All experiments were performed according to the regulations of The Committee for the Ethical Use of Laboratory Animal of Xuzhou Medical College. Male Sprague-Dawley rats provided by the Experimental Animal Center of Xuzhou Medical College (license number, SYXK 2002–0038) were 8–9 weeks old and weighed 250–300 g. The rats were randomly assigned into three groups, each containing six animals. The rats in the visceral pain group were subjected to lower colon instillation of 5% formalin; in the sham group, rats received instillation of saline; in the RP67580 group, a specific NK1R antagonist was injected into the lateral ventricle (LV) of rats 24 h prior to visceral pain induction. All animals were placed in separate rooms, in which conditions and light were controlled (23±1°C; 12/12 h dark/light cycle with light on at 8:00 a.m.), and food and water were available ad libitum.

In the animal experiments, visceral pain was induced by rectal infusion with dilute formalin. In order to allow the rats to regain consciousness as soon as possible, a small quantity of halothane (induction at 3%, then 1.5% in a mixture of 2:3 nitrous oxide and 1:3 oxygen) was used. While under anesthesia, the animal was suspended by its tail (<5 min). Subsequently, a polyethylene tube, which was wrapped in surgical tape at ~30 mm from the edge, was inserted through the anus. A 5% formalin solution or saline (100 µl) was injected through the tube slowly to prevent leakage (15).

All animals were placed in separate observation boxes for adaptation 30 min prior to the behavioral assessments. On regaining consciousness, the rats were observed for 3 h to record pain-associated behaviors. The visceral pain-associated behaviors included licking of the abdomen (A), back stretching (B), contraction of flanks (C), and contraction of whole body (D). Based on the duration of the contraction, the contraction of the whole body was divided into three levels of <30 sec (W1), 30–60 sec (W2) and >1 min (W3). Using the following formula, the visceral pain score (PS) was calculated: PS=L+2B+3C+4W1+5W2+6W3 (16).

The animals were placed in grid-bottomed cages for 30 min for adaption prior to the evaluation of allodynia. Subsequently, to determine the hyperalgesia of the referred area, von Frey hairs (VFHs; Semmes-Weinstein Monofilaments, North Coast Medical, Inc., San Jose, CA, USA) were used. On stimulation of the abdomen with increasing force (0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10 and 15 g), the rats showed withdrawal responses, which included sharp abdominal retraction, licking or scratching the site of application of the hair and jumping with all four paws off the floor (17). Each VFH test lasted for 6 sec, or until a withdrawal response was observed. Once a withdrawal reaction occurred, the next descending force of VFH was applied to retest the area where the former VFH was placed until no response was detected. The minimum force was recorded as the abdomen withdrawal threshold in grams. If the result of allodynia was ≤2 g VFH, it was considered innocuous to the rats.

The animals were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). When immobilized in stereotaxic instrument, the rat was injected with 3 µl of 30% CB-HRP (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) into the LV according to stereotaxic coordinates (Brega, −1.2±0.4 mm; depth, 3.2±0.4 mm; right to median sagittal plane, 1.4±0.2 mm) 48 h prior to the behavioral assessments. The NK1R antagonist, RP67580 (Tocris Biosciences, Bristol, United Kingdom) was dissolved in dimethyl sulfoxide, and 100 pmol was injected into the LV of rats in the RP67580 group 24 h prior to the behavioral assessments.

Following the intracerbroventricular injections, the rats were administered with the formalin instillation and visceral pain behavioral assessments were performed. Subsequently, the rats were deeply anesthetized by injection of pentobarbital sodium (50 mg/kg, i.p.) and then successively perfused with 150 ml of phosphate buffered saline (PBS; 0.01 M; pH 7.4) and 4% paraformaldehyde. Following isolation and overnight fixation, the brainstem was immersed in 30% sucrose solution for 2 days. On a cryostat microtome (Leica CM1900; Leica Microsystems GmbH, Wetzlar, Germany), all tissues were successively cut into 35 µm slices in the transverse plane. The sections were then incubated in donkey serum (1:100 dilution; cat. no. 566460; Merck KGaA) for 1 h at 37°C, following which the sections were treated with goat anti-CB (1:400 dilution; cat. no. 227040; Merck KGaA) and anti-rabbit NK1R (1:100 dilution; cat. no. BA3678; Wuhan Boster Biological Technology Ltd., Wuhan, China). Following reaction with goat anti-IgG coupled to Alexa 546 (1:400 dilution; cat no. A11056; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and rabbit anti-IgG coupled to Alexa 488 (1:400 dilution; cat. no. A21206; Thermo Fisher Scientific, Inc.), the slices were thoroughly rinsed three times with 0.01 mol/ml PBS at the end of each step. The tissue sections were transferred onto slides and allowed to air dry, prior to sealing with glycerol. Finally, ultrathin sections (75×25×1 mm) were prepared and examined under a laser-scanning confocal microscope (TCS SP2; Leica Microsystems). Using Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA), images were captured, cropped and adjusted.

