2.1. In vivo testing

Male Sprague Dawley rats were used for producing the experimental BOO model (Zvara et al., 2002). Briefly, under isoflurane anesthesia, the bladder and the proximal urethra were exposed via a lower abdominal midline incision. A 4-0 silk ligature was placed around the urethra and tied at the urethrovesical junction level proximal to the urethral fenestration with a metal rod (outside catheter diameter of 1.27 mm) placed alongside the urethra, and then the rod was removed. The abdominal wound was closed. This ligation was maintained in place throughout the duration of the experiments. Sham rats underwent similar procedures without urethral ligation.

Twenty four young (3-month old) male rats weighing 370 ± 8.9g, were used in the cystometry (pressure-flow) studies using previously established methodologies (Zvara et al., 2002). Measurements were performed 4 weeks after inducing BOO (n=12) as well as for rats with a sham surgery (n=12). Institutional Animal Care and Use Committee (IACUC) guidelines were observed. Briefly, under isoflurane anaesthesia, a PE-50 polyethylene catheter (Clay-Adams, Parsippany, NJ) was inserted through the bladder dome and a purse-string suture was placed tightly around the catheter. The implanted catheter was exteriorized through the abdominal wall, and the wound was closed with 4-0 silk sutures. The rats were placed in restraining cages (W 80 mm ×L 300 mm ×H 150 mm, Yamanaka Chemical Ind., Ltd, Osaka, Japan) and allowed to recover from isoflurane anaesthesia for 1–2 h before starting cystometry. After recovery, a three-way stopcock was used to connect the intravesical catheter to a pressure transducer (Transbridge 4M, World Precision Instruments, Sarasota, FL, USA) for recording intravesical bladder pressure and to a syringe pump (Harvard Apparatus,Holliston,MA) for infusing saline at a fixed flowrate. Because variability in bladder capacity among BOO rats is typical of this model even when the same obstruction technique was used, the saline injection speed was adjusted to maintain similar micturition intervals among animals. In particular, saline was initially infused at 0.1 ml/min; subsequently, the infusion rate was adjusted to 0.04–0.3 ml/min to obtain an intercontraction interval of approximately 10–15 min (Nishiguchi et al., 2007). Intravesical pressure changes were measured using data acquisition software (AD Instruments, Castle Hill, NSW, Australia) at a sampling rate of 100 Hz using a PowerLab. Saline infusion was continued until stable voiding cycles were established.

The recorded data was used to construct the pressure curves from which the following parameters were evaluated: (i) maximum voiding pressure Pvoidmax; (ii) maximum passive filling pressure Ppassivemax; (iii) maximum active voiding pressure Pactmax; see Table 1 for definitions and Fig. 2 for a illustrative pressure-curve for a healthy bladder. The voided urine was collected using a plastic cup placed underneath the restraining cage and measured to determine the voided volume (V_void). The voiding duration, t_void, was determined through direct observation of urine expelled from the urethral orifice. The post-void residual volume (V_res) was measured by draining the post-void bladder using the bladder catheter with gravity (Takaoka et al., 2018). Bladder capacity, or filled volume (V_F), was calculated as the sum of voided and residual volumes.

Schematic representation of the pressure flow curve during the cystometry studies. The bladder fills under low pressure. Following, the reflex contraction, the SMC develops actives stress and the bladder pressure increases. Initially, the bladder undergoes an isovolumetric contraction until the pressure exceeds the cut-off pressure P_c at which point flow is initiated; flow stops when the bladder pressure reduces below P_c. Immediately, following obstruction, the cut-off pressure increases and the bladder is unable to generate sufficient pressure to actively void and becomes leaky. Subsequent SMC hypertrophy increases the active pressure the bladder can generate and acts to restore voiding functionality.

Summary of definitions of cystometric parameters.