The study was conducted under the guidelines approved by Institutional Animal Care and Use Committee of the Huazhong Agricultural University, Wuhan, China and adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain. (Permit number: HZAUGO-2016-005).

Thirty 1-year-old hybrid male goats, weighing 20 ± 2 kg, were randomly allocated into 4 treatment groups: Saline (n = 9), TNBS (n = 9), TNBS + sham EA (Sham, n = 6), and TNBS + EA (EA, n = 6) groups. All goats were purchased from the Hubei Agricultural Academy of Science Animals, were fed by a dry grass diet supplemented with a cereal-based concentrate, and drank freely. They were dewormed and acclimatized to the surrounding for 2 weeks before the initiation of the study. The experiment was performed in a quiet environment, with a temperature of 20–25°C.

Visceral hypersensitivity (VH) was established with the method described by Hassan et al. (2015). Briefly, the goats were kept off feed to avoid regurgitation and respiratory complications for 24 h prior to the experiment. Baseline cardinal parameters like respiratory rates, pulse rates and body temperature were recorded. The goats were intramuscularly (IM) injected with atropine (0.03 mg/kg, Tianjin Pharmaceutical Group Xinzheng Co., Ltd, China), etamsylate (0.02 g/kg, Shandong Fangming Pharmaceutical Co., Ltd, China) and xylazole (0.1 mg/kg, Hebei Gaocheng Sihai Pharmaceutical Industry Co., Ltd, China) before the anesthesia. Goats were placed on left lateral recumbency. Anesthesia was induced by intravenous (IV) administration of ketamine HCl (0.5 mg/kg/min; Yao Pharma Co., Ltd, China). The anesthetic depth was judged according to eyelid reflex, obtunded corneal reflexes and painful response to the prickle of the abdomen skin and coronary hooves. The trachea was intubated to avoid aspiration of ruminal contents. Lidocaine 2% (Shandong Hualu Pharmaceutical Co., Ltd, China) was used by local infiltration anesthesia. During the surgery, IV infusion (rate 0.05 mg/kg/min) of ketamine was conducted when the goats moved or struggled and until they entered a shallow anesthetic state. Following aseptic surgical procedure, a 6-cm abdominal incision was opened in the right flank to locate the ileum, and its terminal part was exteriorized gently on moist sterile gauze. For TNBS group, 1.2 ml of TNBS (Sigma Aldrich, USA) solution (30 mg TNBS dissolved in 40% ethanol) was injected into the wall of the ileum via five points with a 30 gauge needle, approximately 15 cm proximal to the ileocecal junction. In the case of saline group, the same volume of normal saline (0.9% NaCl) was injected in the same manner as in the TNBS group. The intestine was then returned to the abdominal cavity, the abdominal wall and peritoneum were closed with cat gut sutures, and the skin was closed with silk sutures. The goats were housed individually and monitored during the recovery from anesthesia (1–2 h). To prevent the pain, the goats were given tramadol (5 mg/kg, IM, Hubei Qianjiang Pharmaceutical Co., Ltd, China). The post-operative condition of the goats was monitored at least twice daily. The wound was treated with iodophor (Wuhan Xuehuan Sterilization Goods Co., Ltd, China) and erythromycin ointment (North China pharmaceutical Co., Ltd, China) daily till completely healed. The scheme of experiment is illustrated in Figure 1.

Symptoms such as water/food consumption, mortality, stool consistency, and bloody feces were observed during the experiment. Body weight was recorded at day 0, 7, and 22. Three goats from saline group and another three from TNBS group were taken for a similar laparotomy on day 7. Six-Centimeter terminal ileum of each goat was excised 15 cm proximal to the ileocecal junction, flushed with phosphate buffered saline for detection the macroscopic changes. Macroscopic lesions were scored by two independent observers who were unaware of the treatments according to a scale of 0–10 scores described by Hassan et al. (2015). After that, a 2 × 2 cm tissue block was taken from the approximate part of the ileum segment opposite to the mesentery, and fixed in 4% buffer formaldehyde for hematoxylin-eosin (HE) staining. Another ileal tissue (2 × 2 cm) was weighed, frozen in liquid nitrogen and finally transferred to store in −80°C for measuring the myeloperoxidase (MPO) concentration. Immediately, an end-to-end anastomosis was conducted and the abdomen was closed using standard methods. These six goats received tramadol hydrochloride (5 mg/kg, IM) and ampicillin (20 mg/kg, IM) for 5 days. After two-week recovery, the animals were sent to a goat farm.

