Reagents were purchased from Sigma (St. Louis, MO) unless indicated otherwise. MBP2-18 (ASQKRPSQRSKYLATAS), MBP68-86 (AHYGSLPQKKSHGRTQDENP), MBP84-104 (ENPVVHFFKNIVTPRTPPPSQ), and scrambled MBP84-104 (NKPQTNVVEPFHRTFPIPPVS; sMBP84-104) peptides, derived from human MBP sequence (GenBank #AAH08749), were synthesized by GenScript. To prevent their degradation by exoproteinases, these 97% to 99% purity peptides were N- and C-terminally protected by acetylation and amidation, respectively. Because of the incomplete homology between the human and rodent MBP68-86 sequence, an additional Lys residue was inserted in the MBP68-86 sequence (underlined in the sequence above) to make the resulting peptide more uniformly applicable for our studies. Primers and Taqman oligonucleotide probes for rat MMP-9 (GenBank #NM_031055), GAPDH (GenBank #XO2231), and tumor necrosis factor-α (TNF-α, GenBank #NM_012675) were designed using Primer Express 2.0 software (Applied Biosystems) and obtained from Biosearch Technologies . Similarly, the probes for rat IL-17A (GenBank #NM_001106897.1), were obtained from Applied Biosystems (Assay ID Rn01757168_m1). GM6001, a broad-spectrum MMP inhibitor, was purchased from Millipore. SB-3CT, a selective MMP-2/9 inhibitor, was purchased from EMD Biosciences. The following detection antibodies were used in our studies: rabbit anti-rat MMP-9 (Torrey Pines Biolabs, cat. #TP221, 1:500), goat anti-mouse MMP-9 (R&D Systems, cat. #AF909; 1:250), rabbit anti-S100 (Dako, cat. #Z0311, 1:500), murine anti-CD68 (clone ED1, Abcam, cat. #ab31630; 1:100), rabbit anti-von Willebrand factor (vWF, Abcam, cat. #ab6994, 1:1,000), rabbit anti-Iba1 (Wako, cat. #019-19741, 1:500), rat anti-MBP (Abcam, cat. #ab40390, 1:250), murine anti-human MBP (clone 22, AbD Serotec, cat. #MCA686S, 1:250), murine anti-MHC II (clone OX6, Abcam, cat. #Ab6403, 1:200), mouse anti-T cell receptor alpha/beta (TCR; AbD Serotec, cat. #MCA453G, 1:200), mouse β-actin antibody (Sigma, cat. #A53166, 1:30,000), goat anti-mouse conjugated with Alexa 594 (Molecular Probes, 1:500, red), or goat anti-rabbit conjugated with Alexa 488 (Molecular Probes, 1:500, green). The nuclei were stained with DAPI (Molecular Probes, 1:20,000, blue). We also used a rabbit anti-MBP antibody (Millipore, cat. #AB5864, 1:1,000) that recognizes degraded MBP and that was generated against the YGSLPQKSQRSQDENPVV MBP69-86 synthetic peptide (the guinea pig sequence) as an immunogen. Antibodies were diluted in TBS containing 0.1% Tween-20 and 1% normal goat serum.
Animals, surgery, and therapy
All animals were housed at 22 °C under a 12 h light/dark cycle with food and water ad libitum. Animals were anesthetized with 4% isoflurane (Aerrane; Baxter) in 55% oxygen or a rodent anesthesia cocktail containing Nembutal (50 mg/mL; Abbott Labs) and diazepam (5 mg/mL) in 0.9% PBS (Steris Labs). Sprague–Dawley rats (n = 144, 200–225 g adult females), athymic nude rats (rnu−/−, Hsd:RH-Foxn1
n = 6, 8-week-old females) and their heterozygous controls (rnu+/−, Hsd:RH-Foxn1
n = 6, 8-week-old females) were obtained from Harlan Labs. The common sciatic nerve was exposed unilaterally at the mid-thigh level. Four loosely constrictive chromic gut sutures were tied around the nerve to produce CCI . SB-3CT (10 mg/kg body weight in 10% DMSO) was injected i.p. twice: first at the initiation of CCI and then in 24 h. Sol‐vent alone was used as a vehicle. In a separate group of animals, the exposed naïve sciatic nerves received an intraneural injection of an MBP peptide (50 μg) in 5 μL PBS, or an equal volume of PBS as a vehicle using a 33-gauge needle on a Hamilton syringe. For a sham-operated control, the sciatic nerve was exposed but otherwise not manipulated. The sciatic nerves and ipsilateral dorsal horn of the spinal cords were collected for analyses. FVB.Cg-Mmp9tm1Tvu/J (MMP-9−/−
n = 6; 20 g, adult females) and wild-type FVB/NJ (WT, n = 6; 20 g, adult females) mice were obtained from Jackson Labs. The sciatic nerve was exposed unilaterally at the mid-thigh level and crushed using fine, smooth-surface forceps twice for 2 s each. The animals were sacrificed by an overdose of the Nembutal/diazepam cocktail, followed by Beuthanasia (100–150 mg/mL, i.p., Schering-Plough Animal Health). The animals were handled according to the NIH Guide for the Care and Use of Laboratory Animals and the required protocols were approved by the Institutional Animal Care and Use Committee.
