An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium

Abstract

Key points Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure–volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)‐denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium‐derived mediators at the luminal and anti‐luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine‐scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure–volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate‐ and lipopolysaccharide‐treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM‐denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium–DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

Lamina propria: The connective tissue of rat urinary bladder mucosa

“…So, the lamina propria is seen here as an ovoid balloon of fibrous woven material that is acted upon mechanically by smooth musculature laid on its outer surface. This aspect of the bladder is in agreement with a recent study on the mouse bladder showing that volume‐pressure correlation was identical in the intact bladder and in muscle‐denuded bladders …”

“…This aspect of the bladder is in agreement with a recent study on the mouse bladder showing that volumepressure correlation was identical in the intact bladder and in muscle-denuded bladders. 12…”

The detrusor‐free bladder – it can still hold its water

“…A paper in this issue of The Journal of Physiology by Durnin et al . () has developed an enhanced version of pressure–volume recordings in ex vivo murine bladder, enabling investigation of modulator release from the urothelium into the lumen and the lamina propria. The paper reports a methodology for removal of the outer detrusor layer from the bladder, leaving a detrusor‐denuded bladder comprising only the mucosal layer (confirmed through histological and immunofluorescence observations of AQP‐3 + urothelial cells and PDGFRα + lamina propria interstitial cells).…”

“…Local physiological processes in the bladder wall are typically studied with techniques that include the following: in vitro myography of bladder strips combined with electrical field stimulation and/or pharmacological modulators of membrane receptors or ion channels, recordings of afferent nerve activity perhaps in combination with pressure-volume recordings in ex vivo bladders, Ussing chamber measurements of urothelial physiology and live-cell Ca 2+ imaging in tissue sheets or isolated cells. A paper in this issue of The Journal of Physiology by Durnin et al (2019) has developed an enhanced version of pressure-volume recordings in ex vivo murine bladder, enabling investigation of modulator release from the urothelium into the lumen and the lamina propria. The paper reports a methodology for removal of the outer detrusor layer from the bladder, leaving a detrusor-denuded bladder comprising only the mucosal layer (confirmed through histological and immunofluorescence observations of AQP-3 + urothelial cells and PDGFRα + lamina propria interstitial cells).…”

“…The physiological relevance and cellular origin of detrusor smooth muscle spontaneous activity have been a research focus for many years, in particular the contribution of the mucosa (Drake et al 2018). Durnin and colleagues did not exclude the possibility that mucosal-detrusor cellular signalling was necessary for the generation or modulation of detrusor spontaneous contractions, but importantly, demonstrated that such activity was not necessary for optimal pressure-volume relationships during physiological bladder filling (Durnin et al 2019). Spontaneous activity is considered to be an important determinant of bladder tone during filling, placing the detrusor in an optimal structural position for emptying, and therefore requiring less effort than initiating coordinated contraction from a low-tone baseline (Turner and Brading, 1997).…”