Middle cerebral artery occlusion
A total of 56 Male Wistar-Hanover rats (Møllegaard Breeding Centre, Copenhagen, Denmark) weighing approximately 300–350 g were obtained from Harlan, Horst, Netherlands, and were used for the procedures. The animals were housed under controlled temperature and humidity with free access to water and food. The experimental procedures were approved by the University Animal Ethics Committee (M43-07).
Anaesthesia was induced using 4.5% halothane in N2O:O2 (70%:30%) and was maintained by inhalation of 1.5% halothane by mask. To confirm proper occlusion of the right MCA, a laser-Doppler probe (Perimed, Järfälla, Sweden) was fixed on the skull (1 mm posterior to the bregma and 6 mm from the midline on the right side) to measure local cortical blood flow in an area supplied by the MCA. A polyethylene catheter was inserted into a tail artery to measure the mean arterial blood pressure, pH, pO2, pCO2, and plasma glucose. A rectal temperature probe connected to a homeothermal blanket was used to maintain body temperature at 37°C during the procedure.
An intraluminal filament technique was used to induce transient MCAO . Briefly, an incision was made in the midline of the neck and the right common, external, and internal carotid arteries were exposed. The common and external carotid arteries were permanently ligated with sutures. A filament was inserted into the internal carotid artery via an incision in the common carotid artery and advanced until the rounded tip reached the entrance to the right MCA. The resulting occlusion was visualized by laser-Doppler as an abrupt 80–90% reduction in local cortical blood flow After 2 h of occlusion, the rat was re-anesthetized to allow withdrawal of the filament; reperfusion was verified by laser-Doppler recording. 20–30% of the animals were terminated in conjunction with surgery and MCAO due to imperfect drop in laser-Doppler flow. (They were not included because they did not show the expected reduction in blood flow).
To inhibit MEK1/2, animals were injected intraperitoneal with 30 mg/kg/day of U0126 dissolved in dimethylsulfoxide (DMSO), beginning at reperfusion (0 h), at 6 h, 12 h, or 24 h post occlusion . Rats in the control groups were injected with equal volumes of DMSO. The dose of U0126 was chosen on the basis of previous experiments .
Harvesting cerebral vessels and brain tissue
At 48 h post MCA occlusion, MCAO rats, and MCAO rats treated with U0126, and their respective DMSO controls were anesthetized and decapitated. The brains were removed and immersed in ice-cold bicarbonate buffer solution of the following composition (mM): NaCl 119, NaHCO3, 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. The right and left MCAs and surrounding brain tissue were dissected out using a dissection microscope, snap frozen, and stored at -80°C for immunohistochemical analysis. A large number of proximal MCA segments were also harvested and pooled for protein extraction and western blot analysis.
The animals were subjected to a neurological examination prior to recirculation and immediately before they were sacrificed (48 hours after MCAO), according to an established scoring system [34, 35]: 0, no visible deficit; 1, contralateral forelimb flexion, when held by tail; 2, decreased grip of contralateral forelimb; 3, spontaneous movement in all directions, but contralateral circling if pulled by tail; 4, spontaneous contralateral circling; 5, death.
Brain damage evaluation
The brains were sliced coronal in 2-mm thick slices and stained with 0.5 mg/ml 1% 2, 3, 5-triphenyltetrazolium chloride (Sigma, St Louis, MO) dissolved in buffer solution at 37°C for 20 minutes. The extent of the ischemic brain damage was calculated as a percentage of the total brain volume in the slices using the software program Brain Damage Calculator 1.1 (MB Teknikkonsult, Lund, Sweden). The pictures were evaluated by two independent researchers unknown to the treatment group.
For immunofluorescence analysis, the MCA and the surrounding brain tissue were dissected out, placed into Tissue TEK (Gibo, Invitrogen A/S, Taastrup, Denmark), and frozen on dry ice; thereafter, they were sectioned into 10-μm thick slices. Cryostat sections of the arteries and brain tissue were fixed for 10 minutes in ice-cold acetone (-20°C) and then rehydrated in phosphate buffer solution (PBS) containing 0.3% Triton X-100 for 15 minutes. The tissues were then permeabilized and blocked for 1 hour in blocking solution containing PBS, 0.3% TritonX-100, 1% bovine serum albumin (BSA), and 5% normal donkey serum, and then incubated over night at 4°C with either rabbit anti-phosphoERK1/2 MAPK (ab4376; Cellsignaling, Danvers, MA) diluted 1:50, rabbit anti-rat MMP-9 (ab7299; Abcam, Cambridge, MA) diluted 1:400, or rabbit anti-human TIMP-1 (AB770; Chemicon, Copenhagen, Denmark) diluted 1:200. All primary antibodies were diluted in PBS containing 0.3% Triton X-100, 1% BSA, and 2% normal donkey serum. Sections were subsequently incubated for 1 hour at room-temperature with secondary Cy™2-conjugated donkey anti-rabbit (711-165-152; Jackson ImmunoResearch, Europe Ltd., Suffolk, UK) diluted 1:200 in PBS containing 0.3% Triton X-100 and 1% BSA. The sections were subsequently washed with PBS and mounted with Permafluore mounting medium (Beckman Coulter, PNJM0752). Immunoreactivity was visualized and photographed using a Nikon confocal microscope (EZ-c1, German) at the appropriate wavelength. The same procedure was used for the negative controls except that primary or secondary antibodies were omitted. There was also a know sample as positive control to compare with the present samples to avoided any probability failure in results. Data using blocking peptide (sequence used for the immunization) were provided by the supplier.
