Eighteen eight-week-old adult male Sprague–Dawley rats (300–350 g) were purchased from Charles River Laboratories International, Inc., Wilmington, MA. The rats were housed in regular cages before surgery in a temperature-controlled environment at 22°C, humidity 40%, on a 12-h light, 12-h dark cycle. Animals had free access to a standard rat laboratory diet and water (Murakami et al., 2018). However, they were overnight fasted with ad libitum access to water before surgery. The animal experiment was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the experimental protocol was approved by the Johns Hopkins University Animal Care and Use Committee, Baltimore City, MD, United States. The authors were working at the Johns Hopkins University at the time of the study.

The rats were randomly divided into sham-iVNS group (n = 9) and iVNS group (n = 9; Figure 1). The Nissen fundoplication surgery was performed on both sham-iVNS group and iVNS group. iVNS/sham-iVNS was performed during the first 30-min of the Nissen fundoplication procedure in the corresponding group. Animal behaviors, eating, drinking and feces were monitored at specific postoperative periods. Gastric slow waves (GSWs) and electrocardiogram (ECG) were recorded (Wang et al., 2010; Jin et al., 2017); Rat Grimace Scale (RGS) was used to assess pain (Sotocinal et al., 2011); Blood samples were collected for the assessment of inflammatory cytokines at specific postoperative times. The detailed experimental protocol is shown in Figure 2.

The rat was overnight fasted with water ad libitum, cephalexin (15 mg/kg, S.C.) and Buprenorphine (0.05 mg/kg, S.C.) were administered 1 h before surgery. Anesthesia was induced by 2% isoflurane and oxygen flow of 1–2 L/min in a chamber. The rat was placed on a heating pad, and anesthesia was maintained via a breathing mask attached to the rat. The abdominal wall of the rat was depilated from xiphoid to groin using an electric hair clipper and prepared with an iodine solution followed with 70% isopropanol. All surgical procedures were done aseptically (Sotocinal et al., 2011; Xu et al., 2011; Jin et al., 2017; Murakami et al., 2018).

iVNS was performed using a universal pulse generator (Model DS8000, World Precision Instruments, Sarasota, FL, United States) during the first 30 min of the surgery using following parameters: 5 Hz, 0.5 ms, 2.2 mA, 10s on, 90s off, previously optimized to treat intestinal inflammation (Murakami et al., 2018). In the sham-iVNS group, electrodes were implanted exactly same as the iVNS group but no current was delivered to the vagal nerve.

Observations of post-operative animal behaviors were mainly focused on the first post-surgical time to water intake and food intake (counted starting from wake-up of the animal from anesthesia), the amount of 24-h water intake and food intake, the number of 24-h pellets on postoperative days 1 to 3. The first post-surgical time to water intake and food intake was assessed from a video recording (also used for pain evaluation) that was initiated immediately after placing the rat in wired-bottom cage after surgery (Murakami et al., 2018).

The water and food were weighed at the beginning and ending of every 24-h to calculate the amount of water intake and food intake. Cages were changed every 24 h and fecal pellets were collected. Weight of fecal pellets were measured at collection and at the time when pellets were dried out (kept in an exhaust hood for 3 days) to calculate the water content in feces (Jin et al., 2017).

After the surgery, the rat was housed individually in a wire bottom cage. A digital camera (D600, Canon, Japan) was placed outside the acrylic glass walls of the cage to get clear head shots and record behavioral aspects. The digital movies were taken for 5 min each at 15, 30, 60, 90, 120, 150, and 180 min after the surgery. The Rat Grimace Scale (RGS) for pain evaluation was scored by two evaluator who were totally blinded to this study (Sotocinal et al., 2011). The RGS consisted of four facial “action units” (orbital tightening, nose/cheek appearance, ear and whisker positions) which were scored using still images by the observer. The observer assigned a value of 0, 1, or 2 for each of the 4 RGS action units. The final RGS score was the average score across the 4 action units (Sotocinal et al., 2011).

The autonomic function was assessed by the spectral analysis of the heart rate variability (HRV) signal derived from the ECG (Murakami et al., 2018). The ECG signal was recorded for 30 min using a special amplifier (model 2,283, Fti Universal Fetrode Amplifier, UFI, Morro Bay, CA, United States) at different postoperative time periods. Previously validated software (Xu et al., 2011) was used to derive the HRV signal from the original ECG recording by identifying R waves, interpolating R-R interval data at 100 Hz, and finally down-sampling the interpolated HRV data to 8 Hz for spectral analysis. Two frequency bands in the power spectrum of the HRV were calculated as follows: (1) high frequency (HF): 0.8–4.0 Hz, reflecting purely vagal activity and (2) low-frequency (LF): 0.3–0.8 Hz, reflecting mainly sympathetic activity. The ratio of LF/HF reflected the sympathovagal balance (Xu et al., 2011; Jin et al., 2017; Murakami et al., 2018).