Ileal tissues fixed in 4% buffer formaldehyde were embedded in paraffin. The tissue block was sectioned at a thickness of 5 μm, and mounted on poly-lysine coated slides. Three serial slides were deparaffined, rehydrated sequentially and stained with hematoxylin and eosin. The slides were visualized with the images under a light microscope (Nikon ECLIPSE 80I, Nikon Corporation, and Tokyo, Japan) with a 10 × and 40 × objective lens. Microscopic changes were blindly assessed by two investigators using a scoring system 0–10 as previously described (Hassan et al., 2015). Another ileal tissue (2 × 2 cm) was grinded and homogenized in 1 ml PBS, pH 7.2 at 4°C. The solution was centrifuged at 5,000 g at 4°C for 10 min. The protein concentration of the supernatant was determined using Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc., USA). The MPO concentration was assayed with MPO ELISA Kit (eBioscience, Inc., San Diego, CA 92121, USA) according to the manufacturer's instruments. Each sample was analyzed in triplicate and the values were presented as pg/mg.

Electroacupuncture (EA) treatment started on day 7, then every 3 days for total 6 times according to the method described by Wang et al. (2002). For EA treatment, the set of bilateral Housanli acupoints (equal to Zusanli in humans) was selected for EA groups. Here the acupoints are nominated with Pinyin Naming System instead of the Meridian Numbering System because animal's meridians are not completely recorded. The anatomic location and use of these points have been described in detail in veterinary medicine (Qiu et al., 2015; Hu et al., 2016). Prior to the EA treatment, goats were restrained. The acupoint sites were shaved and disinfected with 75% ethanol. Needles were disinfected and inserted into the points by a skilled acupuncturist. A pair of stainless steel needles was perpendicularly (0.40 mm in diameter and 50 mm in length) inserted at a depth of 20–30 mm into bilaterally Housanli acupoints (in the muscular groove between the long digital extensor and the lateral digital extensor muscles below the head of the fibula on the lateral surface) of each hind leg. The needles in set of EA group were connected by a pair of wires to one output of WQ-6F Electronic Acupunctoscope (Beijing Xindonghua Electronic Instrument Co., Ltd., China). Stimulation with constant wave of 60 Hz (approximate 3.2 V) was applied and lasted for 30 min each time according to Cheng et al.'s method (Cheng et al., 2012). The goats in the other three groups were restrained in the same manner as the EA-treated goats. Sham goats underwent needle placement without electric stimulation.

Visceral hypersensitivity (VH) was reflected by EMG and pain response score. EMG was performed to record VMR to CRD of goats immediately after termination of EA on day 7, 10, 13, 16, 19, and 22 after induction of ileitis as described previously (Hassan et al., 2015). Briefly, prior to EMG record, the goats were standing, two nickel-steel needles (0.45 mm in diameter and 5 cm in length) were inserted 2 cm apart into external oblique muscles at the center of the left abdomen as electrodes, both electrodes were clamped and connected to an EMG recorder (Nanjing Medease Science and Technology Co., Ltd, China). EMG was amplified and filtered with a processing system MedLab-U/4C501H (Nanjing Medease Science and Technology Co., Ltd, China). A manual distension device was made with a T-connector connecting a balloon, a vacuum pump and a sphygmomanometer (Figure 6H). The polyethylene balloon (12 cm) was lubricated and inserted into the distal colon 10 cm from the anus of goats. After goats were acclimatized for 10 min, the balloon was inflated with the pump. The pressure in the balloon was measured with the sphygmomanometer. It continuously increased from 20 to 40, 60, 80, and 100 mmHg by stage and lasted for 6 s at each stage. EMG was recorded during each stage. This procedure was repeated every 5 min for 3 times. The area under curve (AUC) was calculated from EMG data with MedLabV6.3 software (Nanjing medease science and technology Co., Ltd, China) and expressed as millivolt multiply second (mv^*s).

