Tissue preparation and strip tension measurement
Eight adult male Wistar rats (250–300 g, Harlan, The Netherlands) were used. All experiments were performed in accordance with institutional guidelines and were approved by the ethics committee for animal experiments of the VU University Amsterdam. The urinary bladder was removed following isoflurane anaesthesia and heart dissection of the rat. The bladder was immediately placed in Krebs’ solution (mmol/L; NaCl 113, KCl 4.8, MgSO4 1.2, KH2PO4 1.2, CaCl2*2H2O 2.5, NaHCO3 25, glucose 11.5, bubbled with 95% O2 and 5% CO2 to attain a pH of 7.4) at 35 degrees Celsius.
Two longitudinal strips of maximally 0.7 by 7 mm were cut from the dorsal side of the bladder body under a microscope. The urothelial layer of the strips was meticulously removed with micro scissors under dark field illumination. The serosal layer of the bladder was not removed.The two rat bladder strips were mounted between 2 platinum hooks in two parallel organ baths. One of the platinum hooks was connected directly to a force transducer (AE801, SensoNor, Norway) (Figure 1). The baths were perfused with 35°C Krebs’ solution for 60 minutes to stabilize the strips at a passive tension of 10 millinewton. Hereafter the bladder strips were stimulated. Chemical stimulation was performed with 80 mM potassium chloride (KCl) or 1 μM carbachol (CCh). Between the chemical stimulations and during the electrical field stimulation, the organ baths were perfused by Krebs’ solution. Electrical field stimulation (EFS) between two platinum plate electrodes was delivered by a Grass SD9 stimulator. The intrinsic nerves were stimulated with 50 V pulses of 100 shocks at a frequency of 20 Hz. The stimulation interval was 3 minutes.
Before we started the BoNT-A experiments, we incubated two strips in 1 μM tetrodotoxin (TTX) during continuous EFS to verify that the EFS protocol induced primarily nerve cell depolarization[9]. Subsequently, we incubated two strips in Krebs’ solution and gradually lowered the oxygen tension in the organ bath from 95% to 20%. We calculated the critical oxygen tension in the organ bath by a Hill diffusion model previously described by Van der Laarse et al.[10]. Contractions were recorded by a data acquisition program (NEWXLC® software developed by the Department of Physiology, VUmc, Amsterdam).
Drugs and solutions
KCl, CCh, TTX and BoNT-A were obtained from Sigma-Aldrich, Zwijndrecht, The Netherlands. The BoNT-A used was pure BoNT-A (molecular weight of 150 kDa), i.e. toxin without any enveloping proteins. KCL, CCh, TTX and BoNT-A were all dissolved in Krebs’ solution. All solutions were gassed with a mixture of 95% O2 and 5% CO2 (pH 7.4 at 35°C).
Viability of bladder strips
To establish the viability of the bladder strips after preparation and during the stimulation protocol, the strips were excited chemically by adding 80 mM KCl and 1 μM CCh to the organ baths. The organ baths were flushed with Krebs’ solution after chemical stimulation. KCl induces smooth muscle membrane depolarization without the need of activation of any receptor or second messenger system[9]. CCh induces contraction of the bladder strip by direct activation of the muscarinic receptor at the smooth muscle cell membrane. CCh interacts with the same muscarinic receptor as acetylcholine[9]. BoNT-A inhibits the release of acetylcholine and it does not interact with the muscarinic receptor at the muscle cell membrane. CCh still induces strip contraction after BoNT-A incubation. The comparison of CCh-induced strip contraction before and after BoNT-A incubation acts as a viability test of this strip during the experiment.
Stimulation protocol
KCl and CCh were used to verify viability, while EFS provided baseline contractions during phase 1. Organ baths were perfused with 80 mM KCl for 10 seconds, followed by flushing the organ baths with Krebs’ solution for 3 minutes. This cycle was performed three times. Subsequently, organ baths were perfused 3 times with 1 μM CCh solution followed by flushing the organ baths with Krebs’ solution for 3 minutes. Finally, contractions were induced by EFS (50 V, 20 Hz, 100 shocks) for five times, each with an interval of 3 minutes.
In phase 2, strips were incubated in various concentrations BoNT-A (0.03, 0.2, 0.3 nM). Control strips were perfused by Krebs’ solution only. Repetitive EFS was performed.
Phase 3 consisted of perfusion of Krebs’ solution in the organ baths for at least 5 minutes. Subsequently, repetitive EFS and CCh stimulations with the same settings and the same concentrations as used in phase 1 were performed. We refer further to a completed protocol of stimulations as a ‘run’.
BoNT-A incubation
Each BoNT-A concentration was tested 3 times. Two strips of the same bladder were incubated in 2 parallel organ baths. One strip was incubated in 0.3 nM BoNT-A (run 1a, 2a, 4a) whereas the other strip was perfused with Krebs’ solution (=control: run 1b, 2b, 3b). To establish whether there was a minimum concentration necessary to measure an inhibitory effect of BoNT-A in bladder strip contractions, run 3a, 4b and 5a contained strips incubated in 0.2 nM BoNT-A. Run 5b, 6a, 6b contained strips incubated in BoNT-A 0.03 nM.
Data analysis
Three peak forces of strip contraction induced by CCh stimulation were measured and averaged (mPF CCh). The viability of the strip was established by the equation: Contractility = mPF CCh phase III/mPF CCh phase I. A strip is viable if contractility equals 1.
Inhibition of bladder strip contraction by BoNT-A was measured by comparing the mean peak force induced by EFS before and during BoNT-A incubation. Inhibition is defined by: Inhibition = 1 – (mPF incub/mPF max), in which mPF incub is the mean of five repetitive peak forces by EFS measured every 30 minutes during incubation, and mPF max is the mean of five highest repetitive peak forces of strip contractions induced by EFS at the start of phase II.
We analyzed the data by a mathematical model representing diffusion, as we hypothesized that BoNT-A spreads in the bladder strip by diffusion. The model is a log-logistic model[11]: I = I
max
/(1 + exp (- b
*
ln (t/m))), where I = measured inhibition of contraction of bladder strip, Imax = calculated maximum of inhibition, b = shape parameter of the curve, t = time after highest peak forces of EFS at start of incubation (hours), and m = mid inhibition time, at which I = 0.5 Imax (hours). R2 is predictive accuracy of the model, so the closer R2 is to 1.0, the better the data fit to the curve. The curve fits if R2 is more than 0.95, and if the 95% confidence intervals around b and m are significant. Data analysis and curve fitting by non-linear regression was done with SPSS 20®.