Electrophysiology
The mouse urinary bladder and surrounding tissues were dissected and placed in a chamber where it was continuously perfused with oxygenated (95% O2, 5% CO2) Tyrode solution pH 7.4 (content in mM: NaCl 136.9; KCl 2.7; CaCl2 1.8, MgCl2 1; NaH2PO4 0.4; NaHCO3 11.9 and Glucose 5.6). A fine triple-lumen canula (combined diameter 0.9 mm) was inserted into the bladder through the urethra. The canula was connected to: i) a syringe pump (Harvard Apparatus, Holliston, Massachusetts, USA) to infuse Tyrode into the bladder, ii) to a pressure transducer (P75, Hugo Sachs Elektronik - Harvard Apparatus GmbH, March-Hugstetten, Germany) to record intravesical pressure changes and iii) to an outlet tube equipped with a three way stopcock to block or release intravesical fluids from the bladder. An additional canula (MicroFil MF34G 0.1 mm ID, WPI, Sarasota, FL, USA, diameter 0.164 mm) was added along the triple-lumen canula and connected to a 1 ml syringe to allow bolus application of drugs. The urethra and the ureters were ligated to avoid leakage of fluid from the urinary bladder.
Under a microscope, a branch of the pelvic nerve arising from the urinary bladder was dissected. Nerve activity was recorded from very fine filaments teased from the pelvic nerve and sucked inside a glass suction electrode connected to an Axon Instrument head stage (AI 402 × 50 Ultra low noise differential amplifier, Axon Instrument) and an AC/DC amplifier (CyberAmp 380, Axon Instrument). Signals were amplified (250×) filtered (band-pass 10-10000 Hz) and relayed to a noise eliminator (Hum Bug, Quest Scientific, Vancouver, Canada). The electrical activity of the nerve and the intravesical pressure were digitized using a computer connected to a Micro 1401 MK II analog-to-digital interface controlled with Spike 2 (version 6.13) software (Cambridge Electronic Design, Cambridge, UK).
Mechanical stimulation of the bladder from normal or inflamed animals was performed by a slow infusion of Tyrode solution (0.1 ml/min) until the intravesical pressure reached 40 mm Hg, then the pressure was released by opening the outlet valve. Single unit discrimination was performed by using the spike sorting function of Spike2 software.
The effects of inflammation or drug administration on bladder compliance were evaluated by calculating the volume of Tyrode infused in the bladder to reach an intravesical pressure of 40 mm Hg it was measured together with the time required for the intravesical pressure and the flow rate of the pump (0.1 ml/min). The thresholds of firing of each afferent from inflamed bladders were calculated from the ramp of stimulation and compared with our database of afferents from normal bladders.
After several (3-4) stimulations to ensure that the afferent activity was stable, pharmacological assays were done on inflamed bladder. In a first series of experiments, a bolus (intravesical administration with the outlet valve opened) of a non selective cannabinoid agonist AZ12646915 (100 μl, 100 μM) dissolved in Tyrode + 1% DMSO was administered between two inflations, with 20 minutes prior to the second stimulation. This dose was selected as it was shown to be effective in reducing the afferent activity in our previous study. In addition, we observed a recovery and no effect of the vehicle [
3]. To assess the specificity of action of AZ12646915, CB
1 receptor antagonist AM251 (100 μl, 100 μM) or the CB
2 receptor antagonist AM630 (100 μl, 100 μM) were administered 5 minutes prior AZ12646915 in another series of experiments using the same conditions of stimulation as above. Both AM251 and AM630 are selective antagonists with high affinity for CB
1 and CB
2 receptors respectively. The dose of AM251 selected has been shown to be efficient in blocking the effect of AZ12646915 in normal bladders [
3], the same dose was selected for AM630 since AZ12646915 has equal affinity for CB
1 and CB
2 receptors.
Afferent activity was measured as the number of spikes/s counted at every 5 mm Hg intervals during each ramp of stimulation going from 0 to 40 mm Hg. Agonist and antagonists effects were measured using the normalized activity: each point of the ramps of pressure (pre and post-drug) was expressed as a percentage of the maximal activity recorded in the pre-drug stimulation.
Immunofluorescence
A vascular rinse was performed by transcardial perfusion with 50 ml of PBS 0.1 M + 1 UI heparin/ml followed by 200 ml of PBS + 4% paraformaldehyde as a fixative solution. The bladder was harvested and placed in the same fixative solution during 3 hours at +4°C then transferred in PBS 0.1 M + 30% sucrose overnight at +4°C. After permeabilization (PBS, 10% Normal Goat Serum (NGS), 0.25% TRITON, 0.05% NaN
3) and saturation with PBS + 20% NGS, cryostat sections (12 μm) of the bladder were incubated 48 h at +4°C with the primary antibodies solution: anti CB
1 raised in rabbit and anti TRPV1 raised in guinea pig (Table
1). After rinsing, the sections were incubated with goat anti-rabbit IgG (1:600) conjugated with fluorescein isothiocyanate (FITC, 488 nm) and with goat anti guinea pig IgG (1:600) conjugated with Texas red (568 nm) 1 h at RT. Mounted slides were observed under a Zeiss Axioplan 2 Imaging fluorescence microscope and images were captured by a digital camera AxioCam HRc (Zeiss, Canada). Microphotographs were obtained with ×20 objectives. Negative controls were sections incubated with the primary antibodies preadsorbed with their respective blocking peptide (Table
1) and the time of exposure setting was constant during the acquisition of images. Background subtraction and merging of images were performed with the program Image J [
54].
Table 1
Primary antibodies and their respective blocking peptides used for the immunofluorescence study.
CB
1
| 10006590 | Rabbit - Polyclonal | 1:100 | C-terminal MSVSTDTSAEAL | 10006591 | 1:2 | Cayman Chemical |
TRPV1
| GP14100 | Guinea pig - Polyclonal | 1:1000 | C-terminal YTGSLKPEDAEVFKDSMVPGEK | P14100 | 1:5 | Neuromics |
Chemicals
The cannabinoid compound used in this study, AZ12646915 (Astra Zeneca R&D, Montreal, Canada) has equal affinity for both human CB1 and CB2 receptor (Ki = 17 nM and 16 nM respectively). It was selected for its good solubility (up to 150 μM) in aqueous solution in order to prevent as much as possible precipitation in the tubing. The cannabinoid receptors antagonists AM251 and AM630 were purchased from Tocris Bioscience (Ellisville, USA). Cyclophosphamide was purchased from Sigma-Aldrich (Canada). Salts used for the solutions were all purchased from ACP chemicals (Montreal, Canada).