Two-dimensional liquid chromatography/tandem mass spectrometry/mass spectrometry (2D-LC/MS/MS), proteomics, and pathway analysis
The rat sciatic nerves were isolated, snap-frozen in liquid N2 and stored at −80 °C. The samples were homogenized, sonicated, extracted 60 min at ambient temperature in 100 mM Tris–HCl, pH 8.0, containing 8 M urea and the protease and phosphatase inhibitor cocktails, and the insoluble material was removed by centrifugation (16,000xg; 15 min). The supernatant samples (at least 0.5 mg total protein each) were then processed by the Proteomics Core facility of the Sanford-Burnham Medical Research Institute. The samples were reduced (10 mM tris(2-carboxyethyl) phosphine, 37 °C, 30 min), alkylated (20 mM iodoacetamide, 37 °C, 40 min in the dark), and digested using Modified Trypsin, Mass Spectrometry Grade (Promega; 1:100 w/w ratio; 37 °C, 16–18 h). The samples were desalted using a SepPack cartridge, dried using a SpeedVac and re-suspended in 0.1 mL 5% formic acid. The resulting peptides were separated into 24 fractions using an offline Michrom MDLC pump (Michrom) with a Michrom Strong Cation Exchange column. The 1/10 aliquot of each peptide fraction was analyzed using an LTQ-Orbitrap XL mass-spectrometer (Thermo Scientific) and a 15 cm Michrom Magic C18 column coupled with a low-flow Michrom ADVANCED device. The data were analyzed by Sorcerer Enterprise v.3.5 software (Sage-N Research) using the ipi.Rat.v3.56 protein database. 57 Da were added to cysteines to identify carboxyamidomethylated cysteines, 16 Da were added to methionines to identify oxidated methionines. The search results were sorted, filtered, and statistically analyzed using a trans-proteomic pipeline (TPP) (Institute for Systems Biology, Seattle, WA) with a 90% minimum probability score and an error rate ≤2%. An additional search was performed using a Prolucid search algorithm with a DTASelect function via an Integrated Proteomics Pipeline (IP2) server. Relative levels of the proteins in the samples were then analyzed using IP2 for a Label-Free differential peptide/protein analysis. The final data were subjected to bioinformatics analyses using Ingenuity IPA 8.7 software (Ingenuity Systems).
Real-time qRT-PCR, genome-wide transcriptional profiling, and pathway analysis
The rat sciatic nerves were isolated and stored in RNA-later (Ambion) at −20 °C. Primers and Taqman probes were optimized to amplification efficiency of 100.1-100.3% . Total RNA was extracted using TRIzol (Invitrogen) and purified on an RNeasy mini column (Qiagen). The RNA purity was estimated by measuring the OD260/280 and the OD260/230 ratios. The integrity of the RNA samples was validated using an Experion automated electrophoresis system (Bio-Rad). The samples were treated with RNase-free DNAse I (Qiagen). cDNA was synthesized using a SuperScript first-strand RT-PCR kit (Invitrogen). Gene expression levels were measured in a Mx4000™ Multiplex Quantitative PCR System (Agilent Technologies) using 50 ng of cDNA and 2x Taqman Universal PCR Master Mix (Ambion) with a one-step program: 95 °C, 10 min; 95 °C, 30 s; 60 °C, 1 min for 50 cycles. Duplicate samples without cDNA (a no template control) showed no contaminating DNA. Relative mRNA levels were quantified using the comparative delta Ct method  and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a normalizer. The fold change between experimental and control samples was determined using the Mx4000 software.
