Afferent nerve signaling in response to bladder filling is important for normal bladder function, and aberrations in signaling may be associated with voiding dysfunction. McCarthy et. al. demonstrated afferent nerve firing in isolated mouse bladders [1]. An isolated perfused porcine bladder model has been developed for biomechanics experiments [2,3]. The purpose of this study was to quantify afferent nerve signals during filling in the perfused porcine bladder model. To our knowledge nerve signaling in an ex-vivo pig bladder model has not been previously reported.

Porcine bladders were collected fresh from abattoirs in Central Virginia. Ex vivo bladders were stored and transported in physiologic buffer solution. Bladders were prepared for experimentation by intubation of vesicular arteries, ligation of bilateral ureters, and cannulation of the urethra with both a urodynamics catheter for filling and tube for emptying [2,3]. Nerves were isolated and microhook electrodes were placed to record the electroneurogram (ENG). Data were sampled at 20kHz using AD Instruments PL2604 Power Lab (Figure 1A). Vesicular arteries were perfused with an oxygenated physiologic buffer solution during experimentation. Bladders were filled with deionized water at a rate of 100ml/min from a volume of 0 to 1000 ml. Filling was performed in a 1-minute off/1-minute on pattern for a total of 20 minutes. Urodynamic tracings were simultaneously monitored for pressure changes during filling. Bladders were contracted with 50ml of 1M KCl instilled through vesicular arteries upon termination of the experiment. Contractions were observed visually in addition to pressure changes, confirming healthy tissue. Furthermore, ENG data were filtered with a bandpass filter of 200Hz and 2,000Hz. Firing rate was calculated as peaks per minute above threshold (average of first 1 minute off). Average pressure, ENG amplitude and firing rate were calculated for each 2-minute interval (one minute off and one minute on) for the volume range of 200 to 1000 ml and Pearson coefficients were calculated to quantify correlations. Nerves emanating from the posterior of the bladder were removed, and immunohistochemical confirmation of nerve histology was performed using anti-beta tubulin antibody (Abcam, ab15568).

The presence of nerve tissue was confirmed via immunohistochemistry (Figure 1B). Five bladders were tested. ENG activity increased during filling (FIGURE 1C). Average bladder pressure, ENG amplitude and ENG firing rate increased with bladder filling. The Pearson correlation coefficient between pressure and ENG amplitude was 0.92, and the correlation coefficient between pressures and ENG firing rate was 0.70 (Figure 1D).

These results show the direct relationship between intravesical pressure and ENG activity during filling in isolated porcine bladders.

This study demonstrated that an ex-vivo perfused porcine bladder can be used to quantify afferent nerve signaling in response to filling. This novel model could be used to study afferent nerve activity in response to biomechanical changes of the bladder wall due to filling. Future experiments may involve measuring afferent nerve activity in response to changes in bladder shape or induced bladder contractions.

Carly J. McCarthy, Irina V. Zabbarova, Pablo R. Brumovsky, James R. Roppolo, Gerald F. Gebhart, Anthony J. Kanai. Spontaneous Contractions Evoke Afferent Nerve Firing in Mouse Bladders With Detrusor Overactivity. The Journal of Urology, Volume 181, Issue 3, 2009, Pages 1459-1466, ISSN 0022-5347, https://doi.org/10.1016/j.juro.2008.10.139.
Balthazar, A., Cullingsworth, Z., Speich, J., & Klausner, A. P. (2018). A Non-Invasive External Compress-Release Protocol to Measure Dynamic Elasticity in an Isolated Working Pig Bladder. The Canadian Journal of Urology, 25, 5.
Swavely NR, Cullingsworth ZE, Nandanan N, Speich JE, Klausner AP. Phases of decompensation during acute ischemia demonstrated in an ex vivo porcine bladder model. Transl Androl Urol. 2020;9(5):2138-2145. doi:10.21037/tau-20-669