Reagents, drugs and antibodies
General reagents were purchased from Sigma-Aldrich and ThermoFisher Scientific. Rituxan (Rituximab, RTX) was obtained from Genentech. Intravenous immunoglobulin (IVIG) Gamunex-C (immune globulin injection 10%) was purchased from Grifols. Human intact MBP (18.5 kDa isoform) was purchased from Meridian Life Science. Based on human MBP sequence (GenBank, AAH08749), MBP(84-104) (ENPVVHFFKNIVTPRTPPPSQ) and scrambled (SCR, EFPHIKVTVVTPRNGFPNSPP) peptides, N-terminally biotinylated and C-terminally amidated, were synthesized by GenScript. Rabbit polyclonal anti-CD20 antibody was obtained from Abcam (ab85809), mouse monoclonal anti-β-actin antibody was obtained from Sigma (A53166), rabbit anti-degraded (d)MBP antibody, generated against the synthetic peptide encoding guinea pig MBP(69-86), was obtained from EDM-Millipore (AB5864). FluoroMyelin 488 (F34651) was obtained from Molecular Probes. For ELISA, HRP-conjugated goat anti-rat IgM (3020-05) and anti-rat IgG (112-035-175, Jackson ImmunoResearch), a 3,3’,5,5’-tetramethylbenzidine substrate (TMB/E, Surmodics) and BSA (an IgG and protease-free, 30% solution, US Biological) were used.
Animal surgery and sample collection
All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the Veterans Affairs San Diego Healthcare System, the Public Health Service Policy on Humane Care and Use of Laboratory Animals and ethical guidelines of the International Association for the Study of Pain. Sprague-Dawley rats (Envigo, 8-week-old, female and male) were housed in temperature-controlled cages with a 12-h light–dark cycle and free access to food and water. The procedures and behavior testing were performed during the light cycle. Under 3% isoflurane anesthesia, the left common sciatic nerve was exposed at the mid-thigh level with sterile technique. CCI was produced by tying three loosely constrictive chromic gut ligations around the sciatic nerve [44]. Sham operation included nerve exposure with no injury. Animals were sacrificed by intraperitoneal Euthasol (100–150 mg/ml; Virbac Animal Health) after deep isoflurane inhalation anesthesia. Blood, spleen, sciatic nerve, lumbar (L)5 dorsal root ganglia (DRG) and L1-6 dorsal spinal cord tissues were harvested snap-frozen in liquid N2 and stored at -80°C for immunoblotting, or following transcardial perfusion in 4% paraformaldehyde (PFA) in 0.2 M phosphate buffer for immunostaining. For ELISA, 1-2 ml blood aliquots were collected before CCI and 2, 14 and 28 days post-CCI by cardiac puncture in tubes without anti-coagulant, allowed to clot for 30 min at ambient temperature, centrifuged (2,000xg; 10 min; 4°C) and the supernatant (serum) was collected and stored at -80°C.
Drug delivery
At day 7 after CCI, RTX (10 mg/kg, female n=9, male n=9) and IVIG (10 mg/kg, female n=3, male n=4), each diluted in PBS (Steris Labs) vehicle, or PBS vehicle control (female n=3, male n=3), was administered by intravenous (IV) injection through the tail vein in the same volume (10 µl) using a 27-gauge needle.
von Frey testing
Withdrawal threshold to non-noxious mechanical stimuli was assessed by von Frey testing using the Dixon’s up-down method [45]. Briefly, animals were acclimated to the Plexiglas compartments with 6-mm wire grid bottom and habituated to the environment for two days prior to baseline testing. Baseline values were established for three consecutive days before surgery and then up to daily between 2 and 16 days post-CCI, as indicated. Mechanical stimuli were applied using von Frey filaments (0.4–15.2 g, Stoelting, Wood Dale, IL, USA) perpendicularly on the plantar area of the hind paw innervated by sciatic nerve. The stimuli were applied for 2 seconds, with a 10-second interval between each stimulus or until the rat was stable from the pain. The responses were recorded as positive if the paw was rapidly withdrawn. The 50% withdrawal threshold was calculated according to the Dixon up-down method. Testing was performed at fixed times between 8:00 a.m. and 2:00 p.m. by experimenters blinded to the treatment groups.
