Nitric oxide (NO●)-cGMP signaling has been implicated in the pathology of benign prostatic hyperplasia (BPH) and associated lower urinary tract symptoms (LUTS), accordingly, drugs that potentiate NO● signaling demonstrate therapeutic effects for the condition. Cytochrome B5 reductase type-3 (CYB5R3) is a key regulator of soluble guanylate cyclase (sGC) through its redox cycling of the sGC heme moiety which is crucial for maintaining NO● induced cGMP generation. Dysregulation of CYB5R3 can occur in aging and under oxidative stress [1], suggesting it could underlie the decrease in cGMP signaling and may potentially contribute to development of BPH/LUTS. To examine this possibility, we utilized a conditional CYB5R3 knockout mouse to selectively decrease the enzyme in the prostatic lobes through 4-OHT injections. Changes in voiding behavior were monitored by telemetric bladder pressure recordings up to 24 weeks after injections. The aim of the study was to demonstrate the utility of the CYB5R3 KO mouse as a model for BPH/LUTS.

Generation of the CYB5R3 conditional knockout mouse.  A mouse with loxP sites flanking exon3 of the CYB5R3 gene [2] (CYB5R3flox/flox) was crossed with a mouse expressing the tamoxifen inducible Cre recombinase under the β-actin promoter (CAG-Cre, Jackson laboratories, stock#:004682) to generate the conditional CYB5R3flox/flox+CAG-Cre (CYB5R3 KO) mouse (Figure 1A).  Both mouse strains were based on a C57Bl/6 background.  At 8-12 weeks of age, male CYB5R3 KO were anesthetized with isoflurane and using sterile surgical conditions, all eight prostate lobes were injected with 1 mg/ml 4-OHT (20 µl total, dissolved in ethanol/CremophorEL/saline) using a 32-gauge insulin syringe.  At the same time, mice were implanted with pressure telemeters (HD-X10, Data Sciences International, Inc.) where the pressure lines were sutured into the bladder lumen through the dome.  The mice were treated with the analgesic, ketoprofen (3 mg/kg, IM) and antibiotic ampicillin (100 mg/kg) during the recovery period.

Metabolic cage assessments.  Voiding behaviour analysis was also performed using metabolic cages (Columbus Instruments, Inc.) where the mice were maintained in a climate-controlled cabinet with a 12-hour light/dark cycle (7 am - 7 pm). Cages were equipped with receiving units to record bladder pressures simultaneously with urine output measured by highly sensitive load cells. Based on previous experience, mice take a minimum of 28 days to recover voided volumes comparable to pre-telemeter implantation, thus, recordings were initiated after this time point. Recordings were obtained up to 24 weeks after surgery.

Histology. Mice were humanely sacrificed for tissue collection at 12 weeks after 4-OHT treatment.  Urinary bladders were fixed in 10% neutral buffered formalin and processed for paraffin embedding. Tissues were sectioned 3 to 4 µm thick and processed for immunofluorescence of Cre recombinase (Cell Signaling Technology, #15036) and images captured using a widefield microscope (Olympus BX63).

Data and statistical analysis. Data are expressed as mean ± standard error of mean. Pairwise comparisons were performed using Student’s t-test where the null hypothesis was rejected at p<0.05.

Expression of Cre recombinase in the prostate following 4-OHT injections was verified by immunofluorescence where labelling could be detected up to 7 days after injections in CAG-Cre expressing mice (Figure 1B). A representative telemetric bladder pressure and corresponding urine output trace from a load cell under the mouse cage are shown in Figure 2A and 2B, respectively. During voiding the bladder showed a gradual rise in pressure, a brief decrease in pressure which correlated with the start of urine flow that was then followed by rapid phasic contractions resulting in a decrease of overall pressure. This is characteristic for male rodents where voiding efficiency is facilitated by phasic EUS bursting. The CYB5R3 KO mouse did not show differences in voided volume or voiding intervals between 4 weeks and 24 weeks following 4-OHT treatment/telemeter implantation. There was a significant increase in micturition and baseline pressures at 7 weeks and 24 weeks versus 4 weeks (*p<0.05, 7 wks vs 4 wks; **p<0.01, 24 wks vs 4 wks).

CYB5R3 KO mice show a progressive development of high-pressure bladders, a feature of outlet obstruction that surprisingly did not correlate with decreases in voided volumes or changes in voiding frequency between 4 to 24 weeks. Previous studies have indicated CYB5R3 deficiency in the prostate can induce glandular hyperplasia and promote collagen deposition compared to age-matched wildtype mice [3], which may be the underlying cause of obstruction and increase outlet resistance.

These studies support the ability to perform long-term monitoring of bladder pressure and voiding activity in mice as well as support a role for CYB5R3 in BPH/LUTS pathology. Use of telemetric bladder pressure recordings could be an alternative to traditional cystometrograms with the advantage of reducing animal numbers required in preclinical studies.

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Durgin et al.  Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.  JCI Insight, 4(19):e129183, 2019.
Ikeda et al. Glandular Growth, Fibrosis and Lower Urinary Tract Symptoms Induced by Selective Deficiency of Cytochrome B5 Reductase in the Mouse Prostate, Continence, Vol 2(S2), 100200, 2022.

Continence 7S1 (2023) 100828
DOI: 10.1016/j.cont.2023.100828