Fluoro Ruby (FR) and Fluoro Gold (FG) were purchased from Molecular Probes (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Fluorochrome LLC (Denver, CO, USA), respectively. Silastic tubes were purchased from Shanghai Daoguan Rubber Products (Shanghai, China). Polymerase chain reaction (PCR) primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc). Rabbit polyclonal anti-TGF-β1 (cat. no. sc-146) and rabbit polyclonal anti-CTGF antibodies (cat. no. sc-25440) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. 2251-1) and rabbit polyclonal anti-collagen I (cat. no. ab34710) antibodies were purchased from Epitomics, Inc. (Burlingham, CA, USA) and Abcam (Cambridge, UK), respectively. Rabbit monoclonal anti-phosphorylated (p-)ERK (cat. no. 4370) and rabbit monoclonal anti-p-p38 (cat. no. 4511) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal anti-p-c-Jun N-terminal kinase (JNK; ab4821) antibody was purchased from Abcam. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. 074-1506) was purchased from KPL, Inc. (Gaithersburg, MD, USA). Secondary antibodies, including AlexaFluor 488-conjugated goat anti-rabbit IgG (cat. no. ANT030) and AlexaFluor 594-conjugated goat anti-rabbit IgG (cat. no. ANT051) antibodies, were purchased from Antgene Biotech (Wuhan, China).

A total of 100 male Sprague-Dawley rats (age, 7–8 weeks; weight, 250–300 g) were purchased from the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) and housed at 22°C with a 12 h light/dark cycle. Each animal had ad libitum access to food and water. All experimental procedures and protocols were approved by the Board of Ethics of Tongji Medical College, Huazhong University of Science and Technology.

The chronic sciatic nerve compression model was established in accordance with the method described by O'Brien et al (19). The animals were anesthetized using pentobarbital (40 mg/kg intraperitoneal injection) and the right sciatic nerve of the rat was exposed through a gluteal muscle splitting approach. In the model rat group (n=8), a 1 cm-long Silastic tube (internal diameter 1 mm; outer diameter 2 mm; Shanghai Daoguan Rubber Products Factory, Shanghai, China) was cut longitudinally, and placed atraumatically around the sciatic nerve. The longitudinal split in the tube was then closed using 8–0 sutures (Shanghai Pudong Jinhuan Medical Product Co., Ltd., Shanghai, China). The left sciatic nerve was exposed, as above, but remained unbanded and was returned to its original position to serve as a comparative control. In the sham-operated group (n=8), the right sciatic nerves were exposed, using the same procedure as described above for the left sciatic nerve of the model rats, but without Silastic tube placement.

Subsequently, 3 weeks following the above procedures, the ipsilateral and contralateral L4–L6 dorsal root ganglia (DRG) were harvested from the above experimental groups (n=8 for each group). At 3 or 8 weeks after the establishment of the animal model, rats were sacrificed using an overdose of phenobarbital (Wuhan Boster Biological Technology, Ltd., Wuhan, China), and the ipsilateral and contralateral gastrocnemius muscles were collected from the above experimental groups (n=8 for each group).

FR and FG are fluorescent tracers used for the retrograde labeling of afferent neurons in rodents (20). At 2 weeks post-surgery, the rats were anesthetized. The right sciatic nerves were exposed again, and the FR and FG fluorescent tracers were injected intraneurally. In the rats in the chronic sciatic nerve compression model, 5 µl FR (5% dissolved in normal saline) was injected into the proximal end of the compressed nerve, and 5 µl FG (5% dissolved in normal saline) was injected into the distal nerve. In the sham control rats, 5 µl 5% FR and 5 µl 5% FG were injected into the same respective sites as in the model rats. Following injection, the muscle and incision were closed.

