The protein G coated 96-well plates and the mouse monoclonal anti-MMP-9 antibody were purchased from Thermo Fisher Scientific (Cat. Nos. 15157 and MS-817-P, respectively; Rockford, IL, USA). The FRET peptide was purchased from AnaSpec (Cat. No. 60570–01; San Jose, CA, USA). 4-aminophenylmercuric acetate (APMA), GM6001, and SB-3CT were purchased from Sigma-Aldrich (Cat. Nos. A9563-5G, M5939, and S1326, respectively; St. Louis, MO, USA).
Rat and human recombinant MMP-9 were purchased from R&D Systems (Cat. Nos. 5427-MM and 911-MP-010; Minneapolis, MN, USA). Rat and human recombinant MMP-2 were purchased from Abcam (Cat. Nos. ab39304 and ab40964; Cambridge, MA, USA). Crude brain tissue homogenates were prepared from rats with right side focal ischemia. Unfixed frozen cortex samples from both the ipsilateral and contralateral hemispheres were weighed and homogenized in 1% sodium dodecyl sulfate (SDS) buffer containing 150 mM NaCl, 50 mM Tris–HCl pH 7.6, 1% IGEPAL® CA-630, and 1% sodium deoxycholate. Just before the buffer was added to the brain samples, HALT Protease Inhibitor Cocktail (Cat. No. 78430; Thermo Fisher Scientific), HALT Phosphatase Inhibitor cocktail (Cat. No. 78428; Thermo Fisher Scientific) and 0.5 M EDTA were added at 10 μL/mL of homogenization buffer. Chilled tissue was homogenized in the buffer for 15-20 s using a Tissue-Tearor homogenizer (Cat. No. 985370; BioSpec, Inc, Bartlesville, OK, USA) then sonicated twice using a Vibra-Cell™ sonicator (Model VCX130PB; Sonics & Materials, Inc, Newtown, CT, USA). Resulting tissue homogenates were centrifuged at 14,000 xg for 20 min at 4°C in an Eppendorf microcentrifuge Model 5430R, and the supernatants aliquoted and stored at −80°C until used.
Rat stroke model and sample preparation
Focal cerebral ischemia was induced by temporary middle cerebral artery occlusion (MCAO) in male Wistar rats (280–320 g; Harlan Laboratories, Indianapolis, IN, USA) using the intraluminal filament method as described previously by our group [32, 59]. Briefly, rats were anesthetized with isoflurane in medical-grade oxygen and a midline vertical incision was made in the neck to expose the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). The CCA was ligated permanently with a 4–0 silk suture and a vascular clip was temporarily placed in the pterygopalatine artery to prevent incorrect insertion of the occluding filament. A loose tie was placed over the ICA and ECA bifurcation with 4–0 silk suture and vascular clips were placed in the ICA and ECA. A small arteriotomy was made in the CCA approximately 2 mm proximal to the carotid bifurcation. A 4–0 silicone-coated filament (Cat. No. 403523PK10; Doccol Corporation, Sharon, MA, USA) was inserted through the CCA and advanced 18–20 mm inside the ICA until a mild resistance was felt. The occluding filament was left in place for 90 min and animals were allowed to recover from anesthesia. Eight to ten minutes before the end of the occlusion period, animals were re-anesthetized with isoflurane inhalant anesthesia, and the filament was gently retracted to allow reperfusion of the MCA territory.
After 48 h of reperfusion, animals were deeply anesthetized with pentobarbital (150 mg/kg; i.p.) and a blood sample was withdrawn from the vena cava into a heparinized syringe. Blood (1.5 mL) was quickly mixed with 50 μL of heparin (1000 U/mL) and centrifuged for 10 min at 2,000 xg to obtain the plasma. Rats were perfused intracardially with ice-cold saline and brains were harvested and dissected into ipsilateral (stroke side) and contralateral cerebral cortex and striatum. Samples were immediately frozen on dry ice and stored at −80°C until use.
HT-1080 human fibrosarcoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in DMEM:F12 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. 10082–147; Life Technologies), 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2. At 80-85% confluency, cells were washed with Dulbecco’s PBS and fresh media without FBS was added. After 24 h, cell culture media was collected and spun down at 5,000 xg for 10 min at 4°C. Aliquots of the HT-1080 conditioned media were prepared and stored at −80°C until use.
Rat brain endothelial (RBE4) cells were cultured in alpha-MEM/Ham’s F-10 Nutrient (1:1 solution; Cat. Nos. 12571–063 and 11550–043; GIBCO, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Cat. No. F4135; Sigma), 1% penicillin/streptomycin (Cat. No. 15140–122; GIBCO, Life Technologies), and 1% Geneticin (300 μg/mL; Cat. No. ALX-380-013-G001; Enzo Life Sciences). RBE4 cells were seeded in rat tail collagen I (50 μg/mL; Cat. No. C3867; Sigma) coated 6-well plates (20,000-30,000 cells/cm2) and maintained at 37°C, 5% CO2 incubator for 2 days before treatment. When cells reached 80-90% confluency, IL-1β (10 ng/mL; Cat. No. 501-RL/CF; R&D Systems, Inc., Minneapolis, MN, USA) was added to wells as treated groups. After 24 hours incubation, untreated and treated cells were washed once with ice-cold phosphate-buffered saline (PBS), and then lysed in radioimmunoprecipitation (RIPA) buffer consisting of 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate and 1% SDS plus complete protease and phosphatase inhibitor cocktails (Cat. No. 78430 and 78428; Thermo Scientific). Finally, the lysates were spun down at 14,000 xg for 15 min at 4°C. Aliquots of the supernatants were saved and stored at −80°C until use.
