2.3 Organ-bath experiments
All samples were taken from the bladder body. From each patient, 5–10 longitudinal bladder strips (length = 20 ± 1.5 mm; width = 8–9 mm) were studied. Bladder strips were placed in warm Krebs solution (composition in mM: NaCl, 118; KCl, 4.7; CaCl2, 1.9; MgSO4, 1.2; NaHCO3, 24.9; KH2PO4, 1.2; glucose, 11.7; pH = 7.4) and bubbled with 95% O2 and 5% CO2. Strips were tied at each end using a fine thread, mounted in a vertical organ bath in oxygenated Krebs solution (volume = 20 mL), and heated to 37 °C in a circulating warm water-bath. The longitudinal tension of the isolated preparations was recorded continuously with an isometric transducer and then processed with Lab Chart 7 (AD Instruments, New South Wales, Australia). Tissues were stretched to a baseline tension of 15mN (1.5 g). Contractions were allowed to equilibrate for 30–40 min, until the appearance of stable SBCs. In strips with no SBCs, carbachol (1–10nM) was applied to initiate SBCs. Strips that had a spontaneous or carbachol-evoked SBCs were considered as having “good” viability. All the strips from the 26 patients had good viability.
In a pilot study, we found that repeated application of high concentrations (≥100μM) of H2O2 in the same strip induced desensitization (supplementary figure 1). Thus, the noncumulative concentration–response relationship of H2O2was determined. On completion of experiments, threads were removed and strips were weighed after water absorption with tissue paper.