Micro 1401

Mice were anesthetized with intraperitoneal (i.p.) injections of ketamine (80 mg/kg 0.1 ml, Ketaset, Fort Dodge Laboratories Inc., Fort Dodge, IA, USA) and xylazine (12 mg/kg 0.05 ml, Rompun, Mobay Corporation, Shawnee, KS, USA) and electrodes for EMG recordings were chronically implanted as previously described (Kamp et al. 2003 (link); Cenac et al. 2007 (link); Gecse et al. 2008 (link)). A group of three nichrome electrodes (40 AWG Nichrome 80 w/red Poly Enamel, Pelican Wire Company, Naples, FL, USA) was implanted into the external oblique abdominal muscle, then exteriorized in the back of the neck and protected by a plastic tube attached to the skin. Following surgery, mice were housed singly and allowed to recover for 5 days before the experiments.
A PE50 catheter was taped 2 cm below the pressure sensor of a miniaturized pressure transducer catheter (SPR-524 Mikro-Tip catheter; Millar Instruments, Houston, TX, USA). A custom-made balloon (1 cm width × 2 cm length) (Kamp et al. 2003 (link); Arvidsson et al. 2006 (link); Christianson and Gebhart 2007 (link)) prepared from a polyethylene plastic bag was tied over the catheter at 1 cm below the pressure sensor with silk 4.0. Ligature points were covered with parafilm to prevent any air leak (Figure 1(A)). At the start of each experiment, each “balloon-pressure sensor” was calibrated at known pressures of 0, 20, 40, and 60 mmHg using a barostat (Distender Series II, G&J Electronics Inc. Toronto, Canada), and voltage output was converted to pressure using CED digital analog converter (Micro 1401, Cambridge Electronic Design, Cambridge, UK) and Spike 2 software (CED, Ltd., Cambridge, UK).
On experimental day, mice were briefly anesthetized with isoflurane (3% in O₂) and the lubricated “balloon-pressure sensor” was introduced into the colorectum such that the distal end of the balloon was at 0.5 cm past the anus and secured to the tail with tape. Each mouse was placed in an adjustable mouse restrainer (3.3 cm diameter × 9 cm length, #51325, Stoelting Co, Wood Dale, IL, USA), covered with a light tissue blanket and left to rest for 30 min before the CRD procedure. Each balloon was connected to the barostat and the miniaturized pressure transducer to a preamplifier (model 600; Millar Instruments, Houston, TX, USA). The EMG electrodes were inserted into an electrode board connected to an electroencephalograph (Grass Instrument Co., Quincy, MA, USA) where the signal was amplified (×10,000). Both signals were acquired using the CED Micro1401/SPIKE2 program. The EMG signal was filtered (1000 Hz), digitized, and rectified (Million et al. 2006b (link)). The CRD protocol consisted of a set of graded phasic distensions to constant pressures of 15, 30, 45, and 60 mmHg (three times each, 10 s duration, 4 min inter-stimulus interval). A similar CRD paradigm has been used previously to assess pain-related responses in mice (Kamp et al. 2003 (link); Christianson and Gebhart 2007 (link)).
The data analysis of the traces was performed as follows. The phasic component of the ICP (pICP) was extracted from the ICP signal recorded by applying the “DC Remove” process in Spike 2 with a time constant of 1 s, to exclude the slower, tonic changes in ICP resulting from colonic smooth muscle activity, and by applying the “RMS amplitude” process with a time constant of 1 s to the resulting trace. Both EMG and ICP activities were recorded for 10 s before (non-distended ICP), during, and after termination of CRD. The VMR was defined as the increase in area under the curve (AUC) of pICP or EMG during CRD over the mean value of pre- and post-distension periods and was quantified using the “modulus” process in Spike 2. As each CRD pressure was repeated three times, the pre–post CRD and during CRD values were averaged for each pressure. To examine the pressure—response relationship and adjust for inter-individual variations of the signal (Christianson and Gebhart 2007 (link)), ICP amplitudes were normalized for each mouse to the highest pressure (60 mmHg) in the first set of CRD. This value served as 100% response (control) in the baseline period of data collection before exposure to WAS or treatment and represented the baseline VMR. The VMR to the second CRD after WAS or treatment was expressed either as percentage from their normalized control values (percentage control) or mean change from the baseline response (Δ VMR in percentage control) at different distension pressures as validated in our previous studies (Larauche et al. 2009 (link)). It is known that the EMG signal can acquire electrical noise (Raez et al. 2006 (link)). Hence for data to be analyzed, the signal-to-noise ratio should be high: we excluded from the study mice equipped with EMG electrodes showing an EMG signal/noise ratio < 0.5 (Larauche et al. 2008 (link)). The signal/noise ratio was calculated as follows: signal/noise = (value during CRD—mean pre/post CRD value)/mean pre/post CRD value.

This was assessed using the noninvasive manometric method that we have recently developed and validated for use in mice and rats.^{10 (link),13 (link),31 (link)} Briefly, a PE50 catheter was taped 3.5 cm caudal to the pressure sensor of a miniaturized pressure transducer catheter (SPR-524 Mikro-Tip catheter; Millar Instruments, Houston, TX). A custom-made balloon (2 cm wide × 5 cm long),^{31 (link),32 (link)} prepared from an infinitely compliant polyethylene plastic bag was tied over the catheter at 1 cm below the pressure sensor with silk 4.0 (Henry Schein Inc., Melville, NY). At the beginning of each experiment, each “balloon-pressure sensor” was calibrated at known pressures of 0, 20, 40 and 60 mmHg using a barostat (Distender Series II, G&J Electronics Inc, Toronto, Canada), and voltage output was converted to pressure using a digital analog convertor (Micro1401, Cambridge Electronic Design, Cambridge, UK) and Spike 2 software (CED, Ltd., Cambridge). On the day of the experiment, rats were briefly anesthetized with isoflurane (3% in O₂) and the lubricated “balloon-pressure sensor” catheter was introduced into the colorectum such that the distal end of the balloon was positioned at 1 cm from the anus and the catheter was secured to the tail with tape. Each animal was placed in a Bollman cage, to which they had been habituated for 3 consecutive days prior to the experiment (1h/day), covered with a light tissue blanket and left to rest for 30 min before the CRD procedure. Each balloon was connected to the barostat and the miniaturized pressure transducer to a preamplifier (model 600; Millar Instruments, Houston, TX). The intracolonic pressure (ICP) signal was acquired using CED Micro1401/SPIKE2 program. The CRD protocol consisted of two CRDs at 60 mmHg to unfold the balloon, immediately followed by two consecutive series of graded phasic distensions to constant pressures of 10, 20, 40 and 60 mmHg (20 s duration, 4 min inter-stimulus interval within and between series). A similar CRD paradigm has been used previously to assess visceral pain-related responses in rats.^{13 (link),31 (link)}