Immunocapture assay and fluorometric measurement of MMP-9 enzymatic activity
The activity of MMP-9 in rat stroke brain samples was measured fluorometrically using 5-FAM/QXL™520 FRET peptide (Cat. No. 60570; AnaSpec, San Jose, CA). In the intact FRET peptide (QXL™520-Pro-Leu-Gly-Cys[Me]-His-Ala-D-Arg-Lys[5-FAM]-NH2), the fluorescence of 5-FAM (5-carboxy-fluorescein) is quenched by the proximity of QXL™520. Upon cleavage into two fragments by MMP-9, the fluorescence of 5-FAM can be read and monitored at excitation/emission wavelengths of 485/528 nm.
Samples were prepared in 100 μL of TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij® L23). Pierce protein G coated 96-well plates were washed three times with 200 μL TCNB buffer. Mouse monoclonal anti-MMP-9 (1 μg) in 100 μL of TCNB buffer was added to the wells and incubated for two hours at room temperature in a microplate mixer (USA Scientific, Ocala, FL, USA). Plates were then washed three times with 200 μL TCNB buffer and 100 μL of the samples were added to the wells in duplicate and allowed to incubate overnight at 4°C in a microplate mixer. After washing the plates three times with TCNB buffer, 1 mM APMA in 100 μL assay buffer (50 mM Tris–HCl pH 7.6, 200 mM NaCl, 5 mM CaCl2, 20 μM ZnCl, and 0.05% Brij® L23) was added to the wells to activate pro-MMP-9 in the samples to detect total levels of MMP-9, then covered in aluminum foil and incubated for 90 min at 37°C. To detect only endogenously active levels of MMP-9, APMA was not added and only 100 μL assay buffer was added to the wells. Next, 100 μL of assay buffer with 2 μM FRET peptide was added to the wells to bring total FRET peptide concentration to 1 μM. Fluorescence (relative fluorescence units, RFUs) was measured after 24 h of incubation at 37°C at 485 nm excitation and 528 nm emission in a Synergy HT Multi-mode microplate fluorescence reader (BioTek, Winooski, VT, USA) running Gen5™ data analysis software. A substrate control well was used to subtract baseline fluorescence from the sample wells.
Fluorometric MMP-2/-9 activity measurement without immunocapture
Recombinant MMPs were activated by adding APMA for a final concentration of 1 mM and incubated for 90 min at 37°C. Active MMPs were prepared in 100 μL assay buffer and mixed with 2 μM FRET peptide in 100 μL assay buffer for a final FRET peptide concentration of 1 μM. Fluorescence was monitored after a 24 h incubation period at 37°C at the same wavelengths mentioned above.
Substrate-specific zymography for determination of activity of MMP-2 and MMP-9 was done on brain homogenates, HT-1080 conditioned media, and RBE4 cell lysates as described before [27, 60]. Brain homogenates or RBE4 cell lysates (50 μg total protein) were mixed in 10 μL zymogram sample buffer (Cat. No. 161–0764; Bio-Rad, Hercules, CA). 15 μL of HT-1080 conditioned media at 1:10 dilution in zymogram sample buffer was loaded in the gels. Proteins were separated by electrophoresis in a SDS-PAGE gel (8%) containing 0.1% (w/v) gelatin (Cat. No. G-2500; Sigma-Aldrich) at 150 V constant voltage. Gels were then washed twice in 2.5% Triton X-100 to remove SDS for 20 min then incubated for 24 h at 37°C in incubation buffer (50 mM Tris–HCl pH 7.6 containing 200 mM NaCl, 10 mM CaCl2, 0.02% Brij® L23, 0.02% NaN3). Gels were stained with Coomassie Brilliant Blue R solution (Cat. No. B6529; Sigma-Aldrich) for 1 h then destained in 10% acetic acid for one day before being scanned with an HP Scanjet 8300 scanner. Densitometric analysis of lytic zones at 94 and 88 kDa was performed using ImageJ (freely provided by NIH). The rat MMP-9 comprises two bands running in the zymography gels at 94 kDa (glycosylated form) and 88 kDa (intermediate form) .
Recombinant MMP-2 and MMP-9 were mixed in Laemmli sample buffer (Cat. No. 161–0737; Bio-Rad; Temecula, CA) containing 5% 2-mercaptoethanol and boiled for 10 min. Samples were loaded into TGX 4-20% precast gels (Cat. No. 456–1095; Bio-Rad) and subjected to electrophoresis. Proteins were transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes using a Trans-Blot Turbo apparatus (Bio-Rad) at 25V for 30 min. Membranes were blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline (TBS). Membranes were incubated overnight at 4°C with primary antibodies against MMP-9 (mouse anti-MMP-9; Cat. No. MS-817-P; Thermo Scientific) and MMP-2 (rabbit anti-MMP-2; Cat. No. RPCA-MMP2; EnCor Biotechnology Inc., Gainesville, FL, USA) diluted 1:1000 in 5% milk in TBST. After washing with TBST (four times for 5 min each), membranes were incubated for 1 h at room temperature with goat anti-mouse IRDye 800CW secondary antibody (Cat. No. 926–32210; Li-Cor Biotechnology) diluted 1:30,000, and goat anti-rabbit 680RD antibody (Cat. No. 926–68071) diluted 1:40,000 in 5% milk in TBST. Protein bands were visualized using an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE, USA).
GraphPad Prism 5 was used for statistical analysis. Statistical significance was assessed by ANOVA followed by Newman-Keuls corrections for multiple comparisons or with a Student’s t-test. Linear regression analysis was used to determine correlation information and significance. Bar graphs are shown as mean ± SEM. Probability p < 0.05 was considered statistically significant.