A total of 116 male Sprague-Dawley rats (age, 8–10 weeks; weight, 180–250 g) were obtained from the Experimental Animal Center of Jiangsu University (Zhenjiang, China) and acclimatized to the environment for at least 1 week prior to use in the experiments. The environment was maintained at a constant room temperature and humidity level (22°C; relative humidity, 40–60%). All rats were housed with access to food and water ad libitum and maintained in a 12:12 h light/dark cycle. The well-being of the animals was monitored daily. All animal experiments were approved by the Animal Care and Ethics Committee of Jiangsu University.

Surgery was performed according to the method described previously (24). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and a longitudinal incision overlying the lumbar (L)4-sacral (S)1 section was made and the left muscle tissue was separated. The L6 transverse process and L6/S1 posterior interarticular process were exposed and transected. The L5 and L6 spinal nerve was carefully exposed and the distal nerve of L5 and L6 were gently separated, firmly ligated with 5–0 silk suture and transected. The wound was irrigated with sterile saline and inspected for homeostasis, and closed. Sham surgery was performed identically, however, the nerves were not ligated. The animals were allowed to emerge from anesthesia in the warming place. The naive group did not undergo an operation.

The animals were habituated to the behavioral testing apparatus for 30 min to 1 h prior to testing. The testing environment was kept quiet and all tests were conducted in the morning between 8:30 and 11:30. Mechanical sensitivity of the rats' left feet was measured at 24 h prior to (baseline value) and at days 1, 3, 5, 7, 10 and 14 after surgery. The midplantar surface of the rats' left hind paws was stimulated using one of a series of eight von Frey filaments (Stoelting Co., Wood Dale, IL, USA) ranging from 0.4 to 15.0 g (log force 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93, 5.18) using the Up-Down methods (25). The von Frey filament was presented perpendicular to the plantar surface with sufficient force to result in slight buckling against the paw for ≤8 sec. Paw withdrawal was considered a positive response. The 50% mechanical withdrawal threshold (50% MWT) was calculated. If the value of 50% MWT exceeded 4.0 g at day 7 post-surgery, the animal model was considered a failed preparation and excluded from the experiment. A total of 8 rats were included in each group. The person performing the behavioral tests was blinded to the experimental groups.

Rats were deeply anesthetized by inhalation of isoflurane (4%) in 100% O₂ and intracardially perfused with 0.9% saline (between 200 and 250 ml per rat at 4°C), and the spinal cords were subsequently harvested. The lumbar spinal dorsal horns from the L5 to L6 ipsilateral to injury were dissected and rapidly frozen. The tissues were homogenized and total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In the in vitro experiment, total RNA was extracted from astrocytes with 100 ng/ml IL-17 (R&D Systems, Inc., Minneapolis, MN, USA) treatment for three days or without. Interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and/or IL-17 mRNA were measured by RT-qPCR. RT-qPCR was performed with the Verso 1-step RT-qPCR kit, SYBR Green, low ROX (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and the thermocycling conditions were as follows: 5 min at 94°C; followed by 35 cycles of 10 sec at 94°C, 5 sec at 50°C, and 10 sec at 72°C. Relative expression was calculated with normalization to β-actin values (26). The primers used are presented in Table I.

Rats were deeply anesthetized by inhalation of isoflurane (4%) in 100% O₂ and intracardially perfused with 0.9% saline (between 200 and 250 ml per rat at 4°C), followed by 4% formaldehyde in 0.1 M phosphate-buffered saline (PBS; between 300 and 350 ml per rat). The L5 segment of lumbar spinal cord was harvested, postfixed overnight in 4% formaldehyde and then dehydrated in 30% sucrose for at least 72 h at 4°C. Tissue was then freeze-mounted in optimal cutting temperature embedding medium (Sakura Finetek USA, Inc., Torrance, CA, USA) on cork blocks for cryostat sectioning. Spinal cords were sectioned transversely at a thickness of 20 µm. The sections were washed three times with PBS. Sections were then blocked for 1 h at 37°C with blocking solution [PBS containing 5% bovine serum albumin (BSA; Shanghai Universal Biotech Company, Shanghai, China) and 0.25% Triton X-100] followed by incubation with the primary antibody diluted in PBS containing 1% BSA at 4°C overnight. Primary antibodies used were as follows: Monoclonal purified goat anti-rat CD4 molecular complex antibody (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-1140) for the detection of infiltrating CD4⁺ cells; rabbit anti-rat IL-17 (1:100; Santa Cruz Biotechnology, Inc.; cat. no. sc-7927); and mouse anti-rat glial fibrillary acidic protein (GFAP, 1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-135921) for the detection of astrocytes. The sections were then washed five times with PBS and incubated with the appropriate secondary antibody for 1 h at room temperature. Secondary antibodies used were as follows: Fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat (1:100; Santa Cruz Biotechnology, Inc.; cat. no. sc-2042), phycoerythrin (PE)-conjugated donkey anti-rabbit (1:100; Santa Cruz Biotechnology, Inc.; cat. no. sc-3745), and PE-conjugated goat anti-mouse (1:250; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. M30004). Following washing with PBS, the sections were stained with Hoechst 33258 at 37°C for 5 min and the cells were examined with a fluorescence microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).

The ipsilateral (relative to injury) lumbar dorsal horns of L5-L6 spinal cord were harvested as described above. Mononuclear cells from the spinal cord tissue were obtained with discontinuous Percoll gradients method following a previously described method (27). Due to the limited numbers of mononuclear cells from a single animal, each FACS sample was prepared from three spinal cords pooled together. The collected mononuclear cells was suspended in 100 µl PBS containing anti-rat CD3-FITC (1:100; eBioscience, Inc., San Diego, CA, USA; cat. no. 11–0030) and anti-rat CD4-PE (1:100; eBioscience, Inc.; cat. no. 12–0040). Cells were incubated at 4°C for 30 min. Following washing twice with PBS, all stained cells were resuspended in 4% formaldehyde/PBS at 4°C overnight. Data were acquired by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using CellQuest software (version 2.8; BD Biosciences).

