C57BL/6 mice of both sexes (8–12 weeks, 25–35 g) were anesthetized by 2% isoflurane inhalation during all surgical procedures to avoid spontaneous or volitional movement. Throughout the anesthesia phase, mice were placed on a feedback-controlled heating pad (HP-150, Auber Instruments, Alpharetta, GA, United States) to maintain consistent body temperature of ∼37°C. To record abdominal EMG, we used a pair of Teflon-coated stainless wires (Conner Wire, Chatworth, CA, United States), exposed the ∼0.5 mm tip, and sutured them directly to the muscle with hypoallergenic bioabsorbable sutures (polyglactin, Ethicon) with 1 mm separation between both electrodes. The other ends of the electrodes were connected to a differential amplifier (Model 1700, A-M Systems, Sequim, WA, United States) with the ground wire connected to the mouse tail. During surgery, three pairs of Teflon-coated stainless steel wire electrodes were sutured to the rectus abdominis (R.A.) and the external oblique (E.O.) musculature immediately above the inguinal ligament (both sides for E.O.) for measuring the EMG responses to CRD (as shown in Figure 1A). One pair of Teflon-coated stainless steel wire electrodes was sutured to the latissimus dorsi (L.D.) musculature for measuring respiratory rate (RR). In addition, a plastic catheter (Φ 0.2 mm) was placed intraperitoneally beneath the transversus abdominis musculature for the infusion of urethane. Incisions on muscle layers were closed by bioabsorbable sutures (polyglactin, Ethicon) and on the skin layer by nonabsorbable sutures (Vicryl, Ethicon). A flexible lubricated polyethylene-film balloon (Φ 8 mm × 15 mm) was inserted intra-anally into the precise location in the colorectum by measuring and maintaining the 5–30 mm distance between the end of the balloon and the anus (i.e., the balloon depth) via taping the connected plastic inflating catheter to the tail. The balloon was pressurized by a custom-built distension device consisting of four hydrostatic columns of water set at 15, 30, 45, and 60 mmHg pressures (22) (shown in Figure 1A). Computer-controlled solenoid valves were implemented to regulate the onset and termination of CRD, which as shown in Figure 1A consisted of four ascending pressure steps of 5 s duration and 7 s between successive steps (15, 30, 45, and 60 mmHg). EMG responses were recorded using a differential amplifier and a digitizer (CED 1401, Cambridge Electronic Design Limited, Cambridge, United Kingdom).

The new urethane anesthesia protocol consists of an initial bout of infusion right after the catheter insertion as described above and a continuous low-dose infusion of urethane 60 min after terminating isoflurane (shown in Figure 1B). First, the initial dose of 0.6 g/kg urethane was administered intraperitoneally via the catheter inserted during the above surgical preparation. Isoflurane inhalation is removed after completing all the surgical procedures followed by another 60-min clearing period. Then, we started the continuous i.p. infusion of low-dose urethane [0.15–0.23 g per kg weight per hour (g/kg/h)] through the catheter to maintain systemic anesthesia for 2 h. During the 2-h window, VMR to CRD were assessed six times with 10–15 min between successive tests.

The rate of low-dose urethane infusion is critical to maintain optimal mouse anesthesia, i.e., the absence of locomotion and any other volitional movement while the presence of spino-spinal reflex (e.g., plantar reflex) and spino-bulbo-spinal reflex (i.e., the VMR to CRD). Thus, three rates of low-dose urethane infusion (0.15, 0.45, and 1.5 g/kg/h) are objectively assessed not only by the VMR to CRD but also by the respiratory rate as measured from EMG activities of the latissimus dorsi musculature (L.D. musculature in Figure 1A). Each rate of low-dose infusion was applied for 30 min (shown in Figure 2).

Since the distal ∼30 mm of the colorectum is differentially innervated by two distinct afferent pathways between the proximal colonic and distal rectal regions (26), the evoked VMR to CRD are likely different with distending balloon inserted in distal vs. proximal regions of the 30-mm-long colorectum. Thus, we systematically assessed the effect of axial location of the balloon in the colorectum on the evoked VMR by controlling the balloon depth in the colorectum, which is defined as distance between the distal end of the balloon and the anus. We control the balloon depth to be 0, 5, 10, 15, 20, 25, and 30 mm by marking and taping the connected catheter to the mouse tail. VMR to CRD were repeatedly recorded from the same animal with different balloon depths. At least 10 min was given between successive CRD.

Our prior report showed that intracolonic enema of TNBS induced persistent behavioral visceral hypersensitivity in male mice (11). In this study, we implemented the same mouse model to assess the effect of axial location of the distending balloon on VMR to CRD in both male and female mice. Mice were anesthetized by 2% isoflurane inhalation, transanally administered with TNBS (0.2 ml @ 10 mg/ml in 50% ethanol; Sigma-Aldrich, St. Louis, MO, United States) via a 22-gauge feeding needle (#18061-22, Fine Science Tools, Foster City, CA, United States), and held in a head down position (∼30°) for 5 min to preserve TNBS in the colorectum. Dietary gel (NGB-1, Bio-Serv, Flemington, NJ, United States) was provided to mice showing severe weight loss (>5% original body weight). Mice at 10–14 days following TNBS treatment were used in the current study, a time period as characterized by our prior study showing significant behavioral visceral hypersensitivity (11).

