Novel spatiotemporal mapping allows new insight into the modality of myogenic micromotions in the ex-vivo tetrodotoxinised rabbit and pig bladder and their modulation with pharmacological agents.

King Q1, Hulls C2, Lentle R2, Chambers P2, Reynolds G2

Research Type

Basic Science / Translational

Abstract Category

Research Methods / Techniques

PRIZE AWARD: Best in Category Prize - Research Methods / Techniques
Abstract 625
Basic Science: Novel Bladder Insights
Scientific Podium Short Oral Session 31
Friday 31st August 2018
13:30 - 13:37
Hall D
Basic Science Animal Study Overactive Bladder Pharmacology Physiology
1. MidCentral Health, 2. Massey University

Quinten King



Hypothesis / aims of study
To investigate the effect of pharmacological manipulation on micromotions of the tetradotoxinised bladder wall of the rabbit and pig, using a sympathomimetic agent, a parasympathomimetic agent, and agents influencing intrinsic myogenic (ICC and myocyte) elements, using single and two dimensional spatiotemporal maps.
Study design, materials and methods
Investigations were carried out on whole-bladder specimens maintained ex-vivo; pig bladders by arterial perfusion and superperfusion with HEPES solution; rabbit bladders by superperfusion alone. Bladders were filled incrementally with saline. Seven rabbit bladders provided 21 control observations and 21 observations of each experiment (3 replicates each per bladder). One pig bladder provided 3 control observations and one pharmacological observation (a retrospective analysis of previously unreported work).  Neural influences were blocked by tetrodotoxin. Intravesical pressure (pVes) was monitored. Uncompressed high definition video was recorded as AVI files. Spatiotemporal mapping was subsequently conducted in a custom designed suite that allowed changes in surface area (between distinctive visual features) to be mapped on a frame-by-frame basis for all points on part of the region of interest (ROI) on the anterior bladder surface. Indices of contractile activity were calculated using FRAGSTATS v4 (a spatial pattern analysis program). Pharmacological agents were added to the superfusion solution in the rabbit preparation and injected into the arterial perfusate perfusing one side of the bladder in the pig preparation. In the rabbit bath concentrations of 0.1µM carbachol, 1µM isoprenaline, 0.05mM carbenoxolone, and 10µM RhoA Y-27632 were used. In the pig, 5mg of the RhoA inhibitor HA-1077 was used. Contaminated superperfusate was discarded after each replicate.
In the rabbit bladder carbachol increased the size, frequency, and speed of propagation of PPCs, causing the percentage mean total area of bladder wall undergoing contraction (PMTA), and pVes to increase. The addition of isoprenaline temporarily halted the incorporation of individual myogenic contractions (PICs) into propagating patch contractions (PPCs), reduced patch size, PMTA and pVes. The gap junction blocker carbenoxolone reduced the duration of PPCs but increased their frequency and velocity of propagation. In both rabbit and pig RhoA inhibitor caused reduction of PMTA, mean patch size, largest patch index (LPI), and pVes. In Tables 1 and 2, NP is the number of patches of contraction occupying the ROI, and LPI (Largest Patch Index) is the mean of the sizes of the largest patches of contraction in each frame as a percentage of the total area of the ROI.
Interpretation of results
The cholinomimetic agent carbachol and the β-adrenoceptor agonist isoprenaline (sympathomimetic) acted on the temporal organization of individual contractions (PICs) into PPCs indicating the influence of parasympathetic and sympathetic neural stimulirespectively. Carbachol increased the incorporation of PICs into PPCs, and increased pVes, while isoprenaline did the opposite.
Comparison of the effects of the gap junction blocker carbenoxolone and the RhoA inhibitor Y-27632/HA-1077 on the timing and disposition of contraction indicated that the local spatial spread of contractions in PPCs was governed largely by myocytes, whilst the propagation, frequency and duration of PPCs was likely regulated via gap junctions between ICC-IM and myocytes. The fact that the same changes from the RhoA inhibitor myogenic effect were observed in both pig and rabbit validates this effect
Concluding message
Micromotions of the bladder wall have been observed for decades. Recent work suggests that they have a role in the regulation of bladder wall tone and may have clinical relevance, for example in overactive bladder. 
Spatiotemporal mapping (STM) has enabled us to determine that micromotions are cyclic propagating patches of contraction (PPCs) that traverse the bladder in a predominantly vertical direction and that these in turn consist of coordinated groups of relatively brief, propagating individual myogenic contractions (PICs) that grow and propagate in a more random fashion within the PPC. Further, we have observed that the formation of PPCs may cease and be replaced by randomly distributed PICs. 
Consistent effects of pharmacological manipulation, within and between species, indicates that STM offers an objective means of understanding the maintenance of bladder tone and the effects of pharmacological agents on the normal and abnormal bladder. 
Further work is needed to determine whether the patterns of development of PICs, and their incorporation into PPCs, differ in those of normal subjects from those of patients with abnormal bladder function.
Figure 1
Figure 2
  1. Lentle RG, Reynolds GW, Janssen PWM, Hulls CM, King QM, and Chambers J. Characterisation of the contractile dynamics of the resting ex vivo urinary bladder of the pig. BJU Int 116: 973-983, 2015.
  2. Hulls CM, Lentle RG, King QM, Reynolds GW, and Chambers J. Spatiotemporal analysis of spontaneous myogenic contractions in the urinary bladder of the rabbit: timing and patterns reflect reported electrophysiology. American Journal of Physiology-Renal Physiology 313: F687-F698, 2017.
  3. Hashitani H, Brading AF, and Suzuki H. Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder. Br J Pharmacol 141: 183-193, 2004.
Funding Massey University Clinical Trial No Subjects Animal Species Rabbit, Pig Ethics Committee Massey University Animal Ethics Committee (MUAEC approval number 14/50)