Hypothesis / aims of study
Physiology of the detrusor muscle is very complex, as smooth muscle cells (SMC) of detrusor are part of several receptor and signalling pathways. Moreover, detrusor muscle undergoes a series of functional and morphologic changes under influence of different pathophysiological conditions such as bladder outlet obstruction. Considering anatomic and physiologic changes in detrusor muscle of different species, it becomes apparent that investigating detrusor physiology and responses to different stimuli is a challenging task. The aim of our study was to develop a novel methodological approach to study detrusor physiology in mice and investigate the possibilities of its application.
Study design, materials and methods
To prepare tissue slices, adult female NMRI mice were sacrificed utilizing CO2 and cervical dislocation. Abdomen was accessed via lower median laparotomy and bladder was carefully dissected. Dissected bladder was immediately transferred into ice-cold calcium-free Krebs solution (gassed with carbogen). Subsequently, connective tissue and urothelium were gently removed from detrusor muscle under a light stereomicroscope. Isolated detrusor muscle was cut into slices using scissors and transferred into fresh ice-cold calcium-free Krebs solution, where it was incubated for 15-20 minutes. Next, tissue slices were loaded with a membrane–permeable calcium reporter dye fluo-4-AM at 5 µM final concentration in HEPES-buffered solution (HBS) for 35-40 minutes at room temperature on a shaker at 50 strokes per minute. Tissue slices were then transferred into ice-cold HBS and kept on ice until calcium imaging.
Calcium imaging was performed immediately after tissue slice preparation using the inverted confocal microscope Leica TCS SP5 II. Calcium dye was excited using argon laser at 488 nm and emitted fluorescence was detected with HyD detector. For calcium imaging, individual slices were transferred into the recording chamber and perifused with HBS at 37 oC. To investigate responses of the detrusor muscle to pharmacological stimuli, tissue slices were stimulated with increasing concentrations of the cholinomimetic drug carbachol (CCh) in HBS according to the following stimulation protocol: HBS only, 1 µM, 10 µM, 25 µM, 50 µM, and 100 µM CCh, and then again HBS only. Each step of the protocol lasted for 300 seconds. Obtained time series were analysed using custom software, Matlab and MS Excel.
Interpretation of results
Our acute mouse detrusor muscle tissue slice technique is a relatively simple, quick and inexpensive preparation method. Using one adult size female murine bladder, approximately three to four tissue slices and stimulation protocols can be utilized. If kept on ice in cooled HBS, they can be used up to four hours after preparation. We showed that using this technique, SMCs are viable and respond physiologically with oscillatory behaviour of intracellular calcium concentration to stimulation with the cholinomimetic CCh. According to our results, SMCs in detrusor tissue slices exert three types of responses: spontaneous activity with no response to CCh stimulation, induced activity after CCh stimulation, and spontaneous activity prior to and induced activity after CCh stimulation. Percentage of activated SMC increases with increasing concentration of CCh within a relatively narrow concentration range for activation of SMCs. 25 µM CCh resulted in saturation of SMC recruitment and also turned out to be the cut-off concentration at which contraction of the tissue was visible. In our opinion, there are many implications for use of this technique in murine detrusor muscle physiology research, as different pharmacological agents can be tested in situ under well-controlled conditions. Moreover, the method can be used to investigate the effect of different overactive bladder treatment modalities on SMC activity, which could help us towards a better understanding and a more effective treatment of this disorder.