At 1 week post-fluorescent tracer injection, the ipsilateral DRG of L4, L5 and L6 were removed and fixed in 4% paraformaldehyde (Wuhan Boster Biological Technology, Ltd.). The DRGs were then dehydrated, embedded in optimal cutting temperature compound (Applygen Technologies Inc., Beijing, China) and sectioned coronally using a microtome (Leica CM1510S; Leica Microsystems, Wetzlar, Germany). The fluorescence was examined under a fluorescent microscope (Olympus IX71; Olympus Corporation, Tokyo Japan), and the numbers of FR- and FG-positive neurons were analyzed using ImagePro Plus version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The ipsilateral and contralateral gastrocnemius muscles were fixed and embedded in paraffin (Wuhan Boster Biological Technology, Ltd.). The gastrocnemius muscles were stained with Masson's trichrome (Harris hematoxylin, 1% hydrochloric-alcohol solution, acid fuchsin, 1% phosphomolybdic acid and 2% aniline blue). The morphological details of Masson's trichrome staining were examined using a light microscope (CKX41; Olympus Corporation). A total of six images, each from the ipsilateral and contralateral gastrocnemius muscles, were randomly selected to measure the average muscle fiber cross-sectional area (CSA) and collagen volume fraction (CVF). The CVF was calculated as follows: CVF (%) = (collagen area / total area) x 100 in muscle fiber CSA.

At 3 weeks following the establishment of the chronic sciatic nerve compression model, the contralateral and ipsilateral L4–L6 DRG tissues were removed. Total RNA was extracted from the homogenized tissue samples using TRIzol reagent, chloroform and isopropyl alcohol (Wuhan Boster Biological Technology, Ltd.). After centrifugation at 7,500 × g at 4°C for 5 min, samples were collected and dried at room temperature. The total RNA was resolved in RNA-free water. The samples were reverse-transcribed using a First Strand cDNA Synthesis kit (Toboyo Co., Ltd., Osaka, Japan). The RT-qPCR was performed using a sequence detection system (StepOne Real-Time PCR System; Thermo Fisher Scientific, Inc.). PCR amplification was performed using Thunderbird SYBR qPCR mix (12.5 µl; Toboyo Co., Ltd.) with 2 µl specific primers (2.5 µM; Table I), 2 µl cDNA and 8.5 µl double distilled water. The PCR reactions were as follows: Pre-denaturation at 95°C for 1 min, denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 20 sec (for a total of 40 cycles). β-actin was used as the internal control. The relative expression levels from the amplified RNA samples were calculated using the 2^−ΔΔCq method (21).

At 3 weeks following the establishment of the chronic sciatic nerve compression model, the contra-lateral and ipsilateral L4–L6 DRG tissues were removed, embedded in paraffin and sectioned. The samples were blocked with normal goat serum (Wuhan Boster Biological Technology, Ltd.) and probed with anti-TGF-β1 (1:50 dilution), anti-CTGF (1:50 dilution) or anti-collagen I (1:200 dilution) at 4°C overnight. The following day, the slides were washed three times with phosphate-buffered saline and incubated with secondary antibody at 4°C overnight, then washed with Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20). Following washing, the nuclei were counterstained with DAPI (100 µg/ml; Wuhan Boster Biological Technology, Ltd.), and samples were mounted in antifade reagents (Beyotime Institute of Biotechnology, Haimen, China). The fluorescence was examined under a fluorescent microscope (Olympus IX71; Olympus Corporation).

At 3 weeks following establishment of the chronic sciatic nerve compression model, the contralateral and ipsilateral DRG tissues were removed. Total proteins were extracted from tissues using radioimmunoprecipitation assay lysis buffer containing 1% phenylmethanesulfonyl fluoride and phosphatase inhibitor (Wuhan Boster Biological Technology, Ltd.), and lysed on ice for 30 min. After centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was collected and stored at −80°C until used. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) in accordance with the manufacturer's protocol. The samples were resolved using either 10% SDS-PAGE (for CTGF, collagen I and GAPDH; Wuhan Boster Biological Technology, Ltd.) or 15% (for TGF-β1) SDS-PAGE, followed by transfer onto a polyvinylidene fluoride membrane (eBioscience, Inc., San Diego, CA, USA), and blocking with 5% w/v non-fat dry milk. The membranes were then incubated with primary antibodies at 4°C overnight as follows: Anti-TGF-β1 (1:500 dilution), anti-CTGF (1:1,000 dilution), anti-collagen I (1:5,000 dilution), anti-GAPDH (1:10,000 dilution), anti-p-ERK (1:1,000 dilution), anti-p-p38 (1:1,000 dilution) and anti-p-JNK (1:1,000 dilution). Following washing, the membranes were incubated with HRP-labeled secondary antibodies (1:10,000 dilution) at 4°C overnight, then washed with TBS-T (0.1% Tween-20).