Pain response scores were assessed through monitoring pain behavioral responses to CRD. The modified pain behavioral scale of 0–4 was used for this study (Habibullah et al., 2016). The behavior response to CRD distension was observed by two observers blinded to the experimental conditions. The pain behavior responses to different CRD distensions were assessed three times and averaged as pain scores.

Immediately after the last CRD, the goats were anesthetized with intravenously administered xylidinothiazoline at 3 mg/kg. The spinal cord of the eleventh thoracic vertibrae (T11) was removed very carefully and divided into two parts. The anterior part was obtained and stored in 10% formalin for immunohistochemistry (IHC) and the posterior part was weighed, frozen in liquid nitrogen and finally transferred to store in −80°C for western blotting and RT-PCR analysis. Then, the heads of the goats were taken and mounted onto a stereotaxic instrument (Tindal et al., 1968, 1987). The brain was taken out for observing IL-6, pJAK2 and pSTAT3 in the descending pain system (including ventrolateral PAG (vlPAG), RVM (mainly the nucleus raphe magnus, NRM) and SCDH), and nuclei related to visceral motion and sensory regulations (including the nucleus tractus solitaries (NTS) and the dorsal motor nucleus of vagi (DMV)) with IHC. RT-PCR and western blotting methods were not applied for brain tissues because these substances are not evenly distributed in the specific pain-regulated areas and their inaccurate sampling would result in homogenization. The brain was divided into 3 blocks (B1-B3) according to Qiu et al.'s (2015) and Hu et al.'s (2016) methods. The vertical interaural plane represented the zero reference point for the anterior-posterior coordinates, and the horizontal zero plane (H0) intersected the interaural point and a point 25 mm above the lower margin of the orbit. B1-B3 are from anterior 10 mm (A10) to anterior 5 mm (A5), posterior 10 mm (P10) to posterior 15 mm (P15), and posterior 15 mm (P15) to posterior 20 mm (P20), respectively (Figure 2). The locations of the nuclei or areas and the morphological characteristics of the neurons were identified according to the brain atlas of goats and pigs (Dean et al., 1999; Félix et al., 1999).

The blocks and T11 spinal cord were embedded in paraffin with their rostral surface facing up. Each of the blocks was consecutively sectioned at a thickness of 5 μm. Ten serial slides of each nucleus or area were mounted on polylysine coated slides, deparafnized, and rehydrated sequentially. Three of these 10 slides were incubated with rabbit-anti-IL-6 IgG (CST, Inc., USA, #12153, diluted 1:100 in PBS), rabbit anti-pJAK2 IgG (CST, Inc., USA, #3776, diluted 1:200 in PBS) or rabbit anti-pSTAT3 IgG (CST, Inc., USA, #9145, diluted 1:200 in PBS), respectively. One slide was incubated with PBS instead of the corresponding antibody as negative control. Experimental procedures of SABC immunohistochemistry followed the instructions provided by the reagent company (Boster biotech, Wuhan, China). The cytoplasm or nuclei of positive cells was stained as brown yellow. The locations of the observed nuclei (areas) with the representative stained cells are shown in Figure 3.

