A Decentralized (Ex Vivo) Murine Bladder Model with the Detrusor Muscle Removed for Direct Access to the Suburothelium during Bladder Filling

Our bladder model with a removed detrusor muscle is a new experimental approach to studying simultaneously the availability of urothelial mediators in the lamia propria and bladder lumen during bladder filling. This model can provide new insights into the signaling mechanisms between urothelium, suburothelium, and detrusor muscle that regulate bladder excitability during filling. Begin by placing the intact murine bladder specimen into a dissecting dish under a dissection microscope containing 10 degrees celsius oxygenated KBS and cleaning out the connective and fat tissues around the bladder.

Pin a small portion of the dome of the isolated bladder all the way through the outermost edge of the detrusor muscle far from the innermost edge of the muscle that faces the suburothelium lamia propria to a sylgard covered dissecting dish filled with KBS, and pin the ureters and bladder neck to display the intact bladder preparation. Using fine-tipped forceps, gently pull a piece of the serosa at the corner between the ureter and the bladder body and adjust the light of the microscope to increase the transparency and to distinguish the margin of the submucosa underneath the detrusor muscle. To avoid perforating the preparation wall, be sure to cut away the detrusor muscle only while the lateral edge of the bladder mucosa is clearly visible.

Using fine-tipped scissors, begin cutting the bladder wall along the inner surface of the detrusor muscle layer while gently pulling the cut segment away from the preparation taking care that the lateral edge of the mucosa can be observed while avoiding touching the edge of the tissue. Leave a small piece of the detrusor muscle on top of the bladder dome to allow preparation to be immobilized during the remaining steps of the protocol and fill a two centimeter piece of 20 polyethylene tubing with 37 degree celsius oxygenated KBS. After inserting the catheter into the orifice of the bladder urethra, gently push the catheter until the catheter tip reaches the approximate middle of the bladder.

Then secure the catheter in the surrounding tissue of the bladder neck with a double loop 6.0 nylon suture. After checking the tissue for leaks, place the denuded bladder preparation into a three milliliter chamber of 37 degree celsius water jacketed organ dish with a sylgard bottom and secure the catheter to the side of the chamber so that the preparation does not float above the surface of the perfusion solution. Connect the bladder catheter to a piece of polyethylene 20 tubing connected to a three-way stopcock and an infusion syringe filled with 37 degree celsius oxygenated KBS and start the infusion pump to fill the bladder with KBS taking care to monitor the filling volume in the intravesical pressure during the filling.

To detect mediators within the suburothelium lamina propria aspect of the preparation, collect aliquots of the bath solution at various appropriate experimental time points and process the samples according to the planned downstream analysis. The denuded bladder preparation wall contains the urothelium and suburothelium but not detrusor smooth muscle. The pressure volume relationships are remarkably similar in the intact and denuded preparations.

The suitability of the model for measuring urothelium-derived mediators that are released in the suburothelium lamina propria side during filling can be tested by measuring the release of purine mediators into the solution bathing the suburothelium lamina propria of the denuded preparation. As demonstrated, negligible amounts of purines can be detected in a bath containing an isolated bladder preparation with an intact detrusor muscle;whereas the amounts of these purines are significantly higher in samples collected from the bath containing a denuded bladder preparation. Notably, the distribution of purines and metabolites in samples collected from the lumen and the suburothelium lamina propria at the end of filling differ significantly.

Addition of atheno ATP, a highly fluorescent analog of ATP, to the suburothelial side of the detrusor-free preparation results in a decrease in atheno ATP and an increase in the atheno ATP products, atheno ADP, atheno AMP, and atheno ADO. Likewise, the addition of atheno ATP to the lumen preparation results in a decrease in atheno ATP and an increase in atheno ADP, atheno AMP, and atheno ADO in the lumen. Further, the addition of atheno ATP to the suburothelium lamina propria side of the denuded preparation results in the appearance of atheno AMP and atheno ADO within the lumen.

Likewise, adding atheno ATP to the lumen results in the appearance of atheno AMP and atheno ADO in the suburothelium lamina propria. Following this procedure, the availability of excitatory and inhibitory mediators can be measured at the lamina propria and in the bladder lumen at different stages of bladder filling. Bilateral transurethral transport of mediators and metabolism of mediators can also be studied using this model.

Using this new bladder model will result in new understanding of intrinsic, local mechanosensitive signaling between the urothelium and detrusor smooth muscle that control bladder excitability in health and disease.