GSWs were recorded for 30 min at 6 h, 24 h, and 72 h after the surgery using a Biopac system (EOG 100A; Biopac Systems, Santa Barbara, CA; Murakami et al., 2018). Parameters of the GSW were derived from spectral analysis using previously validated software (Liu et al., 2004a,b). The power at the dominant frequency (DF) in power spectrum was defined as the dominant power (DP). The percentages of normal slow waves, bradygastria and tackygastria were calculated according to the percentage of time during which regular 4–6 cycle/min (cpm) waves, waves of <4 cpm and waves of >6 cycle/min (cpm) were presented, respectively (Liu et al., 2004a,b).

Cytokines levels in plasma were measured for assessment of postoperative inflammation. Blood samples were collected one day before the surgery as baseline and recollected at 1 h, 6 h, 24 h, and 72 h after the surgery, and were centrifuged at 3000 × g for 10 min at 4°C. TNF-α, IL-1β, and IL-6 levels in plasma were assessed using corresponding commercial ELISA kits (Abcam, Cambridge, MA) according to the protocols provided by the manufacturer. The absorbance rate was read at 450 nm. The concentrations of the samples were calculated according to the standard curve.

The data are presented as mean ± SD. Student’s t-test was used for the comparison between the two groups and one-way ANOVA was used to compare the difference in any of the measurements among different post-surgical points, followed with Tukey’s test using SPSS. Statistical significance was set at p < 0.05.

iVNS accelerated postoperative recovery by shortening the first post-surgical times of water and food intake, increasing the amounts of water and food intake, and increasing the number of fecal pellets and percentage of water content in fecal pellets. iVNS substantially reduced not only the first post-surgical time of water intake (28.4 ± 3.9 min vs 58.3 ± 15.9 min，p = 0.001) but also the first post-surgical time of food intake (37.6 ± 3.5 min. vs 66.0 ± 20.1 min, p = 0.003) compared to sham-iVNS (no stimulation) (Figure 3A). iVNS increased the amount of water intake (92.0 ± 10.3 vs. 66.3 ± 22.9, p = 0.011, vs. sham-iVNS) and food intake (8.7 ± 2.6 vs 4.2 ± 1.1 g, p = 0.001, vs. sham-iVNS) on postoperative day one (POD1); the effect on food intake remained significant on POD2 (10.3 ± 1.7 vs 5.5 ± 1.7 g, p = 0.001), whereas the effect on water intake was not significant on POD2 but significant on POD3 (86.0 ± 17.7 vs 62.4 ± 17.6 g, p = 0.012; Figures 3B,C).

Compared with sham-iVNS, iVNS increased the number of pellets on POD1-3 (POD1: p = 0.001; POD2: p < 0.013; POD3: p < 0.014; Figure 3D). On POD1-3, the water content in feces was 52.8, 48.9 and 51% in the iVNS group compared with 35.9, 33.8 and 33.4% in the sham-iVNS group (p = 0.01, p = 0.005, p = 0.001), respectively (Figure 3E). No diarrhea was observed in any of the rats. No correlation was noted between the food intake and number of fecal pellets (r = 0.256, p = 0.306) on POD3.

iVNS reduced postoperative pain assessed by the analysis of RGSs. There were significant differences in the RGSs at 15 and 30 min after surgery between iVNS and sham-iVNS (15 min: 0.83 ± 0.13 vs. 0.97 ± 0.15, p = 0.049; 30 min: 1.19 ± 0.21 vs. 1.44 ± 0.24, p = 0.032). These differences became more significant at 45 min to 120 min (45 min: 1.44 ± 0.13 vs. 1.86 ± 0.18, p = 0.004; 60 min: 1.56 ± 0.33 vs. 1.86 ± 0.18, p = 0.004; 90 min: 1.50 ± 0.25 vs. 1.83 ± 0.18, p = 0.005; 120 min: 1.17 ± 0.18 vs. 1.39 ± 0.13, p = 0.008). However, there were no significant differences between iVNS and sham-iVNS at post-operative time 150 min and 180 min (Figure 4).

iVNS improved gastric pace-making activity at 6 h after surgery: a higher percentage of normal slow waves was observed in the iVNS group compared to the sham-iVNS group (67.9 ± 12.7% vs. 52.2 ± 11.8%, p = 0.015, vs. sham-iVNS; Figure 5A). There was a decrease in the percentage of tackygastria and an increase in bradygastria with iVNS at the same time period. However, these differences were no longer significant at 24 h or 72 h after the surgery.

The DF in the iVNS group was significantly lower than that in the sham-iVNS group 6 h after surgery (4.33 ± 0.34 vs. 5.81 ± 0.89, p = 0.001). At 24 h and 72 h after the surgery, there were no significant differences in DF between iVNS and sham-iVNS (Figure 5B). There were no significant differences in dominant power at any post-operative periods between the two groups.

