Cystometry in conscious rats

Conscious cystometry was recorded following the measurement of body weight in all rats by 8 weeks post spinalization. First, the bladder dome was exposed via a midline abdominal incision and the ureters were ligated at the level of the aortic bifurcation under 2% isoflurane anesthesia, then, cystometric parameters was recorded according to previous report after two stable micturition cycles at an infusing speed of 0.08 mL/min with 0.9% saline after the rats recovered from anesthesia for 1 h. Cystometric parameters included bladder volume, non-voiding contraction (NVC) frequency and NVC amplitude (9). Bladder volume was defined as the infusion volume when the fluid releases from urethra, and NVCs were defined as rhythmic intravesical pressure increases over 7 cmH₂O from baseline pressure without release of fluid from the urethra (9). After cystometry, the rats were sacrificed and the bladders were weighed after removal and divided into 4 pieces via the sagittal and coronal planes. Three pieces of the detrusor were separated and used for Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis, while one piece was used for paraffin embedding.