Animals
Male Sprague Dawley (SD) rats (180–230g) were obtained from the Changsheng biological company and housed in Shengjing Hospital Benxi experiment base. This study was in accordance with the ethical guidelines of China Medical University for the use of laboratory animals and approved by the Animal Ethics Care and Use Committee of China Medical University Shengjing Hospital (approved number:2016PS013K). All surgery was performed under 1% pentobarbital sodium anesthesia (35mg/kg i.p.). Rats were randomly separated into different groups. n=6 rats per group. Timeline of experimental procedure was demonstrated as Figure 1.
Spinal nerve ligation (SNL)
Spinal nerve ligation procedure was carried out as described previously(29). Rats were placed in the prone position under anesthesia. A 2 cm incision was made on the left lumber 5 level, 0.5 cm approximately to midline. The left L5 and L6 spinal nerve were separated and ligated tightly with 4-0 silk thread, distal end of ligation was transected.
Behavior tests
Mechanical withdrawal threshold (MWT)
To examine the mechanical hypersensitivity of rats, the mechanical withdrawal threshold (MWT) test was measured with Von Frey filaments (Stoelting Company, Wood Dale, IL, USA) as demonstrated previously(10, 30). Rats were habituated 30 min before MWT test in plexiglass chamber. A positive withdrawal threshold of the hind paw was measured with up and down procedure. For each MWT trial, stimulation duration was 5s approximately; interval time was 5 min; cut-off value was 15g.
Open field test (OFT)
The anxiety and locomotor level was evaluated with open field test as reported previously(31). The open field arena consisted of an aluminum plate base (100 cm×100 cm) surrounded by walls of 45 cm. The interior was painted black. Open field arena was equipped with infrared detectors and analyzed by Noldus software. Rats were put in open field for 10 min. The distance, the proportion of the path in the center, travel trace and heat map were demonstrated. The field was cleaned with 75% ethanol after each trial.
Drug delivery
5μg 2’3’-cGAMP (STING agonist, Cat: #tlrl-nacga23, InvivoGen, USA) was intrathecal injected with 5 μl Hamilton microsyringe at 4h before operation and postoperative day 2, 4, 6. Single intrathecal injections were administrated via percutaneous lumbar puncture through 5th or 6th intervertebral space. A rapid tail flick manifested that the microsyringe penetrated the dura mater. The injection speed was 0.5s/μl approximately and microsyringe was hold still for 30 s after injection.
Ketamine (20 mg/kg, Cat. #1709291, Fujian Gutian Pharmaceutical Co., Ltd. China) or vehicle was intraperitoneally (i.p.) injected at 4 h before operation and postoperative day 2, 4, 6.
Dexmedetomidine (20 μg/kg, Cat. #181017BP, Jiangsu Hengrui Pharmaceutical Co., Ltd. China) or vehicle was intraperitoneally (i.p.) injected at 4h before operation and postoperative day 2, 4, 6.
Western blot
At postoperative day 7, SD rats were deeply anaesthetized and sacrificed. The L4~L6 spinal cord were rapidly dissected and frozen at −80°C. The tissues were homogenized in RIPA buffer (p0013B, Beyotime, China) and phosphatase inhibitors (1:100, Solarbio, China) for 30 min on ice, then centrifugation at 14,000 rpm for 40 min at 4 °C. The supernatant fraction after centrifugation was collected. The lysate with loading buffer (Beyotime, China) were separated with 12% SDS/PAGE gel and transferred to PVDF membranes (GE, USA). Primary antibodies were incubated at 4 °C overnight (12h at least) respectively after 1h room temperature blocking in 5% BSA with TBST (0.1%Tween 20 in Tris-buffered saline). Bands were incubated with HRP-conjugated second antibodies for 1.5 h at room temperature. After 3 times TBST washing, signal was detected with ECL plus kit (Tanon, China bands), captured with the chemiluminescence imaging systems (GE, USA; c300, Azure biosystems, USA) and quantified with Image J software (NIH, USA).
The following antibodies were used in this study: rabbit Grp78 (1:2000, Abcam, USA), rabbit LC3 (1:1000, CST, USA;), rabbit p62 (1:2000, CST, USA), rabbit FAM134B (1:1000, Abcam, USA), rabbit p-STING (1:1000, CST, USA), rabbit STING (1:1000, CST, USA), rabbit p-TBK (1:1000, CST, USA), TBK (1:1000, CST, USA), mouse GAPDH (1:8000, Solarbio, China), goat anti-rabbit/goat anti-mouse IgG horseradish peroxidase (1:5000, Beyotime, China).
Immunofluorescence staining
Rats were deeply anesthetized and perfused with 0.9% NaCl solution transcardially, followed by cold 4% paraformaldehyde in 0.1M PBS. L5 spinal cords were removed, post-fixed in fixative solution for 24h, and dehydrated with 30% sucrose in ddH2O at 4°C for 24h. The brains were embedded with optimal cutting temperature (OCT, SAKURA, USA) compound. Embedded L5 tissue was coronal sectioned in cryostat at 10μm thickness. For confocal immunostaining, sections were incubated with anti-NeuN (neuronal marker, 1:200, MAB377, Millipore, USA)/GFAP (glial cell marker, 1:200, Abcam, China) and anti-Grp78 (ER stress marker, 1:200, Abcam, USA)/p-TBK (1:200, CST, China). Cell nuclei were counterstained with DAPI (Beyotime, China) for 5 minutes.
Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM). Analysis was measured with IBM SPSS Statistics 22 software (SPSS Inc., Armonk, New York, USA). Western blot and open field test results were analyzed by one-way analysis of variance (ANOVA) following post hoc multiple comparison; MWT data were analyzed by two-way analysis of variance (ANOVA) following post hoc multiple comparison. P values < 0.05 were considered significant.