Characterization of a method to study urodynamics and bladder nociception in male and female mice

Abstract

Objectives: Abdominal electromyogram or visceromotor response (VMR) elicited by bladder distension is a validated as a measure of bladder nociception in mice, however it is not without its limitations. The aim of this study is to address some of these limitations and validate voiding evoked VMR as a measure of bladder nociception mice.

Methods: Using both male and female C57BL/6 mice we assessed the VMR response to cytometry- induced voiding before and after instillation of 0.5% acetic acid into the bladder. We then delivered intravesical lidocaine to confirm the VMR response as nociceptive. VMR and correlative cystometric bladder pressures were analyzed.

Results: We found that the VMR can be evoked by continuous fluid infusion into the bladder of both male and female mice. This response is potentiated after bladder injury and can be attenuated by administration of a local anesthetic, providing strong evidence that this method can be used to evaluate bladder nociception. Further, evaluation of cystometric pressure traces obtained during VMR recording revealed that intercontraction intervals were not altered after bladder injury in either male or female mice. However, we did observe a decrease in peak threshold pressures after bladder injury in female mice, which could be rescued by lidocaine administration.

Conclusions: In conclusion, this technique can measure the VMR and bladder nociception associated with voiding in both female and male mice. Although confounds still exist with the use of anesthesia, further exploration of non-anesthetized voiding-evoked VMR is warranted.

Keywords: bladder pain; mouse cystometry; visceral motor response (VMR).

Figure 1.. Mouse voiding-evoked VMR recording experimental system.

Illustration and block diagram of the experimental apparatus (A) and characteristic responses post-bladder inflammation (Bladder pressure – top, VMR – bottom; B) for voiding evoked VMR. Peak pressure (3) and baseline pressure (4) were determined by maximum and minimum pressures during a voiding cycle. The threshold pressure was visually determined as the point that bladder pressure rapidly increases to initiate a void (2). The cursor (1) used to determine the pre-distension response was placed so that the time difference between cursor 1 and 2 was equal to the time difference between cursor 2 and 4 (threshold pressure and subsequent baseline pressure). The intercontraction interval was defined as the time difference between the maximum pressure of a void and maximum pressure (cursor 3) of the subsequent void. DAQ – Data acquisition hardware.

Figure 2.. Mouse voiding-evoked VMR.

Example traces of voiding-evoked VMR in naïve mice (A) and after instillation of acetic acid (0.05%) in to the bladder (B). The middle trace represents the raw EMG response and the top is the rectified and smoothed (0.1 sec bins) EMG to more easily visualize the area under the curve. The bottom trace is the corresponding bladder pressure trace. Acetic acid treatment increases the VMR compared to naïve response. EMG- Electromyogram

Figure 3.. Traces of voiding-evoked VMR in female mouse.

(A-C) The VMR induced by bladder voiding is increased after 0.05% acetic acid treatment (B, top). This increased VMR is attenuated after lidocaine (0.1%) instillation (C, top). Non-voiding bladder contractions are increased after .05% acetic acid treatment (B, bottom). These traces are representative of the responses seen in both male and female animals. Voids are indicated by the black triangle. All other pressure peaks are considered non-voiding contractions. EMG- Electromyogram

Figure 4.. Intravesicular lidocaine attenuated the acetic acid-induced increase in voiding evoked VMR.

(A) Intravesicular acetic acid significantly increased the VMR in both female (P = 0.033) and male (P = 0.0004) mice compared to baseline. Intravesicular lidocaine significantly attenuated the acetic acid-induced increase in the VMR. (B) The ICI was not significantly altered comparing baseline to acetic acid or acetic acid to lidocaine treatment. After lidocaine treatment the ICI was significantly less than compared to baseline in female mice (P=0.04) (C) Threshold pressure (TP) and peak pressure (PP) were significantly decreased with intravesicular acetic acid in female mice but not male mice. Lidocaine restored TP and PP to near baseline pressures in female mice but had no effect in male mice. For all graphs data are represented as mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons (A and B); two-way ANOVA with Sidak’s multiple comparisons (C); * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001; n = 6–7. VMR – Viseromotor response