A method to study bladder urothelial cellular function in situ

Abstract

OBJECTIVE:The bladder urothelium is comprised of basal, intermediate, and apical urothelial cells. Ability to functionally study an individual urothelial cell while preserving its in situ location would represent an advance in bladder urothelial biology. MATERIALS AND METHODS:Mice were euthanized and cardiac perfused with phosphate-buffered saline. Bladders were then excised. Urothelial sheets were dissected off the lamina propria using forceps with a microscope (5 × magnification). Sheets were stained with H&E. In separate experiments, cell-attached electrophysiology was performed by placing urothelial sheets in Ringer's bath solution, with either basal or apical surface down. Using 40 × magnification, individual urothelial cells from different layers were identified. Potassium currents on these cells were measured in situ using single channel patch-clamp technique. RESULTS: CONCLUSIONS:A novel approach was developed in which individual urothelial cells within the multi-layered urothelium were identified and functionally studied in situ. Electrophysiologic characterization revealed different phenotype between the cells from different layers. Single cells from an identified layer can be harvested off the urothelium allowing for other studies. This technique allows investigators to study various cellular functions while preserving cellular location within the urothelium.

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is released by female mouse bladder urothelial cells and expressed by the urothelium as an early response to lipopolysaccharides (LPS)

“…The pyrogen‐free saline or LPS was allowed to dwell for 30 min before animals were awakened, followed by harvest of mouse bladder urothelium 3, 6, and 18 hr later. The dissection of each of the bladder layers followed previously published methods in which investigators were able to dissect off pure urothelium (“urothelial sheet”) that was devoid of underlying lamina propria and muscularis . Thus, we could conduct experiments on three distinct and separate tissue types—urothelium, lamina propria, and muscularis.…”

“…The dissection of each of the bladder layers followed previously published methods in which investigators were able to dissect off pure urothelium (''urothelial sheet'') that was devoid of underlying lamina propria and muscularis. 21 Thus, we could conduct experiments on three distinct and separate tissue types-urothelium, lamina propria, and muscularis. Quality assurance histology of the urothelial sheet dissections confirmed pure urothelium without other layers.…”

Mucosal signaling in the bladder

“…A new technique, not involving cell cultures, was developed to isolate a pure sheet of bladder urothelium devoid of all underlying LP (Lu and Chai, 2014). The dissected urothelial sheet was used to perform single urothelial cell patch-clamp electrophysiology in situ within the tissue allowing denotation of whether the cells patched were apical, intermediate or basal in location within the urothelium.…”

“…It is likely that the "urothelium" removed is the "mucosa". The ability to remove a pure urothelium without the underlying LP has been demonstrated in mice (Lu and Chai, 2014), but it is uncertain whether pure urothelial tissue can be dissected off other species such as guinea pigs or pig. Photomicrographs obtained after muscle organ bath experiments of porcine mucosal strips showed the presence of smooth muscle, but interestingly, loss of urothelium (Sadananda et al, 2008).…”

“…3). A technique was recently described to dissect only the urothelium off the underlying LP in mice bladders (Lu and Chai, 2014). Fig.…”