Double immunofluorescence labelling was performed for TIMP-1, MMP-9, and phosphorylated ERK1/2 versus smooth muscle actin or glial fibrillary acidic protein (GFAP), an astrocyte/glial cell marker. In addition to the antibodies described above, we used mouse anti-rat smooth muscle actin antibodies (SC-53015; Santa Cruz Biotechnology, Inc, Santa Cruz, CA) diluted 1:200 and mouse anti-GFAP (G3893; Sigma) diluted 1:600 in PBS containing 0.3% Triton X-100, 1% BSA, and 2% normal donkey serum. The secondary antibodies were Cy™2-conjugated donkey anti-rabbit (Jackson ImmunoResearch) diluted 1:200 and Texas Red-labeled donkey anti-mouse (Jackson ImmunoResearch Europe) diluted 1:300 in PBS containing 3% Triton X-100 and 1% BSA. The antibodies were detected at the appropriate wavelengths using a confocal microscope (EZ-cl, Germany).
Fluorescence intensity was measured using ImageJ software http://rsb.info.nih.gov/ij/. Measurements were made in 4 different preset areas (located on the clock at 0, 3, 6 and 9 h) from 4 vessel sections from each vessel sample and the investigator was blinded to the treatment group of each sample. The fluorescence intensity of each treatment group was given as the percentage change relative to control; the control value was normalized to 100%. The mean value for each was used for comparisons [33, 36].
Proximal MCA segments (n = 12 rats in each group; vessels from 3 rats were pooled for each measurement and experiments were done in total 4 times) were harvested and frozen in liquid nitrogen and homogenized in cell extract denaturing buffer (BioSource, Carlsbad, CA) that contained both phosphatase inhibitor and protease inhibitor cocktails (Sigma). Whole cell lysates were sonicated on ice for 2 min, centrifuged at 15 000 × g at 4°C for 30 min, and the supernatants were collected as protein samples. Protein concentrations were determined using standard protein assay reagents (Bio-Rad, Hercules, CA) and stored at -80°C awaiting immunoblot analysis. The protein homogenates were diluted 1:1 (v/v) with 2× sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad). Protein samples (25–50 μg of total protein) were boiled for 10 min in SDS sample buffer and separated on 4–15% SDS Ready Gel Precast Gels (Bio-Rad, USA) for 120 min at 100 v and transferred to nitrocellulose membranes by electroblotting (Bio-Rad) at 100 v for 60 min. The membrane was then blocked for 1 hour at room temperature with PBS containing 0.1% Tween-20 (Sigma) and 5% non-fat dried milk and incubated with primary antibodies, as appropriate [rabbit anti-rat MMP-9 (ab7299; Abcam) and rabbit anti-human TIMP-1 (AB770; Chemicon)], diluted 1:200-1 000 overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies (Amersham Biosciences, Piscataway, NJ) diluted 1: 5 000–10 000 for 1 hour at room temperature. The labeled proteins were developed using the LumiSensor Chemiluminescent HRP Substrate kit (GenScript Corp., Piscataway, NJ). To detect multiple signals on a single membrane, the membrane was incubated in Restore Plus western blot stripping buffer for 5–15 min at room temperature (Pierce Biotechnology, Inc., Rockford, IL) between the various labeling procedures. The membranes were visualized using a Fujifilm LAS-1000 Luminescent Image Analyzer (Stamford, CT), and band intensity was quantified using Image Gauge Version 4.0 (Fuji Photo Film Co., Ltd., Japan). Three independent experiments were performed in duplicate.
Calculations and statistical analyses
Data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using the nonparametric Kruskal-Wallis test with Dunn's post hoc test for quantitative immunohistochemistry and western blot evaluation. One-way analysis of variance (ANOVA) with Dunnett's test was used for infarct volume studies (using GraphPad Prism v 4). P-values less than 0.05 were considered significant; "n" refers to the number of rats.