EMG activities evoked by CRD were recorded from the abdominal oblique musculature, digitized at 2000 Hz, and processed off-line using customized MATLAB scripts. The EMG signals were rectified for calculating the area under the curve (AUC), which was used to evaluate the level of VMR to CRD (27). VMR evoked by CRD was quantified as the AUC values during the 5 s CRD subtracted by the AUC of the 5 s pre-distending baseline recording. Results are expressed as means ± standard error (SE). One-way ANOVA and Tukey's repeated post-hoc comparisons were performed as appropriate using SigmaStat v4.0 (Systat Software, San Jose, CA, United States). Differences were considered significant when p < 0.05.

The main objective of the study is to establish a new anesthesia protocol for robust recordings of VMR to CRD in mice, the critical parameter of which is the rate of continuous infusion of low-dose urethane (Figure 1B). As shown in Figure 1A, VMR to CRD was recorded by the EMG electrode pair on the right E.O. musculature. RR was recorded by the EMG electrode pair on the L.D. musculature. The balloon depth in the colorectum was maintained at 5 mm in this study. In six mice (three females and three males), three ascending rates of continuous urethane infusion were assessed (0.15, 0.45, and 1.5 g/kg/h), each with a duration of 30 min (Figure 2A). The VMR and RR were recorded at the end of each 30-min duration as indicated by T1, T2, and T3 in Figure 2A. The VMR evoked by the 60-mmHg stepped pressure recorded at T1, T2, and T3 in each mouse were normalized by the VMR recorded at T1 with the lowest infusion rate (0.15 g/kg/h). As shown in Figure 2B, the infusion rate of 1.5 g/kg/h resulted in a significant increase in RR recorded at T3 (one-way ANOVA with repeated measure, F_2,15 = 6.82, p < 0.05; post-hoc comparison, p = 0.015 vs. T2, p = 0.016 vs. T1). Also as shown in Figure 2C, the rate of 1.5 g/kg/h significantly suppresses the VMR to CRD as recorded at T3 (one-way ANOVA with repeated measure, F_2,15 = 9.06, p < 0.5; post-hoc comparison, p = 0.004 vs. T2, p = 0.009 vs. T1). There was no significant difference in RR or VMR between the urethane infusion rates of 0.15 and 0.45 g/kg/h measured at T1 and T2, respectively. Thus, we maintained the rate of low-dose urethane infusion of no more than 0.3 g/kg/h for the rest of the study.

The EMG magnitude as a metric of VMR is affected not only by the muscle recorded from but also the spacing between the two differential electrodes (∼1 mm). As shown in Figure 1A, we implanted three pairs of electrodes to simultaneously record from bilateral E.O. and the medial R.A. musculatures in 16 mice (8 females and 8 males). The balloon depth in the colorectum was set to be 5 mm in all recordings. The VMR recordings were processed as AUC values from four distending pressures (15, 30, 45, and 60 mmHg), which were normalized by the AUC value of 60 mmHg CRD (100%) recorded from the right E.O. Representative VMR from the right E.O., left E.O., and R.A. musculatures are displayed in Figure 3A, and the pooled data from 16 mice are displayed in Figure 3B. The VMR recorded from left and right E.O. musculatures were not significantly different (two-way ANOVA with repeated measure, F_2,510 = 33.262, p < 0.0001; post-hoc comparison p = 0.19 for left vs. right E.O.), both of which are significantly higher than from the R.A. (post-hoc comparison, p < 0.0001 for right E.O. vs. R.A.; p < 0.0001 for left E.O. vs. R.A.). Thus, we only recorded VMR from the right E.O. for the rest of this study.

We aim to advance the VMR recordings from anesthetized mice as a reliable test bench to objectively assess various neuromodulatory interventions for treating visceral pain. Thus, we systematically determine the repeatability of VMR recordings evoked by multiple CRD within the 2-h time window during the low-dose urethane infusion in eight mice. From four mice (two females and two males), VMR to CRD was recorded six times during the first 60 min period at the low-dose urethane infusion rate of 0.15 g/kg/h and recorded for another six times during the second 60 min period at the infusion rate of 0.23 g/kg/h. The interval between successive CRD was maintained at 10 min throughout the test. Similar tests were conducted on another four mice (two females and two males) while switching the sequence of urethane doses, i.e., 0.23 g/kg/h of urethane in the first 60 min and 0.15 g/kg/h in the second 60 min period. Within each mouse, the AUC of all VMR was normalized by the response to the 60-mmHg distension in the first VMR. Figure 4A shows representative VMR recorded 12 times from one mouse at 0.15 g/kg/h and 0.23 g/kg/h urethane infusion. VMR recordings showed no within-sample differences in each of the eight mice recorded under the same rate of urethane infusion (two-way ANOVA with repeated measures, F_5,360 = 1.925, p > 0.05 for all). In each of the eight mice, the VMR to CRD recorded at the same urethane infusion rates were averaged and plotted in Figure 4B, which showed no significant difference between 0.15 and 0.23 g/kg/h urethane in all of the eight mice tested (two-way ANOVA with repeated measures, F_1,56 = 0.005, p > 0.05 for all). These results indicate that the new anesthesia protocol with low-dose urethane infusion (0.15–0.23 g/kg/h) enables robust and repeatable VMR recordings to CRD with 10 min intervals for at least 2 h.