Optical images of the stained nuclei or area in the CNS were obtained under a light microscope (Nikon ECLIPSE 80I, Nikon Corporation, and Tokyo, Japan) connected to a video-based and computer-linked system (high-resolution pathological image analysis system 1000, Wuhan Qianping Ltd., Wuhan, China). Three fields of each nucleus or brain area on each side were observed with a 40× objective lens. The optical density values of the IL-6-like, pJAK2-like and pSTAT3-like immunoreactive cells were calculated with the Image-Pro plus 6.0 system (Media Cybernetics, Inc., Bethesda, MD, USA).

The posterior segment of T11 spinal dorsal horn was weighed, grinded in liquid nitrogen, then protein extracted from the grounded spinal cord using the RIPA buffer according to the manufacturer's instruction (Beyotime Biotech, Nantong, China). The protein concentration was measured by Nano Drop Spectrophotometer (Thermo Fisher Scientific, Inc., USA). Equal amounts of protein (40 μg) sample was loaded in 15% SDS polyacrylamide gel and transferred to a PVDF membrane using the Mini-PROTEIN Tetra Cell (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 2 h at the room temperature in 5% BSA and was subsequently immunolabeled overnight at 4°C with primary antibodies, rabbit anti-IL-6 IgG (1:500), rabbit anti-pJAK2 IgG (1:500), rabbit anti-pSTAT3 IgG (1:500) (CST, Inc., USA) or rabbit anti-beta-actin (1:300, Santa Cruz, CA, USA), respectively. The membrane was washed and treated with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (1:5,000, Boster biotech, Wuhan, China) for 1 h at the room temperature. Visualization of the antigen-antibody complex was conducted with a horseradish peroxidase substrate (Millipore, Billerica, MA, USA) using the ImageQuant LAS 4,000 min CCD camera (GE Healthcare, Piscataway Township, NJ, USA). The bands were analyzed by Quantity One software (Bio-Rad). Beta-actin was used as the internal control. Values of these substances were represented as the ratio of the optical density of the bands to the density of the related beta-actin band.

Total RNA was extracted from dorsal spinal cord of each group by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, cDNA was synthesized from 900 ng of total RNA using a First Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan). The primer sequences of IL-6, JAK2, STAT3 and GAPDH are shown in Table 1. RT-PCR was performed with Step One Plus™ Real-Time PCR System (Applied Biosystems, CA, USA) using SYBER Green RT-PCR kit (Takara Dalian, China). The mRNA of IL-6, JAK2 and STAT3 relative to GAPDH mRNA were quantified with the 2^−ΔCt method, where ΔCt = Ct _{target gene} - Ct _GAPDH.

All data were expressed as mean ± SD. Statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). The statistical comparisons for parametric data (body weight changes, MPO, VMR, IHC, RT-PCR and western blot) were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The statistical differences of non-parametric values (macroscopic scores, microscopic scores and pain behavior response) among groups were identified using Kruskal-Wallis ANOVA followed by the rank-based Mann-Whitney U-test. A difference was considered significant if P was less than 0.05.

The TNBS-treated goats showed inappetence, sluggishness, mucoid diarrhea and weight loss at day 7. The body weight of goats in TNBS, sham and EA groups was decreased (P < 0.05) as compared to goats in saline group at day 7. No difference in body weight was found among all groups at day 22 (Figure 4A).

The saline-treated ileums showed no any apparent histopathological changes at day 7 and 22. However, TNBS-treated ileums revealed light red color, sporadic adhered pseudo-membrane, localized necrosis and apparent wall thickening at day 7. No apparent lesions were observed in the adjacent visceral organs and tissues such as large intestines, jejunum and mesentery (Figure 4B). Macroscopic lesions in TNBS group were more severe (P < 0.05) than those in saline group at day 7. However, no difference in macroscopic lesions among all groups was observed at day 22 (Figure 4C). Microscopically, TNBS-treated ileal wall showed extensive infiltration of neutrophils and lymphocytes in lamina propria, apparent submucosal and muscular layer ulceration and blood vessel congestion at day 7, and moderate inflammatory cell infiltration and granuloma in the submucosa and muscular layer at day 22. TNBS-treated goats showed severe (P < 0.05) microscopic lesions as compared with saline-treated goats at day 7 and 22. Sham or TNBS-treated goats showed severe (P < 0.05) microscopic lesions as compared with EA-treated goats at day 22. No difference in microscopic lesions between saline and EA group was observed at day 22 (Figure 5). TNBS-treated goats showed elevated (P < 0.05) MPO concentration as compared with saline-treated goats at day 7. However, no difference in the MPO concentration was observed among all groups at day 22 (Figure 4D).

