Powerlab device

The animal was weighed and anaesthetized by an intraperitoneal injection with a combination of fentanyl (3.313 mg/kg), fluanisone (104.8 mg/kg), and midazolam (52.44 mg/kg), and if necessary the anesthesia was maintained every 30 min. The neck area was shaved and the animal was placed on a heating pad. A 1 cm incision was made on the right side of the neck. By blunt dissection, the right external jugular vein was located and isolated. A hole was cut in the vein and through this, a catheter (SPC-1000, Millar Instruments Inc, USA) was inserted and secured by a suture. The catheter was led into the right ventricle and the position was confirmed by observing the characteristic ventricular waveform. The pressure profile was recorded over a three minute period using a Quad Bridge amplifier connected to a PowerLab device (ADInstruments, Oxfordshire, England) and recorded with Chart 5.5 (ADInstruments). The animal was euthanized after blood pressure measurement by cervical dislocation.

Abdominal pain was assessed by measuring the visceromotor responses (VMRs) of mice to colorectal distension (CRD) following a previously described protocol^{17 ,35} . Briefly, electrodes were implanted in the abdominal external oblique muscles and exteriorized onto the back of the necks of mice at least 15 days prior to VMR measurement. Conscious mice were habituated to a plexiglass cylinder for 30 min per day for 3 consecutive days before VMR measurement to accustom the animals to partial constraint. For recording, the electrodes were connected to an electromyogram (EMG) acquisition system (AD Instruments). The colon was distended by inflating a balloon catheter inserted intra-anally such that it ended 1.5 cm proximal to the anus. The mice were subjected to four 10-second distensions (15, 40, and 65 mmHg) at 3-minute intervals. EMG activity was amplified and digitized using a transducer connected to a P511 AC amplifier (Grass Instruments) and a Powerlab device with Chart 5 software (AD Instruments). EMG activity was rectified, and responses were recorded as the increase in the area under curve (AUC) of the EMG amplitude during CRD versus the baseline period.