Characterization of mouse neuro-urological dynamics in a novel decerebrate arterially perfused mouse (DAPM) preparation

Abstract

Aim: To develop the decerebrate arterially perfused mouse (DAPM) preparation, a novel voiding model of the lower urinary tract (LUT) that enables in vitro-like access with in vivo-like neural connectivity.

Methods: Adult male mice were decerebrated and arterially perfused with a carbogenated, Ringer's solution to establish the DAPM. To allow distinction between central and peripheral actions of interventions, experiments were conducted in both the DAPM and in a "pithed" DAPM which has no brainstem or spinal cord control.

Results: Functional micturition cycles were observed in response to bladder filling. During each void, the bladder showed strong contractions and the external urethral sphincter (EUS) showed bursting activity. Both the frequency and amplitude of non-voiding contractions (NVCs) in DAPM and putative micromotions (pMM) in pithed DAPM increased with bladder filling. Vasopressin (>400 pM) caused dyssynergy of the LUT resulting in retention in DAPM as it increased tonic EUS activity and basal bladder pressure in a dose-dependent manner (basal pressure increase also noted in pithed DAPM). Both neuromuscular blockade (vecuronium) and autonomic ganglion blockade (hexamethonium), initially caused incomplete voiding, and both drugs eventually stopped voiding in DAPM. Intravesical acetic acid (0.2%) decreased the micturition interval. Recordings from the pelvic nerve in the pithed DAPM showed bladder distention-induced activity in the non-noxious range which was associated with pMM.

Conclusions: This study demonstrates the utility of the DAPM which allows a detailed characterization of LUT function in mice.

Keywords: external urethral sphincter; lower urinary tract; micturition; mouse; neural control.

Figure 1

Schematic of decerebrate arterially perfused mouse (DAPM) in situ preparation for bladder studies (A). The perfusion cannula is shown with a knuckle at the tip to secure its placement in the aortic arch (B). The cannula is inserted under direct vision through the left ventricle (C) and pushed through the aortic valve until the knuckle fits through the aortic valve tightly and the tip is visible in the proximal aorta. (D) Lower urinary tract showing a needle inserted into the bladder dome allowing infusion of fluid and monitoring of intravesical pressure. A suction electrode enabled simultaneous EUS‐EMG activity recording. (E) Pelvic nerve of the bladder held in a suction electrode for recording of afferent activity. (F) Consecutive micturition cycles recorded with EUS‐EMG activity. Expanded time scale shown below with EUS bursting activity during voiding. The following micturition parameters (Figure 1F) were measured (averaged over at least three voiding cycles):

1. Baseline was taken as the lowest bladder pressure reached immediately following a void.

2. Voiding threshold was the bladder pressure when the EUS‐EMG changes its activity to bursting, indicating the initiation of voiding.

3. Micturition pressure was the absolute value of the peak bladder pressure achieved during voiding (bursting phase of the EUS‐EMG).

4. Non‐voiding contractions (NVCs) were identified as discrete increases in bladder pressure (>1 mmHg) observed during the filling phase in voiding preparations.

5. In preparations without voiding (ie, pithed DAPM) the small rhythmical pressure fluctuations with amplitude of more than 0.4 mmHg were termed putative micromotions (pMM).

6. Bladder compliance was defined as bladder capacity/(threshold—basal pressure) (μL/ΔmmHg) during filling at a rate of 25 μL/min

Figure 2

Relationship between NVCs, pMM, pelvic nerve activity and the phase of the micturition cycle. Both NVC amplitude and frequency increased with bladder filling (A‐1). The NVC amplitude and frequency showed a clear relationship to the phase of the micturition cycle (A‐2 and A‐3), normalized to the largest event/interval observed in each DAPM preparation, assessed over four micturition cycles, n = 7 mice). Similar relationship was found for pMM amplitude and frequency and micturition phase (n = 7 mice, B‐1, B‐2, and B‐3). Recording of pelvic afferent neural activity in pithed model (C) showing relationship to bladder pressure (Cc) and to pMM (Cd). * P < 0.05, significant difference compared to baseline with repeated measures ANOVA with Dunnett's post hoc test for multiple comparisons

Figure 3

Effect of vasopressin on LUT function in the DAPM. (A) Representative recording of perfusion pressure, phrenic nerve activity, bladder pressure, and EUS‐EMG activity before and after administration of vasopressin (2 and 10 nM) showing the loss of bladder—sphincter co‐ordination manifesting as an increase in tonic activity on the EUS. (B) Direct effects of vasopressin (80 pM, 400 pM, 2 nM, and 10 nM) added to the perfusate on perfusion and bladder pressures in pithed and DAPM model (shown graphically below). *^{, #} P < 0.05, ⁻ P < 0.01, significant difference compared to baseline with repeated measures ANOVA and Dunnett's test for multiple comparisons (*—DAPM and ^#—pithed DAPM)

Figure 4

Urinary retention caused by administration of the neuromuscular blocker, vecuronium bromide (2 μg/mL) (A) and ganglion blocker, hexamethonium (330 μM) (B) to the perfusate. Effect of intravesical infusion of acetic acid (0.2%) on phrenic nerve activity, bladder pressure and EUS‐EMG activity (C‐A; Control, C‐B; After acetic acid)