Middle cerebral artery occlusion
Male Wistar-Hanover rats (Møllegaard Breeding Centre, Copenhagen, Denmark) weighing approximately 300–350 g were used. Experimental procedures were approved by the Lund University Animal Ethics Committee (M43-07). The animals were initially anaesthetized using 4 % halothane in N2O:O2 (70 %:30 %). Thereafter the rats were kept anaesthetized through a mask with 2 – 2.5 % halothane during the operation. To confirm a proper occlusion of the proximal middle cerebral artery (MCA), a laser Doppler probe (Perimed, Järfälla, Sweden) was fixed on the skull (1 mm posterior to bregma and 6 mm from the midline of the right side), measuring regional cortical blood flow. A polyethylene catheter was inserted into a tail artery for measurement of mean arterial blood pressure (MAP), 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 operational procedure.
An intraluminal filament technique was used to induce transient MCAO as outlined before [9]. 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 by sutures. A filament was inserted into the internal carotid artery via an incision in the common carotid artery and further advanced until the rounded tip reached the entrance of the right MCA. The resulting occlusion was visible by laser-Doppler as an abrupt reduction of cerebral blood flow of approximately 80–90 %. The anesthesia was then discontinued and the animals allowed recovering.
Two hours after the occlusion, the rat was re-anesthetized to allow for withdrawal of the filament and subsequent reperfusion. In conjunction with the reperfusion (0 h), at 6 h or at 12 h afterwards, and at 24 h after the start of the reperfusion in the same animal, the rats were injected intraperitoneal with 30 mg/kg U0126 dissolved in dimethylsulfoxide (DMSO) while control groups received the same volume of vehicle (DMSO). In preliminary experiments we evaluated U0126 doses varying between 10 mg/kg and 100 mg/kg (n = 3–6) (unpublished data); 30 mg/kg was the lowest that elicited a clear significant effect on infarct volume. Control rats were injected with an equal volume of DMSO at the same time points. The rats were neurologically examined immediately before sacrifice according to an established system [26]. For details of the neurology scale see [20]. Forty-eight hours after MCAO, rats were anesthetized and decapitated; the brain was removed and placed in cold buffer solution. The right and left MCAs and associated brain tissue were dissected, snap frozen in cold isopentane, and maintained at -80°C for further examination with immunohistochemistry.
The specificity of U0126 has been tested in numerous studies previously [13, 18] on isolated cells and in vivo; we have performed this on cerebral blood vessels with and without the MEK1 inhibitor [20, 27]. In addition, MCAO and organ culture elicited an early increase in the pERK1/2 activity but not in pP38 or in pJNK [28].
Evaluation of brain damage and edema
Brains were sliced coronal into 2-mm thick sections and stained by 1% 2, 3, 5-triphenyltetrazolium chloride dissolved in buffer solution at 37°C for 20 min. The size of ischemic brain damage and the degree of edema (swelling of the right part of the brain relative to the left contra lateral side) were calculated as a percentage of the total brain volume using the software program Brain Damage Calculator 1.1 (MB Teknikkonsult, Lund, Sweden).
Neurological examination
The animals were examined neurologically before recirculation and immediately before they were sacrificed, 48 hours after MCAO according to an established scoring system [26, 29].