Within the same mouse, VMR to CRD was assessed with a distending balloon positioned at different depths in the colorectum as illustrated in Figure 5A. The representative VMR to CRD at balloon depth of 5 vs. 15 mm from the same mouse is displayed in Figure 5B, indicating the dramatic reduction of VMR with increased balloon depth in the colorectum. The VMR were recorded with balloon depths up to 30 mm into the anus in male mice and 25 mm in female mice. VMR from six male mice recorded at different balloon depths are normalized by the AUC evoked by the 60-mmHg pressure step with 5-mm balloon depth. Figures 5C,D show the average responses from six male and six female mice, respectively. VMR to CRD is significantly higher when recorded at the lowest balloon depth (5 mm) as compared to deeper distension with increased depth (10–20 mm) in both male (two-way ANOVA, F_3,560 = 5.151, p < 0.05; post-hoc comparison, 5 mm vs. others, p < 0.05) and female mice (two-way ANOVA, F_3,560 = 3.038, p < 0.05; post-hoc comparison, 5 mm vs. others, p < 0.05).

We previously reported that intracolonic TNBS treatment produced prolonged behavioral visceral hypersensitivity in male mice from 7 to 14 days post-treatment (11), which is evidenced by increased VMR to CRD recorded in conscious mice. VMR to CRD were recorded in mice under the newly developed urethane anesthesia protocol (0.3 g/kg/h urethane infusion) with multiple balloon depths into the colorectum in both male (5–30 mm depth) and female mice (5–25 mm depth). Tests were conducted in mice receiving intracolonic saline (control) or TNBS. As shown in Figure 6A for 10 mm balloon depth, the VMR to CRD is significantly higher in male mice receiving TNBS than those receiving saline (two-way ANOVA, F_1,40 = 4.391, p < 0.05) but is not significantly different between female mice receiving TNBS and saline (two-way ANOVA, F_1,40 = 0.62335, p = 0.434). This sex difference in TNBS-induced increase in VMR to CRD is also observed with 15 mm balloon depth as shown in Figure 6B, i.e., sensitized response by TNBS only in male mice (two-way ANOVA, F_1,40 = 14.43158, p < 0.05) but not in female mice (F_1,40 = 0.64, p = 0.428). VMR to CRD in saline-treated groups showed no significant sex differences for both 10 and 15 mm balloon depths (F_{1, 40} = 1.946, p > 0.05 for 10 mm, F_{1, 40}= 3.723, p > 0.05 for 15 mm). CRD recorded in each mouse were normalized by the response to 60 mmHg distension at 5 mm balloon depth. All responses to 45 mmHg distension at different balloon depths from saline and TNBS groups were pooled together and displayed in Figure 6C, and all responses to 60 mmHg displayed in Figure 6D. In male mice, VMR to distension of 45 mmHg was significantly higher at balloon depth of 10 and 15 mm in the TNBS group than in the saline group (Two-way ANOVA, F_1,20 = 26.659, p < 0.05; post-hoc comparison), whereas female mice showed no difference between the TNBS and the saline groups at all tested balloon depths. Similarly, VMR to 60 mmHg distension were significantly increased in male mice in the TNBS group as compared to the saline group at balloon depths of 10, 15, 20, and 25 mm (two-way ANOVA, F_1,40 = 36.421, p < 0.05; post-hoc comparison) while female mice showed comparable VMR between the TNBS and the saline groups.

We set the threshold for positive VMR response as five times the root mean square of the baseline noise level of the EMG signals. As illustrated in Figure 7A, mice with positive response to stepped distension are considered responders and those with negative response are non-responders. As shown in Figure 7B, mice of either sex receiving TNBS or saline are all responders to 45 and 60 mmHg distension at balloon depth of 5 mm. As summarized in Figure 7C for 10-mm balloon depth, only 50% mice are responders to 60 mmHg distension in male mice receiving saline, in comparison to 100% responders in male mice receiving TNBS. In contrast, female mice had comparable proportions of responders (83.3%) between the saline and the TNBS groups. At a balloon depth of 20 mm shown in Figure 7D, no male mice in the saline-treated group responded to 60 mmHg distension, whereas 83.3% in the TNBS-treated group did. Again, the proportions of responders in female mice were not different between saline (16.7%) and TNBS groups (16.7%).