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

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.

SUBMITTER: Serdinsek T

PROVIDER: S-EPMC7021722 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Serdinšek Tamara T Lipovšek Saša S Leitinger Gerd G But Igor I Stožer Andraž A Dolenšek Jurij J

Scientific reports 20200214 1

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 concentration ...[more]

PMID: 32060298

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Project description:Introduction: The tissue slice technique offers several benefits compared to isolated cells and cell clusters that help us understand the (patho)physiology of several organs in situ. The most prominent features are preserved architecture and function, with intact homotypic and heterotypic interactions between cells in slices. In the pancreas, this technique has been utilized successfully to study acinar and endocrine islet cells. However, it has never been used to investigate ductal function. Since pancreatic ductal epithelial cells (PDECs) play an essential role in the physiology of the pancreas, our aim was to use this technique to study PDEC structure and function in situ. Materials and methods: Eight- to sixteen weeks old C57BL/6 mice were used for preparation of pancreas tissue slices. Low melting point agarose was injected into the common bile duct and the whole organ was extracted. For morphological studies, pieces of tissue were embedded in agarose and cryosectioned to obtain 15 μm thick slices. In order to visualize pancreatic ducts, (i) the Giemsa dye was added to the agarose and visualized using light microscopy or (ii) immunostaining for the cystic fibrosis transmembrane conductance regulator (CFTR) was performed. For functional characterization, agarose-embedded tissue was immediately cut to 140 μm thick tissue slices that were loaded with the cell permeant form of the Oregon Green 488 BAPTA-1 dye and used for confocal calcium imaging. Results: Giemsa staining has shown that the injected agarose reaches the head and body of the pancreas to a greater extent than the tail, without disrupting the tissue architecture. Strong CFTR expression was detected at the apical membranes of PDECs and acinar cells, whereas islet cells were completely negative for CFTR. Stimulation with chenodeoxycholic acid (CDCA, 1 mM) resulted in a robust transient increase in intracellular calcium concentration that was readily visible in >40 ductal cells per slice. Conclusion: Our results confirm that the acutely-isolated pancreas tissue slice technique is suitable for structural and functional investigation of PDECs and their relationship with other cell types, such as acini and endocrine cells in situ. In combination with different genetic, pharmacological or dietary approaches it could become a method of choice in the foreseeable future.

Project description:Although patients with lower urinary tract symptoms constitute a large and still growing population, understanding of bladder detrusor muscle physiology remains limited. Understanding the interactions between the detrusor smooth muscle cells and other bladder cell types (e.g. interstitial cells, IC) that may significantly contribute to coordinating and modulating detrusor contractions represents a considerable challenge. Computer modeling could help to elucidate some properties that are difficult to address experimentally; therefore, we developed in silico models of detrusor smooth muscle cell and interstitial cells, coupled through gap junctions. The models include all of the major ion conductances and transporters described in smooth muscle cell and interstitial cells in the literature. The model of normal detrusor muscle (smooth muscle cell and interstitial cells coupled through gap junctions) completely reproduced the experimental results obtained with detrusor strips in the presence of several pharmacological interventions (ryanodine, caffeine, nimodipine), whereas the model of smooth muscle cell alone (without interstitial cells) failed to reproduce the experimental results. Next, a model of overactive bladder, a highly prevalent clinical condition in both men and women with increasing incidence at older ages, was produced by modifying several processes as reported previously: a reduction of Ca(2+)-release through ryanodine receptors and a reduction of Ca(2+)-dependent K(+)-conductance with augmented gap junctional coupling. This model was also able to reproduce the pharmacological modulation of overactive bladder. In conclusion, a model of bladder detrusor muscle was developed that reproduced experimental results obtained in both normal and overactive bladder preparations. The results indicate that the non-smooth muscle cells of the detrusor (interstitial cells) contribute significantly to the contractile behavior of bladder detrusor muscle and should not be neglected. The model suggests that reduced Ca(2+)-release through ryanodine receptors and Ca(2+)-dependent K(+)-conductance together with augmented gap junctional coupling might play a major role in overactive bladder pathogenesis.

Dataset Information

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

Publications

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

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