The resulting immunobands were visualized using an Enhanced Chemiluminescence kit (EMD Millipore, Billerica, MA, USA), and the densitometric values were used for statistical analysis. The housekeeping protein, GAPDH, was used as an internal control.

The data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. Statistical significance between two groups was determined using Student's t-test.

The present study examined the pathological changes in the rat chronic sciatic nerve compression model. At 3 weeks and 8 weeks post-surgery, Masson's trichrome staining showed normal gastrocnemius muscle fiber structure and collagen fiber morphology on the contralateral side. However, at 3 weeks, collagen fiber accumulation was detected on the ipsilateral side and, at 8 weeks, excessive collagen fiber formation and muscle atrophy were observed (Fig. 1A).

The chronic sciatic nerve compression in the model group was associated with lower levels of gastrocnemius muscle fiber CSA ag 3 weeks post-surgery, which was significantly lower at 8 weeks, compared with the contralateral side (Fig. 1B). In the model rats, the percentage of gastrocnemius muscle fiber CVF was significantly higher on the injured side, compared with the contralateral side at 3 weeks, and this difference was more marked at 8 weeks (Fig. 1C). In the subsequent experiments, the molecular and cellular changes in the DRG neurons 3 weeks following nerve compression.

In the present study, the FR-labeled neurons exhibited red fluorescence, predominantly in the cytoplasm, and the FG-labeled neurons appeared bright blue under the fluorescent microscope (Fig. 2). The majority of the DRG neurons were stained with FG, and no significant difference was detected in the number of FG-labeled neurons between the sham control group and the group of rats with compression on the injured side (P>0.05).

By contrast, the number of FR-labeled DRG neurons was significantly higher in the rats with chronic sciatic nerve compression, compared with the sham control group (P<0.05). Few neurons were stained with both FR and FG. No significant differences were observed in the total number of neurons labeled with FR, FG or the two together, between the two groups (P>0.05).

In the model rats, the present study found that the mRNA expression levels of TGF-β1, CTGF and collagen type I were significantly higher in the ipsilateral DRG neurons, compared with the contralateral DRG neurons (P<0.05; Fig. 3A). Consistent with this, the protein levels of TGF-β1, CTGF and collagen type I were also higher in the ipsilateral DRG neurons and surrounding tissues, compared with the those on the contralateral side (P<0.05; Fig. 3B and C).

Immunohistochemical analysis showed low levels of expression of TGF-β1, CTGF and collagen type I in the contra-lateral DRG neurons and surrounding tissues, whereas their levels of expression were higher in the ipsilateral DRG neurons and surrounding tissues (Fig. 3D). Additionally, TGF-β1 and CTGF were predominantly distributed in the cytoplasm and axons of the DRG neurons, and collagen type I was expressed in the tissues surrounding the neurons.

Activation of the three major MAPK members, ERK1/2, JNK and p38 MAPK is known to modulate the pathogenesis of fibrosis in multiple organs (22–24). In the present study, immunoblotting revealed that, in the model rats, the protein levels of p-ERK1/2 were higher in the ipsilateral DRGs following chronic nerve compression, compared with the levels in the contralateral DRGs (P<0.05; Fig. 4). By contrast, chronic nerve compression was associated with significantly lower levels of p-JNK and p-p38 in the DRGs (P<0.05).