Serum IL-17 was measured by an ELISA kit (Rat IL-17A Platinum ELISA; eBioscience, Inc.) following the manufacturer's protocols. All samples were measured in triplicate, and the mean concentration was calculated from the standard curve.

As previously described (28), astrocyte cultures were prepared from 1–2 day-old postnatal rats. Briefly, in a sterile environment, spinal cords were dissected and cell suspensions were prepared. These were subsequently transferred to culture flasks and incubated at 37 °C in a CO₂ balanced incubator. After 7 days, the flask was agitated at 200 rpm at 37°C overnight to remove oligodendrocytes, neurons, and microglial cells. Following three passages, the astrocytes were divided into IL-17-stimulated and naive groups. Briefly, 1×10³ cells/well were seeded into 96-well plates in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) and following cell adherence to the plate, IL-17 was added to the wells at concentration of 10, 50 or 100 ng/ml, and incubated at 37°C for 24–72 h. Subsequently, 10 µl (5 mg/ml) MTT was added to each well and the plate was further incubated for 4 h to deoxidize MTT under light-blocking conditions. Following removal of the MTT dye solution, the cells were treated with 100 µl DMSO and the absorbance at a wavelength of 490 nm was measured using the ELx800 UV microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Cells from the naive group were not stimulated by IL-17.

All data were expressed as the mean ± standard deviation. Figures and statistical analyses were made with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). For comparison of mechanical sensitivity, spinal inflammatory cytokines, infiltrated-cellular phenotypes in the spinal cord and the MTT assay, appropriate two-way analysis of variance was performed followed by Bonferroni's multiple comparison post-hoc test. Immunohistochemistry data were analyzed by t-test or one-way analysis of variance. Inflammatory cytokines data in vitro were evaluated using t-test. P<0.05 was considered to indicate a statistically significant difference.

The mechanical sensitivity (indicated by the 50% threshold force for paw withdrawals) was measured in the injured ipsilateral hind paws prior to (baseline values) and up to 2 weeks after surgery. Baseline values were not different between the groups. Following SNL, rats developed pain hypersensitivity at day 3 up to day 14 compared with the other two groups. Furthermore, no significant differences were observed between the sham group and the control group at any time point (Fig. 1).

Following spinal nerve ligation, IL-17 mRNA increased significantly in the ipsilateral dorsal horn of L5-L6 spinal cord at day 7 (P<0.05). No differences were detected between the sham and control group at any time (Fig. 2A). Serum IL-17 in naive, sham-operated, and SNL rats at days 7 and 14 post-surgery was analyzed by ELISA to determine whether the changes of IL-17 in spinal cord was associated with the changes in the peripheral serum. Though spinal nerve ligation induced a moderate increase in IL-17 expression, no differences were determined amongst the three groups at any time point (Fig. 2B). Furthermore, IL-17 localization was detected in the spinal cord at day 7 after surgery. As IL-17 is preferentially expressed by CD4⁺ T cells, double immunofluorescence labeling was used to demonstrate that IL-17 immunoreactivity was colocalized with CD4 in the ipsilateral dorsal horn, and the immunostaining density of IL-17 and CD4 markedly increased when compared with the sham-operated rats (Fig. 2C).

To further confirm IL-17 was produced by spinal cord-infiltrating CD4⁺ T cells, CD4⁺ T cells were detected in the lumbar spinal cord using FACS at days 7 and 14 post-surgery. SNL induced a significant increase in the infiltration of CD4⁺ T cells into the ipsilateral side of lumber spinal cord at day 7 (P<0.001), while no marked changes were observed at day 14 compared with the naive and sham groups (Fig. 2D).

SNL was demonstrated to induce marked expression of GFAP at the ipsilateral side compared with either the contralateral spinal cord or either side of the sham group. The level of GFAP in the ipsilateral side was consistent with the contralateral side in the sham group. GFAP activation was predominantly localized to the superficial laminae of the lumbar spinal dorsal horn. The immunoreactivity of GFAP notably increased compared with the sham-operated rats (Fig. 3A). Furthermore, the qPCR indicated significant increases in the expression levels of IL-6 and IL-1β in the ipsilateral spinal dorsal horn following SNL at day 7 (P<0.05 and P<0.01) and IL-6 remained at high levels at day 14 (P<0.05; Fig. 3B and C). No change in expression of TNF-α was demonstrated at days 7 and 14 in all groups (data not shown).

To investigate the effect of IL-17 on astrocytes, 10, 50 or 100 ng/ml IL-17 was used to stimulate astrocytes in vitro. The results of the MTT assay demonstrated that astrocytes proliferated successively over time, but not significantly higher than that at 24 h in each group. IL-17 induced significant astrocyte proliferation at 48 h compared with the naive group (P<0.05). However, at 72 h, only 100 ng/ml IL-17 significantly promoted proliferation (P<0.01; Fig. 4A). Thus, the expression levels of IL-1β, IL-6 and TNF-α mRNA at 72 h between the group with 100 ng/ml IL-17 stimulation and the naive group were investigated. Treatment with 100 ng/ml IL-17 elevated IL-1β and IL-6 mRNA expression levels (Fig. 4B), but not TNF-α (data not shown). These results demonstrate that IL-17 enhanced astrocytic activity.