For the genome-wide transcriptional profiling, the samples of total RNA (50 ng) from the wild-type and athymic nude rat nerves and spinal cord tissues were labeled using LowInput QuickAmp Labeling Kit and Cy3-CTP (Agilent Technologies). The labeled RNA samples were hybridized 17 h at 65 °C to SurePrint G3 Rat GE 8x60K slides (Agilent Technologies). Slides were scanned using an Agilent C Scanner. The raw data were processed using Feature Extraction software version 10.5. The initial analysis and normalization to the median were performed using GeneSpring GX software (Agilent). Differentially expressed mRNAs with signal intensities higher than two-fold over the background standard deviation were filtered by t-test. The statistically significant data only (P < 0.05) were used to calculate gene expression ratios in the samples. The gene expression data have been deposited to GEO database (accession # GSE34868, http://www.ncbi.nlm.nih.gov/geo/info/linking.html). The final data were analyzed using Ingenuity IPA 9.0 software.
MMP-9 purification and proteolysis of MBP in vitro
The recombinant pro-form of MMP-9 was purified from the serum-free medium conditioned by the stably transfected HEK293 cells using the gelatin-column chromatography. The purity of the isolated MMP-9 samples was confirmed using SDS-PAGE in a 4-20% gradient acrylamide gel followed by Coomassie staining. Only the samples with purity over 95% were used in our studies. Purified pro-MMP-9 was activated using 4-aminophenylmercuric acetate. The concentration of the catalytically active MMP-9 was determined using a fluorescent assay by active site titration against a standard solution of a GM6001 of known concentration. (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (Bachem) was used as a fluorescent substrate [27, 37].
Human MBP (4 μg; approximately 11 μM) was co-incubated with activated MMP-9 (1–100 nM; an enzyme-substrate ratio 1:100–1:10,000) in 50 mM HEPES, pH 6.8, supplemented with 10 mM CaCl2 and 50 μM ZnCl2, for 1 h at 37 °C. The total volume of the reactions was 20 μL. Where indicated, GM6001 (2.5 μM) was added to the reactions to inhibit MMP-9. The cleavage reaction was stopped using a 5xSDS sample buffer. The digest samples were analyzed by SDS-PAGE and by MALDI-TOF MS using an Autoflex II MALDI TOF/TOF instrument (Bruker Daltonics). For MS analysis, the reactions were cooled on ice and equal volumes (2 mL) of a sample and of a sinapic acid (20 mg/mL) in 50% acetonitrile-0.1% trifluoroacetic acid solution were mixed, spotted directly on a MALDI target plate, air-dried, and co-crystallized for 10 min. Mass spectra were processed with FlexAnalysis 2.4 software (Bruker Daltonics). The singly charged cleavage products, which were observed only in the cleavage reactions but not in the controls, were recorded and processed further.
Sciatic nerves were isolated, snap-frozen in liquid N2, and stored at −80 °C. Proteins were extracted in 50 mM Tris–HCl, pH 7.4, containing 1% Triton-x 100, 150 mM NaCl, 10% glycerol, 0.1% SDS. Extract aliquots (10–70 μg total protein as determined by BCA Protein Assay, Pierce) were analyzed using 10% acrylamide gels co-polymerized with 0.1% gelatin. After electrophoresis, gels were washed in 2% Triton X-100 for 30 to 60 min at ambient temperature, incubated for 16 to 18 h at 37 °C in 50 mM Tris–HCl buffer, pH 7.4, containing 10 mM CaCl2 and 1 μM ZnCl2 and 0.2 mM sodium azide, and stained with Coomassie Blue R250 to visualize the gelatinolytic activity bands.
