A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice

Tamara Serdinšek,Saša Lipovšek,Gerd Leitinger,Andraž Stožer,Igor But,Jurij Dolenšek

doi:10.1038/s41598-020-59337-0

Abstract

The aim of our study was to develop a novel approach to investigating mouse detrusor smooth muscle cell (SMC) physiological activity, utilizing an acute tissue dissection technique and confocal calcium imaging. The bladder of a sacrificed adult female NMRI mouse was dissected. We used light and transmission electron microscopy to assess morphology of SMCs within the tissue. Calcium imaging in individual SMCs was performed using confocal microscopy during stimulation with increasing concentrations of carbamylcholine (CCh). SMCs were identified according to their morphology and calcium activity. We determined several parameters describing the SMC responses: delays to response, recruitment, relative activity, and contraction of the tissue. CCh stimulation revealed three different SMC phenotypes: spontaneously active SMCs with and without CCh-enhanced activity and SMCs with CCh-induced activity only. SMCs were recruited into an active state in response to CCh-stimulation within a narrow range (1–25 µM); causing activation of virtually all SMCs. Maximum calcium activity of SMCs was at about 25 µM, which coincided with a visible tissue contraction. Finally, we observed shorter time lags before response onsets with higher CCh concentrations. In conclusion, our novel in situ approach proved to be a robust and reproducible method to study detrusor SMC morphology and physiology.

Highlights

Investigating detrusor muscle physiology and responses to different stimuli is a challenging task because of its complex physiology, occurrence of functional and morphological changes under pathophysiological conditions, and anatomic and physiologic differences in detrusors of different species[1,2]
To characterize morphology of smooth muscle cell (SMC) obtained with our approach, we resorted to light and transmission electron microscopy
We developed a novel approach to study detrusor muscle physiology utilizing a combination of acute detrusor tissue preparation and confocal calcium imaging

Summary

Introduction

Investigating detrusor muscle physiology and responses to different stimuli is a challenging task because of its complex physiology, occurrence of functional and morphological changes under pathophysiological conditions, and anatomic and physiologic differences in detrusors of different species[1,2]. Several different in vitro techniques for investigating urinary bladder physiology have been described, each with inherent drawbacks They can be broadly divided into (i) techniques utilizing freshly isolated smooth muscle cells (SMCs), (ii) cell culture techniques, and (iii) tissue dissection techniques. Tissue dissection techniques are especially useful in detrusor contractility investigations In these methods, the urinary bladder is dissected and the urothelial and the serous layer are usually removed[4,5], since responses to pharmacological stimuli can be altered in the presence of mucosa[6]. After an additional equilibration period of 20 minutes, strips were stimulated with CCh or electrical field[7] This particular method has been used several times in investigation of bladder physiology and determining the effect of different drugs on the detrusor contractility[7,10,11,12]

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