Experiments were conducted on male adult Sprague-Dawley rats (8-week-old at the experiments). The number of animal use permits is SYSU-IACUC-2018-000241. Surgical and animal care procedures were carried out under the provisions outlined in the National Health and Medical Research Council animal ethics and ARRIVE guidelines. All procedures were performed with the approval of the Animal Care and Utilization Committee of Sun Yat-sen University. Rats were housed in ventilated Plexiglas cages in a climate-controlled room (20℃) under a 12-hour light/dark cycle (lights off at 8:00 P.M). Animals were allowed to acclimatize to the housing conditions and handling before the start of each experiment. Food and water were available ad libitum or otherwise indicated.
C8 and T1 spinal roots avulsion
The microsurgery procedure for the BPRA injury was minorly modified based on what we previously described [47, 48]. Briefly, the anesthesia was induced in rats with ketamine/xylazine (80/20 mg/kg, i.p.) and maintained with 1% isoflurane. Each animal was laid out in the supine position, following the retraction of the paravertebral muscles. The right side C6 and C7 laminectomy were performed under a surgical microscope, and then the dura mater was opened. After identifying the C8 and T1 segments of the spinal cord for the BPRA group, their spinal roots were selectively avulsed one by one. The proximal residual rootlets and the distal parts of the C8 and T1 avulsed roots were cut away to ensure that spinal MNs would not regrow axons into the damaged nerves in the avulsed rats. All the avulsed distal parts of roots were cut away, and the success of the avulsion model was confirmed under the microscope for the animals in the sham control group. The lower trunk was exposed and isolated but not avulsed. Finally, the muscle, fascia, and skin were sutured successively in layers in all animals. The body temperature of rats remained at 36.5±0.5℃ throughout the procedure. The pain behaviors were evaluated 24 h after surgery.
Western blot assay
The western blot assay was performed as described . Briefly, at 7 days post-injury (dpi) followed BPRA injury or 2 h after pharmacological behavioral tests, the rats were perfused transcardially with ice-cold saline under deep anesthesia with sodium pentobarbital (50 mg/kg, i.p.) and dorsal part of the C8-T1 spinal segments was collected under a microscope, then stored at −80 °C until processing. After that, the lysis and protein extraction of spinal cord tissues was performed using the RIPA lysate buffer (P0013, Beyotime). The concentration of the extracted protein was determined by the BCA Protein Assay Kit (Cat No: 23225, Pierce Biotechnology). The 40 μg of protein extracts from each sample were denatured and subjected to 4–15% Tris–HCl precast Mini Protean® TGX™ gels (BioRad) for electrophoresis. Protein-separated PVDF membranes (Bio-Rad, Philadelphia, PA) were blocked with 5% non-fat milk (Bio-Rad) in TBST (in mM, 24 Tris, pH 7.4, 137 NaCl, 2.7 KCl, and 0.05% Tween 20) for 1 h at room temperature and incubated with antibodies against GLT-1 antibody (1:1000, ab41621, Abcam), α2-AR (1:1000, ab 85570, Abcam), GLAST (1:1000, ab 41751, Abcam), EAAC1 (1:1000, ab 124802, Abcam), total PKA (1:1000, ab216572, Abcam), phosphorylated PKA Thr197 (1:1000, ab75991, Abcam) and GAPDH (1:3000, G8795, Sigma Aldrich, St. Louis, MO) overnight at 4 °C. After washing with TBST, the membranes were incubated for 2 h with goat anti-rabbit or mouse IgG-HRP conjugated antibody (1:2500, Jackson Immuno-Research, West Grove, PA) at room temperature and developed with ECL solutions (PerkinElmer, Waltham, MA). All protein signals were analyzed by scanning densitometry using Image Lab software (Bio-Rad) and normalized to GAPDH.
Immunofluorescence and cell counting
The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused with 4% PFA. Then, the cervical spinal cord was dissected under the surgical microscope. Notably, the C8-T1 spinal segments were defined as the region between the uppermost and the lowermost rootlets of the contralateral C7-C8 spinal nerves. The tissue was post-fixed with 4% PFA and dehydrated by overnight immersion in 30% (v/v) sucrose in phosphate buffer solution at 4 °C, followed by a frozen section at 30μm.
Next, every third section of the spinal cord sections was chosen for the standard immunostaining as we previously described . The sections were rinsed three times in PBS and then treated with 0.3% Triton X-100 and 0.1% bovine serum albumin (BSA) in PBS at room temperature for 30 min. These sections were further incubated overnight at 4°C. The following primary antibodies were used: mouse anti-NeuN (1:500, ab104224, Abcam, UK), rabbit anti-GFAP (1:500, 80788S, CST, USA), rabbit anti-IBA-1 (1:500, 01919741, Wako Bioproducts, USA), rabbit anti-pCREB S133 (1:200, ab32096, Abcam, UK). Then, following washing three times with PBS, the sections were incubated with respective secondary antibodies. The following antibodies were used: Goat anti-Mouse IgG (H+L), Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1500, A32723, Invitrogen, USA), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (1:1000, A32732, Invitrogen, USA). Incubation was 2 h at room temperature in the dark after rinsing the sections three times with PBS. In addition, the staining without primary or secondary antibodies served as negative controls. The immunofluorescence (IF) was evaluated under a fluorescence microscope (Nikon Eclipse 90i, Japan). The average fluorescence intensity (AFI) and the number of GFAP- or IBA-1-immunoreactive (IR) cells in each rat for 8–10 sections were evaluated with the Nikon N.I.S. software package under 10 × magnification.
