers in the bladder wall. In addition, to demonstrate for the first time that increases in C-fibers can actually carry excess evoked potentials, we have activated these fibers by instilling acetic acid in the bladder and assessed pERK expression, a well-established proxy of nociception, in the dorsal root ganglia (DRG).

- Male and female wild type and Akita mice at 15 weeks were used. For 3D analysis of C-fibers, bladders were pinned, fixed, cut into sagittal strips (≈3mm) and cleared for 3 days in CUBIC Reagent 1 (25% urea, 25% N,N,N’,N’-tetrakis (2-hydroxypropyl) ethylenediamine, 15% Triton X-100). Strips were then stained for calcitonin gene-related peptide (a marker of C-fibers) using lengthy incubation times, ending with an overnight incubation in CUBIC Reagent 2 (50% sucrose, 25% urea, 10% triethanolamine, 0.1% Triton X-100). Strips were mounted in CUBIC Reagent 2 (8.3 µM chamber) and z-stacks (10 µm z-stacks, 1 µm slices) obtained by confocal microscopy (lower 1/3 of bladder) beginning just beneath the urothelial layer. Images were imported into Imaris 3D software (ver. 10) and filaments traced using the filaments tool.  For analysis of evoked potentials, mice were anesthetized, the urethra ligated and a suprapubic tube implanted. 100 ul of 0.5% acetic acid was instilled through the catheter for 30 min. DRGs (L6-S1) were fixed, paraffin-embedded and stained for pERK, a well-established proxy of nociception. Entire cross sections were evaluated in ImageJ. Density of positively staining cell bodies in areas of the section containing cell bodies was then calculated.

There was a significant increase in combined length of C-fibers in diabetic bladders compared to nondiabetics in both male and female mice. This increase was associated with an increase in branching (sprouting) but not in average filament length nor number of individual filaments. Following instillation of acetic acid, DRGs from diabetic mice demonstrated an increase in pERK+ cell bodies compared to nondiabetics.

Our novel 3D assay has shown the increased C-fibers in the diabetic bladder is due to sprouting, not proliferation as had been previously supposed. This result will direct studies of the mechanism of diabetic bladder overactivity, and perhaps other types of overactivity, towards well-known pathways that drive neuronal sprouting, a result that may quickly advance our understanding of this complication and identify critical targets for intervention. Our demonstration of increased evoked potentials emanating from the bladder in response to acetic acid verifies that the C-fiber networks in the diabetic bladder are more sensitive than nondiabetics, a result due, at least in part, to the changes in neuronal morphology we have demonstrated.

Diabetic bladder overactivity is likely due to sprouting of C-fibers which subsequently carry an increase in invoked potentials.