Neuropathology, immunohistochemistry, and microscopy
Plastic-embedded transverse nerve sections (0.75 μm each) were used for neuropathologic evaluation. Sciatic nerves were isolated and placed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, osmicated, dehydrated, and embedded in araldite resin. Sections were cut with a glass knife on an automated Leica RM2065 microtome and stained using methylene blue Azure II. Immunohistochemistry was performed in tissues fixed in 4% para‐formaldehyde, embedded in paraffin, or cryoprotected in graded sucrose and embedded into OCT compound in dry ice. The 10-μm sections, when required, were deparaffinized using xylene and rehydrated in ethanol and PBS, immersed in 0.5% sodium borohydride followed by treatment with the antigen retrieval reagent (Dako) for 5 min at 95 °C, then for 20 min at ambient temperature. Teased nerve fibers were prepared from the transected and de-sheathed sciatic nerves. Nerve bundles were separated using a pair of fine smooth microforceps. Individual fibers were teased out using 0.20-0.22 mm acupuncture needles (Vinco, Oxford Medical Supplies) on a glass slide, dried at ambient temperature and stored at −20 °C. Non-specific binding was blocked using PBS containing 5% normal goat serum and 0.25% Triton X-100. The sections were incubated with a primary antibody (4 °C, 16–18 h) followed by an Alexa 488-conjugated (green) or Alexa 594-conjugated (red) species-specific secondary antibody (Invitrogen, 1 h, ambient temperature). The nuclei were stained with DAPI (5 min). Sections were mounted using a Slowfade Gold antifade reagent (Molecular Probes). The images were acquired using a Leica DMR microscope and Openlab 4.04 imaging software (Improvision).
Sensitivity to non-noxious mechanical stimuli was measured by von Frey testing . Rats were acclimated to being on a suspended 6-mm wire grid. The plantar surface of the hindpaw was stimulated within the spinal nerve innervation area using calibrated von Frey filaments (Stoelting). Stimuli were applied for 4 to 6 s with a 0.4 to 15.0 g buckling force to the mid-paw plantar surface. In the event of a positive response, the next weaker stimulus was chosen for the next measurement. In the absence of a response, a stronger stimulus was presented. This consecutive way of applying filaments was continued until six responses in the immediate vicinity of the 50% threshold were obtained. The resulting sequence of positive and negative responses was used to interpolate the 50% withdrawal threshold, determined using the up-down method. Stimuli were separated by several seconds or until the animal was calm with both hind paws placed on the grid. Paw withdrawal latency to a thermal stimulus was measured by a Hargreaves testing device . The hind paw was stimulated by a radiant heat source. Withdrawal of the paw from the heat source was measured four times to calculate the mean withdrawal latency. A maximal cut-off of 20 s was used to prevent tissue damage. The interval between two trials on the same paw was at least 5 min. Spontaneous pain-like behavior was measured as described in . Each animal was placed in a 19 x 31 cm plexiglass cylinder and allowed to habituate. A 2 min testing period included continuous pressing of one of six (0–5) numerical keys on a computer keyboard, corresponding to the instantaneous behavior of the animal, rated by the positions of the injured hind paw as follows: 0, the paw is placed normally on the floor; 1, the paw is placed lightly on the floor and the toes are in a ventroflexed position; 2, only the internal edge of the paw is placed on the floor; 3, only the heel is placed on the floor and the hind paw is in an inverted position; 4, the whole paw is elevated; 5, the animal licks the paw. The measurements were repeated twice within 2 h. An index for noxious behavior was calculated by multiplying the time that rat spent in each behavior by a weighting factor for that behavior, and divided by the length of the observational period, using the formula: [0 t0 + 1 t1 + 2 t2 + 3 t3 + 4 t4 + 5 t5]/120 s, where t0-t5 are the time in sec spent in behaviors 0–5, respectively. The three values corresponding to three blocks of 120 s were averaged to determine the spontaneous pain score for each rat. All tests were performed daily for 3 days before peptide injection and then daily thereafter by an investigator blinded to the experimental groups.
Statistical analyses were performed using KaleidaGraph 4.03 (Synergy Software) or SPSS 16.0 (SPSS) software by a two-tailed, unpaired Student’s t-test for comparing two groups, or analyses of variance (ANOVA) for repeated measures for comparing three or more groups, followed by the Tukey-Kramer post-hoc test, unless specified otherwise. P values ≤ 0.05 were considered significant.