Mechanical and thermal tests were carried out before BPRA injury and at different time points after surgery. Each behavioral test was carried out at 1 h intervals. The paw withdrawal thresholds (PWT) in rats in response to mechanical stimuli (calibrated von Frey filaments) were measured as we previously described [50-53]. Briefly, the animal was placed in an individual Plexiglas chamber on an elevated mesh screen. For rats, calibrated von Frey filaments (Stoelting Co., Wood Dale, IL, USA) in log increments of force (0.69, 1.20, 2.04, 3.63, 5.50, 8.51, 15.14, and 26 g) were used to stimulate the plantar surface of the rats' left and right hind paws. The 3.63-g stimulus was used first. If a negative response occurred, then the next larger von Frey hair was applied, if a positive response was seen, then the next smaller von Frey hair was applied. The application was terminated when (i) a negative response was seen with the 26-g stimulation or (ii) three stimuli were used after the first positive response. Based on a formula provided by Dixon , the PWT was calculated by converting the pattern of positive and negative responses to a 50% threshold value.
Paw withdrawal latencies (PWL) to thermal stimulation were then examined with a Model 336 Analgesia Meter (IITC Inc. Life Science Instruments. Woodland Hills, CA), as described previously [50, 53]. In brief, the animal was placed in an individual Plexiglas chamber on a glass plate. A beam of light through a hole in the lightbox of Model 336 Analgesia Meter and thence through the glass plate was used to stimulate the middle of the plantar surface of each hind paw. The light beam was automatically turned off when the animal withdrew its foot. The PWL was defined as light beam onset to paw withdrawal. Each test was repeated five times at 5-min intervals for the paw on each side. The cut-off time of 20 s was set up to avoid tissue damage.
The open-field test was utilized to screen the sub-anesthetic dose of DMET by assessing its effect on the animal locomotor activity as we previously described . The apparatus consisted of four fixed walls around a plastic square floor (43.2×43.2×30.5 cm). The illumination was 30 - 50 lux. Each animal was gently placed in the open field center, and its behavior was recorded. The total distance traveled of animals was measured by software (SOF-811, Med Associates Inc, St. Albans, VT).
Chemicals and application
All common salts were purchased from Sigma Aldrich (USA), including the DMET (1-100 μg/kg, i.p. 1μg/10μl, i.t.), selective α2AR antagonist BRL-44408 (100μg/10μl, i.t.), α2CR antagonist JP-1302 (0.1μg/10μl, i.t.), PKA inhibitor H89 dihydrochloride (H89, 5μg/10μl, i.t.), and PKA activator 8-Bromo-cAMP (8-Br-cAMP, 300μM/10μl, i.t.). The doses of these chemicals were selected based on our pilot study and previous reports . All chemicals were dissolved in a mixture of sterile aCSF and suspended in 10% Dimethyl Sulfoxide (DMSO), 40% PEG 300, 5%Tween-80, 45% Saline, solution following pilot work that confirmed this concentration by allowing passage of high concentrations of this hydrophobic drug through the fine-gauged microinjectors.
Cell lines and cell treatments
We studied the influence of DMET/PKA signaling on proinflammatory stimulation of human astrocyte 1321N1 cells (Cat No: 86030402, Sigma-Aldrich) with lipopolysaccharides (LPS). The cells were maintained in DMEM (Gibco, Invitrogen, USA) supplemented with 10% fetal bovine serum in a 5% CO2 incubator at 37 °C for 2 weeks. The cells were then seeded in 6-well plates and subjected to the following 30 min pretreatment before stimulation with 100 ng/ml LPS for 24 hours. 1) DMET (1 µM) alone, 2) DMET combined with α2A receptor antagonist BRL-44408 (5µM), 3) DMET with α2C receptor antagonist JP-1302(1µM), 4) DMET with PKA activator 8-Br-cAMP (250 μM), 5) selective PKA inhibitor H89 (20 μM). The doses of the above chemicals and treating time were chosen based on previous studies [57, 58]. After that, the cells were harvested for histology, western blotting, and ELISA measurement.
Enzyme-linked immunosorbent assay (ELISA)
Tissue levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-18, IL-33, as well as anti-inflammatory cytokines such as IL-4 and IL-10 in the spinal dorsal horn were quantified via enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by the manufacturer (USCN Life Science, Wuhan, China). As we previously reported, the detailed procedure was modified . The right dorsal horn of the C8-T1 spinal segment was dissected on ice and then immediately transferred into the liquid nitrogen. Tissue samples were homogenized in a lysis buffer containing protease and phosphatase inhibitors. Homogenates were centrifuged at 4 °C for 10 min at 2,500 rpm, and the supernatants were collected. The Bradford method was used to quantify the concentration of each cytokine. Using an optical density (OD) of 450 nm, standard curves of the analyzed cytokines were generated, and the concentrations of inflammatory factors were calculated.
All data were expressed as a mean ± SEM (standard mean error). Animal sample sizes chosen to ensure adequate statistical power were determined based on our preliminary studies. Animals were randomly assigned to different studies. Investigators were blinded to group allocations in the pharmacological behavioral experiments. Before the analysis, all data were checked for normality and homogeneity of variances by using Sigma-Aldrich, Plot 12.5. For histology staining and protein expression, the relative fluorescence intensity and protein expression were analyzed using the two-tailed unpaired student's t-test between two groups or one-way ANOVA followed by Turkey's for multiple comparisons. The data of the behavioral tests, including pain and open field tests, were analyzed using a one- or two-way repeated-measures ANOVA, followed by Bonferroni or LSD. Posthoc tests. Statistical significance was declared at p < 0.05.