Several clinical studies have shown that phosphodiesterase type 5 inhibitors (PDE5-I) improve male lower urinary tract symptoms (LUTS), including both storage and voiding components, without the impairment of sexual function seen with other BPH/LUTS treatments. However, none of these studies showed an improvement in peak urinary flow rate, raising the question of how they act to improve LUTS. Recently, we have reported an in situ voiding mouse model, the decerebrate arterially-perfused mouse (DAPM), allowing assessment of lower urinary tract (LUT) function of mice without the confounding effects of anaesthesia [1]. This has many of the advantages of accessibility afforded by in vitro approaches but retains functional neural connectivity. Using this approach, we showed that low doses of the PDE5-I, sildenafil, at subnanomolar concentrations, increased bladder compliance by directly relaxing the bladder [2]. Interestingly, sildenafil strikingly increased the number of bursts of external urethral sphincter (EUS) activity per void (to 130.1 ± 6.9 % of control at 30pM). This follow-on study aimed to investigate the influence of sildenafil on mouse bladder afferent activity in situ, lumbosacral spinal cord in vivo and electrical field stimulation (EFS)-induced contractions of EUS in vitro to determine the mechanisms underlying the increased bursting activity of EUS.

Adult CD1 male mice were decerebrated and arterially perfused with a carbogenated Ringer’s solution to establish the DAPM. To allow distinction between central neural and peripheral actions of sildenafil, recordings of the pelvic nerve were conducted in a ‘pithed’ DAPM which has no central nervous system or spinal cord. The nerve was identified, cut and recordings were made from the distal end with a suction electrode. A polyethylene catheter (PE-50, Clay-Adams, Parsippany, NJ, USA) was sutured into the dome of bladder. Bladder distention-induced pelvic neural activity which increased exponentially over the range of pressures normally associated with the micturition cycle (<15mmHg). Nerve activity was AC amplified (5-10k), band pass filtered (100 Hz to 3 kHz) and digitised at 10Khz. Multifibre afferent nerve activity was quantified by thresholding to count the number of action potentials (Spike2, CED, Cambridge, UK). After clamping the urethra, saline was infused (25ul/min) until the intra-vesical pressure reached 15mmHg (filling phase) and then the infusion was stopped for 3 minutes (isovolumetric phase) (Figure 1A). At the end of each cycle the bladder was emptied by aspiration. 
To investigate the action of sildenafil on the lumbosacral spinal cord, sildenafil was intrathecally injected to urethane anaesthetized mice (1.0-1.2 mg/g i.p). To allow intrathecal access, the vertebral spines (L4 – L6) were exposed through a midline skin incision. To allow EUS-electromyography (EMG), stainless steel insulated wires (0.075mm, diameter) were inserted into the sphincter muscle percutaneously. The bladder was cannulated with PE-50 catheter to monitor the intra-vesical pressure. The bladder was filled (at 25ul/min) and once a stable pattern of micturition was established, baseline cystometric parameters were measured (more than 4 micturition cycles). Sildenafil was administered intrathecally (final concentration of 30 and 300pM, each in 5ul – assuming an intrathecal volume of 20uL) and CMG parameters and EUS-EMG activity were monitored. 
Mice were euthanised by cervical dislocation and the whole EUS was removed through a midline laparotomy. The whole EUS (with internal urethral sphincter and urethra intact, approximately 10 mm length, 3 mm width) were tied to an isometric force transducer in a perfusion trough, and superfused longitudinally with Tyrode’s solution at 37°C. Nerve-mediated tetanic contractions were generated by electrical field stimulation (EFS) (0.1 ms pulses, 40 Hz, 3-s train). The amplitude of peak force of contractions and area under the curve (AUC) were normalised for tissue preparation weight (mN / g). After sildenafil (10 and 30pM) and sodium nitroprusside (SNP 100uM) application, EFS were repeated.

Sildenafil (10 and 30 pM) produced an increase in pelvic afferent activity during the filling (from 16.1 ± 5.1 Hz at baseline to 23.0 ± 5.2 Hz at 30pM at 15mmHg, P=0.020, n=9) and during the isovolumetric phase (to 205.4 ± 30.2 % of control at 30pM, P=0.034, n=9) (Figure 1B). The vehicle control showed no change in pelvic afferent activity (n=9). Intrathecal application of sildenafil (5ul, 30 and 300 pM) did not alter cystometry and EUS-EMG parameters in urethane anaesthetised mice (n=5). EFS (40Hz) induced tetanic contractions of EUS. Sildenafil (10 and 30 pM) did not alter maximum amplitude (124.5 ± 28.2 mN / g at baseline) and AUC (424.4 ± 110.1 mN.sec / g at baseline)) of the tetanic contractions of whole EUS induced by EFS (n=6). In contrast, a control experiment with the NO donor SNP (100uM) reduced the tetanic contractions of whole EUS at maximum amplitude (to 66.1 ± 6.3 % of baseline, P=0.014) and AUC (to 53.9 ± 9.1 % of baseline, P=0.014).

We propose that the increased afferent activity produced by sildenafil in the normal range of micturition pressures (& bladder distension) might facilitate better co-ordination of bladder and sphincter function. It is known that external urethral sphincter motor neurons can be activated via segmental afferents, and some studies in rats and cats showed that electrical stimulation of pelvic afferents elicited reflex firing in pudendal nerve efferents or the external urethral sphincter [3]. Although these studies used rats and cats not mice, the augmented bladder afferent activity we have observed might increase the EUS-EMG bursting in DAPM after sildenafil.

Sildenafil acts at picomolar concentrations to increase pelvic nerve afferent activity and then augment the bursting activity of the external urethral sphincter. We propose that these novel actions may underlie some of its beneficial activity in LUT dysfunction.

Ito H, Drake MJ, Fry CH, Kanai AJ, Pickering AE. Characterization of mouse neuro-urological dynamics in a novel decerebrate arterially perfused mouse (DAPM) preparation. Neurourol Urodyn. 2018 Jan 15. [Epub ahead of print]
Ito H, Drake MJ, Kanai AJ, Fry CH, Pickering AE. Effects of Sildenafil on bladder and urethral function in a novel voiding mouse model: The decerebrate arterially-perfused mouse (DAPM) preparation. Moderated poster, 47th Annual meeting of International Continence Society (ICS) 2017 in Florence
Thor KB, de Groat WC. Neural control of the female urethral and anal rhabdosphincters and pelvic floor muscles. Am J Physiol Regul Integr Comp Physiol. 2010;299:R416-38.