All experimental protocols were approved by the Animal Experimentation Ethics Committee of China Pharmaceutical University and adhered to the guidelines of the International Association for the Study of Pain (IASP). All experiments were conducted in a blinded manner.
Sprague-Dawley rats (180–220 g) were obtained from the Experimental Animal Center of Yangzhou University (Jiangsu, China). All animals were group-housed, six per cage, and acclimated to a temperature- and humidity-controlled environment with a 12:12 h light-dark cycle. Rodent chow and water were accessible ad libitum. Animals were randomly assigned to the following groups: 1) control; 2) CCI; 3) sham-operated; 4) CCI + Vehicle; 5) CCI + l-CDL (1.5, 3, and 6 mg/kg); or 6) CCI + NGF (0.1 μg/30 μL); 7) CCI + NGF (0.1 μg/30 μL) + l-CDL (6 mg/kg); 8) CCI + anti-NGF mAbs (6 μg/30 μL); or 9) CCI + TAK1 inhibitor (10 μg/30 μL). Six animals were assigned to each group for the behavior test, and four animals were assigned to each group for molecular testing.
Chemicals and reagents
l-CDL (purity ≥99.0%, assessed by high performance liquid chromatography (HPLC)) was obtained from China Pharmaceutical University (Nanjing, China). Recombinant β-NGF and IL-1β were obtained from R&D Systems (Minneapolis, MN). Anti-NeuN antibody was purchased from Abcam (Cambridge, MA). Anti-NGF and anti-IL-1β antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other primary antibodies and secondary antibodies for western blotting were obtained from Cell Signaling Technology (Beverly, MA). Secondary antibodies for immunofluorescence were obtained from Jackson ImmunoResearch Laboratories (Beverly, MA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Biological Industries (Israel) and FBS was obtained from Gibco (Inc., PA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). l-CDL was ground and dispersed in corn oil for animal experiment.
Chronic constriction injury of the sciatic nerve
Chronic constriction injury of the sciatic nerve was conducted as described by Bennett and Xie . All surgical instruments were disinfected in advance. The rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and the left sciatic nerve was exposed. Four loose ligatures (4/0 catgut plain) were placed around the sciatic nerve at 1 mm intervals until a brief twitch in the hind paw was observed. For each ligature, we grasped the two ends close to the loop and tightened until the loop was just barely snug and the ligature did not slide along the nerve. We immediately stopped if a brief twitch was observed, to prevent arresting of epineural blood flow. Finally, the incision was sutured in layers. Penicillin was intramuscularly injected at a dose of 40,000 IU.
Behavioral tests for pain states
The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded for 2 days before surgery (day − 2, − 1). Behavioral tests were performed at 0, 0.5, 2, 4, 8, and 24 h after drug delivery on postoperative day 15. Each rat was tested three more times.
Von Frey hairs (Woodland Hills, Los Angeles) were used as previously described . A series of Von Frey hairs with logarithmically incremental stiffness (1.4 ~ 15.0 g) were used to stimulate the hind paws of rats, and each hair was held for approximately 6 s. A positive response was defined as quick withdrawal or licking of the hind paw upon the stimulus. Whenever a positive response to a stimulus occurred, the next lower Von Frey hair was applied, and whenever a negative response occurred, the next higher hair was applied. Each rat was tested three more times, and the applied force (g) was recorded. Then the average of the threshold was determined to be the MWT.
Thermal hyperalgesia was determined by paw withdrawal latencies to radiant heat (model 37,370; Ugo Basile Biological Instruments). The radiant heat source was positioned at a fixed distance below the glass plate, and the left hind paw on the operated side was stimulated. The intensity of the light source was adjusted to produce withdrawal latencies of 10–13 s in control animals, which was 45 °C in our experiment, and the time until the rats licked and/or raised their foot was recorded. TWL was defined as the elapsed time (in seconds) between the delivery of the heat source and the withdrawal of the paw. A cutoff time of 25 s was set to prevent tissue damage.
RSC96 and RAW264.7 cells were purchased from CHI Scientific, Inc. (Jiangyin, Jiangsu) and cultured in DMEM containing 10% v/v fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2. Both cells lines were plated in 6 well plates at 4 × 106 cells per well and grown to approximately 80% confluence.
Analysis of mRNA levels by quantitative real-time PCR (qPCR)
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA concentration was determined by a spectrophotometer at 260 nm and 280 nm. Equal amounts of RNA (1 μg) was reverse transcribed into cDNA, and the cDNAs were used as templates for PCR amplification. A QuantStudio 3 Real-Time PCR System and fast gene-expression method were used with the following cycling conditions: 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 57 °C for 20 s, and 72 °C for 20 s. Then, melt curve analysis was performed by raising the temperature from 61 °C to 95 °C at a rate of 0.15 °C/s. GAPDH was used as an internal control to normalize the variability in expression levels. The 2−∆∆CT (cycle threshold) method was used to calculate the results, and mRNA expression levels are presented as fold-induction relative to Ctrl cells, which were set as 1. The specific primer sequences were as follows,
NGF Forward 5′- CCAGTGAAATTAGGCTCCCTG − 3′
Reverse 5′- CCTTGGCAAAACCTTTATTGGG -3′
GAPDH Forward 5′- TGATGGGTGTGAACCACGAG − 3′
Reverse 5′- GCCCTTCCACAATGCCAAAG − 3′
Tissue collection and immunofluorescence
After 2 h of drug administration, the rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and the ipsilateral L4-L6 DRGs and sciatic nerves were collected after the rats were perfused with PBS, followed by 4% paraformaldehyde. The tissues were postfixed with the same 4% paraformaldehyde and then transferred to 30% sucrose. Tissue sections were blocked with 10% normal donkey serum containing 0.3% Triton-X-100. After incubation, the tissue sections were observed under a laser-scanning microscope (Carl Zeiss LSM700, Germany). Eight images for each group were evaluated and photographed with the same exposure time to generate the raw data. Fluorescence intensities were analyzed using Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA), with n = 4 in each group.
In brief, the rats were anesthetized with pentobarbital (50 mg/kg, i.p.) 2 h after drug administration, and the ipsilateral L4-L6 DRGs, and sciatic nerves were collected and extracted using RIPA buffer . The proteins in the cell culture supernatants were extracted by methanol-chloroform precipitation . The proteins were separated on sodium dodecyl sulfate-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes. After incubation with antibodies, immunoreactivity was detected using ECL reagents (PerkinElmer, Waltham, MA). The data were analyzed with the associated Quantity One-4.6.5 sofeware (Bio-Rad Laboratories), with n = 4 in each group.
Sample size estimation was determined by G*Power 3.1  and the powers (1-β err prob) were greater than 0.9 which was sufficient to detect differences between two different groups. All values are depicted as the mean ± SEM and the statistical analyses were performed using SPSS Rel 15 (SPSS Inc., Chicago, IL, USA). Data from western blotting, immunofluorescence and behavioral tests were statistically analyzed by one-way analysis of variance (ANOVA) and two-way ANOVA, followed by Bonferroni’s post-hoc tests with significance set at P < 0.05.