The EMG of abdominal muscles for assessment of VH is shown in Figure 6. The VMR was elevated with CRD stimulation intensity increasing. Compared to the goats in saline group, the goats in TNBS or sham group showed higher (P < 0.05) VMR to 20–100 mmHg distension pressures at day 7–22. Compared with saline-treated goats, EA-treated goats showed increased (P < 0.05) VMR to 40–100 mmHg distension pressures at day 7, to 20–100 mmHg distension pressures at day 10 and 13, and to 60–100 mmHg distension pressures at day 16. There was no difference in VMR between TNBS-treated and sham-treated goats during the experiment. Compared with sham or TNBS-treated goats, EA-treated goats showed decreased (P < 0.05) VMR to 20 mmHg distension pressure at day 7, to 80-100 mmHg distension pressures at day 10, to 40–100 mmHg distension pressures at day 13, and to 20–100 mmHg distension pressures at day 16–22.

The experimental goats showed distinct signs such as restlessness, rapid breathing, guarding, tail wagging, curling of the lips, neck movement and change in posture when distension was applied. Compared with the saline-treated goats, Sham or TNBS-treated goats exhibited apparently increased (P < 0.05) behavioral responses to 20–100 mmHg distention pressures at day 7–22. EA-treated goats showed higher (P < 0.05) pain response scores than saline-treated goats to 20–100 mmHg distention pressures at day 7 and 10, and to 40–100 mmHg distention pressures at day 13 and 16. There was no difference (P > 0.05) in pain response scores to all distention pressures between TNBS and sham group during the experiment. Goats in EA group showed lower (P < 0.05) pain response scores than goats in TNBS group to 60–100 mmHg distention pressures at day 10, to 40–100 mmHg distention pressures at day 13–19, and to 20–100 mmHg distention pressures at day 22 (Figure 7).

The changes in IL-6, pJAK2 and pSTAT3 immunoreactivity-like cells were observed in vlPAG, NRM, NTS, DMV and SCDH. Their representative distributions are displayed in Figure 3. The expressions of IL-6, pJAK2 and pSTAT3 in the different treatments are shown in Figures 8B–D. Compared with saline-treated goats, TNBS-treated goats showed an increased (P < 0.05) expression in IL-6, pJAK2 and pSTAT3. Compared with the goats in saline group, the goats in EA group showed enhanced pJAK2 and pSTAT3 in vlPAG, NRM and NTS. There was no difference in the expression of these three substances between sham-treated and TNBS-treated goats. Compared with sham or TNBS-treated goats, EA-treated goats showed decreased (P < 0.05) IL-6, pJAK2 and pSTAT3 in the measured nuclei or areas except that in DMV where no change in pSTAT3 was observed.

Compared with saline-treated goats, TNBS-treated goats showed increased (P <0.05) gene expression of IL-6, JAK2 and STAT3. There was no difference in the gene expression of IL-6, JAK2 and STAT3 among sham-treated, TNBS-treated and EA-treated goats (Figure 8A).

The protein level of IL-6, pJAK2 and pSTAT3 in the spinal cord are shown in Figure 9. Compared with saline-treated goats, TNBS-treated goats showed increased IL-6, pJAK2 and pSTAT3. There was no difference in the three proteins between saline-treated and EA-treated goats. IL-6, pJAK2 and pSTAT3 of EA goats were decreased compared with those of TNBS or Sham goats. There was no difference in the three proteins between TNBS and Sham goats.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.