The concentrations of TNF-α, IL-1β, and IL-6 in plasma were increased after the surgery in both sham-iVNS and iVNS group, and this increase was reduced in the iVNS group compared to the sham-iVNS group (Table 1; TNF-α: 311.9 ± 48.9 vs. 392.9 ± 34.7, p = 0.001; IL-1β: 167.2 ± 28.4 vs. 196.5 ± 26.3, p = 0.037; IL-6: 152.9 ± 18.2 vs. 182.5 ± 16.4, p = 0.002; Figures 6A–C).

iVNS improved vagal activity that suppressed by the surgical procedure. iVNS increased vagal tone compared to sham-iVNS at 6 h (HF: 0.58 ± 0.12 vs. 0.38 ± 0.09, p = 0.001) and 24 h (HF: 0.57 ± 0.08 vs. 0.49 ± 0.05, p < 0.017) after the surgery (Figure 6A). Similarly, there was a decrease in LF/HF with iVNS at 6 h and 24 h after the surgery (Figure 6B). These effects were diminished as the vagal tone spontaneously recovered. Most interestingly, the iVNS seemed to be able to prevent the surgery-induced decrease in vagal tone as its value at 6 h was even higher than that at 72 h after the surgery.

The increased vagal tone was correlated with a faster postoperative recovery to start water and food intake; the correlation was stronger at earlier postoperative time periods. The vagal tone at 6 h after the surgery was negatively correlated with the first post-surgical time of water (r = −0.623, p = 0.006) and food intake (r = −0.594, p = 0.009). The vagal tone at 24 h after the surgery was moderately and negatively correlated with the first post-surgical time of water intake (r = −0.511, p = 0.03). No significant correlations were found at 48 and 72 h after the surgery (Figures 7, 8).

Increased vagal tone was also correlated with the increased number of fecal pellets; the correlation was stronger at earlier postoperative times. Vagal tone at 6 h after the surgery was positively correlated with the number of fecal pellets on POD1 (r = 0.552, p < 0.018) and POD2 (r = 0.482, p < 0.044). Vagal tone at 24 h after the surgery was positively correlated with the number of fecal pellets on POD1 (r = 0.572, p = 0.013) and POD3 (r = 0.477, p = 0.045). No significant correlations were found between the vagal tone at 48 or 72 h and the number of fecal pellets.

Interestingly, increased vagal tone was also correlated with the percentage of water content in the fecal pellets. Vagal tone at 6 h after the surgery was positively correlated with the percentage of water content in the fecal pellets on POD1 (r = 0.588, p < 0.01), POD2 (r = 0.528, p = 0.024) and POD3 (r = 0.669, p = 0.002).

Moreover, increased vagal tone was correlated with increased amount of food intake and the correlation was stronger at earlier postoperative times. Vagal tone at 6 h was positively correlated with the amount of food intake at POD1 and POD2 (POD1: r = 0.733, p = 0.001; POD2: r = 0.542, p = 0.02). Vagal tone at 24 h was positively correlated with the amount of food intake at POD1 and POD2 (POD1: HF: r = 0.506, p = 0.032; POD2: r = 0.635, p < 0.005). No significant correlations were found between food intake and vagal tone at 48 h or 72 h.

Increased vagal tone was correlated with the decreased level of inflammatory cytokines in plasma on the entire postoperative period (HF with TNF-α: r = −0.775, p = 0.001; HF with IL-1β: r = −0.856, p = 0.001; HF with IL-6: r = −0.888, p = 0.001).

The findings of this study suggest that brief intra-surgical vagal nerve stimulation may lead to an accelerated recovery from the surgical procedure. The abdominal surgical procedure may benefit most from iVNS due to its relatively higher prevalence of postoperative ileus. The surgical procedure with easy access to the vagus nerve is best suited for applying iVNS as no extra procedure would be required. Clinical studies are needed to prove the translatability of the animal findings to humans. However, several previous studies with the use of noninvasive auricular vagal nerve stimulation have demonstrated the promotility effect of vagal nerve stimulation. In one study, transcutaneous cervical vagal nerve stimulation applied a few minutes daily gastric emptying and symptoms of gastroparesis (Gottfried-Blackmore et al., 2020). In another study, daily transcutaneous auricular vagal nerve stimulation improved symptoms and gastric motility functions in patients with functional dyspepsia (Zhu et al., 2021). The similar method of transcutaneous auricular electrical stimulation also improved constipation in patients with irritable bowel syndrome (Yin et al., 2010; Shi et al., 2021). Both of these studies demonstrated improvement in vagal activity with transcutaneous auricular vagal nerve stimulation (Shi et al., 2021; Zhu et al., 2021).

In summary, brief intra-surgical vagal nerve stimulation accelerates postoperative recovery reflected as a shortened time for the first defecation and increased food intake. The accelerative effect might be attributed to improved gastrointestinal motility mediated via the enhancement of vagal activity.