Immunostaining
Tissues (sciatic nerves and spleen) were excised, postfixed in 4% PFA, rinsed in 0.2 M phosphate buffer solution (pH 7.4) for 16-18 h. Tissues were embedded in the optimal cutting temperature compound (Sakura Finetek) following the 15-30% gradient sucrose treatment or paraffin and cut into transverse, 10-µm-thick sections [14, 28, 29, 31]. The paraffin sections were deparaffinized in xylene and rehydrated in ethanol. Non-specific binding was blocked with 5% goat serum for 30 min at ambient temperature, followed by polyclonal rabbit anti-CD20 (ab85809, Abcam, 1:200) or dMBP (AB5864, EDM-Millipore 1:2000) antibody application at 4°C overnight. After rinsing in PBS, the sections were incubated with the goat anti-rabbit Alexa-conjugated secondary antibody (red, ThermoFisher Scientific) for 1 h at ambient temperature. In select frozen sections, FluoroMyelin 488 (F34651, Molecular Probes, 1:500) was applied for 20 minutes at ambient temperature. Slides were mounted in Slowfade Gold anti-fade reagent containing DAPI (4’,6-diamidino-2-phenylindole, ThermoFisher Scientific, blue). Staining specificity was confirmed by a primary antibody omission. The images were acquired using All-In-One Fluorescence Microscope BZ-X700 (Keyence, Itasca, IL) and Leica fluorescence microscope.
Immunoblotting
Tissue extracts were prepared in TBS supplemented with 1% Triton X-100, 10% glycerol, 0.1% SDS, 5 mM EDTA, 1 mM PMSF, aprotinin, pepstatin and leupeptin (1 μg/ml each). Insoluble material was removed by centrifugation (14,000×g; 15 min). Extract aliquots (25-50 μg of total protein) were separated by SDS-gel electrophoresis in 15% Tris-glycine gels (Bio-Rad). Separated proteins were transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat milk (Bio-Rad) and incubated for 16-18 h at 4°C with the rabbit polyclonal anti-CD20 antibody (Abcam, ab85809) followed by incubation for 1 h at ambient temperature with the rabbit-specific horseradish peroxidase-conjugated goat secondary antibody (Cell Signaling; 1:10,000 dilution). For loading control, the membranes were re-probed using mouse β-actin antibody (Sigma, A53166). The blots were developed using an enhanced chemiluminescence system (GE Healthcare) or SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific). The membranes were re-probed using a β-actin antibody (loading control). The bands were digitized and quantitated using Image J.
ELISA
Rat serum was subjected to the anti-MBP(84-104) epitope IgG and IgM ELISA [42]. The wells of a 96-well Maxisorp ELISA plate were coated with ExtrAvidin (3 μg/ml in 0.125 ml 15 mm bicarbonate buffer, pH 9.6) or BSA (3 μg/ml, control) for 18 h at 4°C. The wells were blocked for 1 h at 37°C using 1% IgG and protease-free BSA (0.4 ml) in 50 mM Tris-HCl buffer, pH 7.8, containing 1 M NaCl and 0.1% Tween-20 (TBS/T). Incubation with the biotin-labeled MBP(84-104) and SCR peptides (5 μg/ml in 0.1 ml TBS/T-1% BSA, each) continued at 4°C for 16-18 h. Serum samples (diluted 1:50 in 0.1 ml TBS/T-1% BSA) were allowed to bind to the wells for 3 h. The secondary HRP-conjugated goat anti-rat IgG or IgM antibodies (diluted 1:10,000 in 0.1 ml TBS/T-1% BSA, each) were added for 1 h. Six washes (5 min each; 500-700 rpm) in TBS/T at ambient temperature were done after each step. After a wash with H2O, the TMB/E substrate (0.1 ml) was added to the wells. The reaction was stopped using 1 m H2SO4 (0.1 ml) and estimated at A450 using a plate reader. Data represent means ± SE from at least 3 individual experiments performed in triplicate. The A450 values for MBP(84-104) peptide were calculated relative to SCR peptide. The threshold values for MBP (A450 = 0.298) and SCR (A450 = 0.237) peptides were determined as described [42].
Statistical analyses
GraphPad Prism 6.0 software (Synergy Software) was used to conduct two-way repeated measures analysis of variance (ANOVA) or a one-way ANOVA with Bonferroni, Holm-Sidak, or Tukey’s post hoc test, or nonparametric Mann-Whitney rank sum test, as detailed in the figure legends. Data are presented as the mean ± SEM, and differences are considered significant at a P value < 0.05.