Burn injury model
Animal experiments were approved by the Ethics committee of Henan province. Zhengzhou, Henan, China.
Male SD rats (weight 180–220 g) were purchased from Animal Experiment Center of Henan Province, housed 4 per cage and maintained on a 12 h light/dark schedule in a temperature-controlled at 25 °C environment with available access to food and tap water. The rats were randomly divided into 2 groups (n = 16): Burn injury group: A metal plate cover floated on which water circulated from 85 °C water bath was arranged. One hind paw of each rat was held in contact with the metal plate cover for 15 s to establish the burn model. Sham group: Sham-treated animals went through the identical treatment except that the hind paw was placed on the room temperature metal plate instead of 85 °C. Burn injury was performed n the rats as described previously [19]. Anesthesia was induced with 5 % isoflurane and maintained at 2.5 % (V/VO2 2 l.min-1) before the process and remained throughout the duration of the process. Rats were allowed to recover from anesthesia in their home cages within 10 min after operation. According to the behavioral test results, animals displayed the extreme acute pain on 7 d after operation. Therefore, animals were sacrificed on 7 d to collect DRGs.
Behavioral test
Rats were habituated to the test apparatus for at least 30 min before test on test days.
Behavioral test was performed blindly to treatment group. Hind paw radiant heat (Hargreaves’) test: Rats were placed in plastic chambers on a glass surface maintained at 25 °C. A radiant heat source was focused on the hind paw and latency to respond was recorded in three trials per paw, separated by at least 10 min. Heat intensity was 30 % and cutoff to avoid tissue damage was 30 s. von Frey test of mechanical threshold: rats were placed in plastic chambers on a wire mesh grid and stimulated with von Frey filaments according to the up-down method described previously [1].
Application of lentiviral vector-mediated shRNA
Thirty six male SD rats were randomly divided into 2 groups (n = 18): Burn injury + empty lentiviral vector -SCN9AsiRNA-GFP (LV3- SCN9AsiRNA-GFP group), burn injury + lentiviral vector negative control (LV3-NC-GFP group). Recombinant Lentivirus were offered by Shanghai GenePharma Co.,Ltd. On the zero day, burn injury model rats in LV3-NC group and LV3- SCN9A group received DRG microinjection [14] of 4 μl empty lentiviral vector and LV3- SCN9A vector, respectively. On the day before burn injury and 4 d, 7 d, 14 d, 21 d after burn injury (Post Operation Days, POD4, POD7.POD14, and POD21), mechanical withdrawal threshold and thermal withdrawal latency were measured. Rats were scarified on 7 d after operation and DRG was collected.
Immunofluorescence
Under deep anesthesia with isoflurane, the rats were perfused with normal saline followed by cooled 4 % paraformaldehyde in 1 M phosphate buffer. L4 -6 DRGs were collected at 4 d, 7 d, 14 d, and 21 d. DRG was paraffin embedded and sectioned at 20 mm. For single labeling, 3 sets of DRG sections (4–5 sections/DRG) were collected. After being dewaxed by gradient with xylene ethanol, the sections were rinsed by PBS 5 min/times. After Antigen repair, the sections were blocked with goat serum for 2 h at room temperature. Then, the sections were incubated with Nav1.7 mouse monoclonal antibody (1 mg/mL; abcam) and C-fos goat anti-mouse antibody (1:500; Beijing Zhongshan biotechnology) for 30 min at 37 °C then at 4 °C overnight. The sections were then incubated with goat anti-rabbit antibody conjugated with FITC (1:80; Shanghai Weiao biotech) or donkey anti-mouse antibody conjugated with Cy3 (1:200; Jackson ImmunoResearch) for 2 h or DAPI (1:1000; Sigma) at room temperature (RT) and covered with BSA (10 %; Shanghai Weiao biotech). All stained sections were viewed with an epifluorescence microscope (Olympus Corporation, Japan).
Western blot analysis
Total protein was extracted from L4–6 DRGs of the rats using tissue protein extraction reagent (Weiao Biotech, Shanghai, China). The proteins were loaded and separated by sodium dodcyl sulfate (SDS)- polyacrylamide gels, electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, MA, US). The membranes were then blocked with the blocking buffer (5 % fat free dry milk with TBST) for 2 h at RT and incubated with rabbit anti-Nav1.7 (1:600, Abcam, England) at 4 °C for 2 nights. Finally, the membranes were incubated with goat anti-rabbit antibody (1:800, Zhongshan Jinqiao, China) for 2 h at RT and signal was detected with ECL detection reagents (Alphalmager proteinsimple, San Jose, USA).
Statistical analysis
For statistical analysis, GraphPad Prism software was used. Behavioral data and immune fluorescence intensity were analyzed by either the Student’s t-test to compare 2 groups or ANOVA followed by planned comparisons of multiple groups. In both cases, when significant main effects were observed, P < 0.05 was considered to be statistically significant.