Following the behavior assessments, the rats were sacrificed and the CSF-CN region of rat brain was removed and stored at −80°C. The tissues were homogenized by an electric homogenizer for 20 sec at room temperature. Following centrifugation at 13,523 × g for 15 min at 4°C, supernatant collection and denaturation, the lysates were electrophoresed on 8% SDS-polyacrylamide gel. According to the analysis, the semi-dry western blot transfer method was used, and the polyvinylidene difluoride membranes were blocked with 5% skim milk for 1 h at room temperature. The membranes were then incubated with anti-rabbit NK1R (1:500 dilution; cat. no. BA3678; Wuhan Boster Biological Technology Ltd.) and anti-rabbit GAPDH (1:5,000 dilution; cat. no. sc-25778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. The membranes were washed with TBS-Tween 3 times and incubated with secondary anti-rabbit IgG antibody (1:1,000 dilution; cat. no. A0545; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. The densities of the bands were analyzed using ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD, USA).

Quantitative immunohistochemistry was used to assess the expression of NK1R in the CSF-CN. Image analysis software (Image-Pro Plus Version 6.0; Media Cybernetics, Inc.) was used for digital image analysis. On examination of every tissue section, details were recorded on the number, area and density of immunoreactive cells. Dual labeling of neurons with CB-HRP/NK1R enabled counting via a yellow fluorescence reaction, with areas between 40 and 1,000 µm². Data are expressed as the mean ± standard deviation and were imported into Microsoft Excel 2003 (Microsoft Corporation, Redmond, WA, USA). Using SPSS v.13.0 (SPSS, Inc., Chicago, IL, USA), all data were analyzed using one-way analysis of variance or Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Prior to surgery, the PS of visceral pain behavior at baseline was determined in untreated rats, which was 0.8±0.7. Following formalin instillation, a series of discrete behavioral episodes were observed, including licking of the upper abdomen, stretching of the whole body and arching of the back against the floor. Compared with the PS of rats in the sham group, the PS of rats in the visceral pain group presented with a typical biphasic time course. The first peak occurred at ~15 min post-instillation (PS=87.8±5.4), which was 15 times higher, compared with that in the saline instillation group (P<0.05). The second PS peak occurred at 60 min post-instillation (PS=36.7±3.3), which was 12 times higher, compared with that of the sham group (P<0.05). The PS of the RP67580 group was significantly decreased at 60 min post-formalin instillation, compared with that of the visceral pain group (P<0.05; Fig. 1A).

In the present study, the von Frey assessment was used to measure the abdomen withdrawal threshold. The values of the abdomen withdrawal threshold in rats of the visceral pain group were significantly lower 60 min post-formalin instillation, compared with that of the sham group (P<0.05). The values were significantly upregulated in rats pretreated with RP67580 (P<0.05; Fig. 1B).

The CSF-CN was first identified by injecting CB-HRP, as a fluorescent tracer, into the LV of the rats, which was used as a reliable method to label CSF-CN. As shown in the images in Fig. 2, the neurons labeled by CB-HRP were predominantly located around the midline of the aqueduct midbrain and ventral periaqueductal gray matter of the brainstem. In order to determine whether the CSF-CN expressed NK1R, a double-labeled immunofluorescence technique was used. The results indicated that NK1R-immunoreactive neurons (green) were distributed in the CSF-CN. The total CSF-CN sample of the visceral pain group did not differ from that of the sham group (P>0.05). The number of simple labeled NK1R-containing neurons counted in the visceral pain group was significantly higher, compared with that in the sham group (P<0.05). Compared with the sham group, then number of dual-labeled CB-HRP/NK1R neurons counted in the visceral group was significantly upregulated (P<0.05; Fig. 2; Table I).

Based on the above-mentioned findings, the present study investigated the role of NK1R in visceral pain. Western blot analysis was used to examine the protein expression levels of NK1R, the result of which indicated that NK1R was upregulated 60 min following formalin instillation, compared with that of the sham group (P<0.05).

Following LV injection of RP67580, the rats experienced significant relief from pain on formalin instillation, which suggested that NK1R in the CSF-CN may be associated with the alleviation of visceral pain. Western blot analysis was performed to observe the expression of NK1R 24 h following the injection of RP67580. Compared with the rats in the visceral pain group, there was a significant downregulation in the expression of NK1 in the rats pretreated with RP67580 (P<0.05; Fig. 3).