Immunostaining
For immunostaining, the middle cerebral artery and the surrounding brain tissue were dissected, placed on Tissue TEK (Gibo, Invitrogen A/S, Taastrup, Denmark), and frozen on dry ice [17]. Thereafter, they were sectioned into 10 μm-thick slices. Cryostat sections of the arteries and tissue were fixed for 10 min in ice-cold acetone (-20°C) and rehydrated in PBST (phosphate buffer solution containing 0.3% Triton X-100) for 15 min at room temperature. The tissues were then permeabilized and blocked for 1 h in blocking solution containing PBS, 0.3% TritonX-100, 1% bovine serum albumin (BSA), and 5% normal donkey serum to ensure antibody specificity. They were incubated overnight at 4°C with primary antibodies: rabbit antihuman ETB (Abcam, ab1921) 1:400, rabbit antihuman AT1 (Santa Cruz, sc-1173), goat antihuman 5-HT1B (Santa Cruz, sc1460) and goat antihuman ETA (Santa Cruz, sc-21194) 1:100, rabbit antiphospho ERK 1/2 MAPK (Cellsignaling #4376) 1:50, and rabbit anti phospho Elk-1 (Cellsignaling #9181) 1:50 (diluted in PBS containing 0.3% Triton X-100, 1% BSA, and 2% normal donkey serum). Sections were subsequently incubated for 1 h at room temperature with secondary antibodies: Cy™2-conjugated donkey anti rabbit (JacksonImmunoResearch, 711-165-152) and Cy™2-conjugated donkey anti goat (JacksonImmunoResearch, 705-225-003) diluted 1:200 in PBS containing 0.3% Triton X-100 and 1% BSA. The sections were washed with PBS and mounted with Permafluor mounting medium (Beckman Coulter, PN IM0752). Immunoreactivity was visualized and photographed with a Nikon confocal microscope (EZ-c1, German) at the appropriate wavelength. The same procedure was used for the negative controls, but primary antibodies were omitted. The fluorescence intensity was measured with the software image J http://rsb.info.nih.gov/ij/ The fluorescence was measured in 4–6 areas in each tissue (in a blinded fashion), and the mean value was used.
Double immunostaining
Double immunostaining was done for endothelin ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptor protein, and phosphorylated ERK1/2 and Elk-1 proteins versus smooth muscle actin, expressed in the smooth muscle cells. The same antibodies were used as above but in addition mouse anti rat smooth muscle actin antibodies (Santa Crus, SC-53015) 1:200, diluted in PBS containing 0.3 % Triton X-100, 1 % BSA and 2 % normal donkey serum. The secondary antibodies used were donkey α-rabbit Cy™2- (JacksonImmunoResearch,) 1:200, donkey α-goat Cy™2- (Jacksson,) 1:200 and donkey α-mouse Texas Red (JacksonImmunoResearch,) 1:250 diluted in PBS containing 3 % Triton X-100 and 1 % BSA. The antibodies were then detected at the appropriate wave lengths in a confocal microscope (EZ-cl, Germany).
Western blot experiments
The proximal MCA segments (n = 12 in each group) were harvested and frozen in liquid nitrogen and homogenized in cell extract denaturing buffer (BioSource, USA) with addition of a phosphates inhibitor cocktail and protease inhibitor cocktail (Sigma, USA). Whole cell lysates were sonicated for 2 min on ice, centrifuged at 15,000 × g at 4°C for 30 min, and the supernatants were collected as protein samples. The Protein concentrations were determined using the protein assay reagents (Bio-Rad, Hercules, CA, USA) and stored at -80°C until immunoblotting assay. The protein homogenates were diluted 1:1 (v/v) with 2 × SDS sample buffer (Bio-Rad). 25–50 μg of total proteins were boiled for 10 min in SDS sample buffer and separated by 4–15 % SDS Ready Gel Precast Gels (Bio-Rad, USA) for 120 min at 100 v, and transferred electrophoretically to nitrocellulose membranes (Bio-Rad) at 100 v for 60 min. The Membrane was then blocked for 1 h at room temperature with phosphate buffered saline (PBS) containing 0.1 % Tween-20 (Sigma) and 5 % non-fat dried milk, and incubated with primary antibody, Rabbit polyclonal to endothelin B receptor (ab 1921), diluted 1:200 overnight at 4°C, followed by incubation with anti-rabbit IgG, horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) diluted 1: 5000–10.000 for 1 h at room temperature. The probed proteins were developed by .LumiSensor Chemiluminescent HRP Substrate kit (GenScript Corp., Piscataway, NJ, USA). To detect multiple signals using a single membrane, the membrane was incubated for 5–15 min at room temperature with restore plus Western blot stripping buffer (Pierce Biotechnology, Inc., Rockford, IL. USA). The membranes were visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer (Stamford, CT, USA.). The quantification of band intensity was analyzed with Image Gauge Ver. 4.0 (Fuji Photo Film Co., LTD., Japan). Three independent experiments were performed in duplicate.
Statistical analysis
Data are expressed as the mean ± s.e.m. Statistical analyses were performed using the nonparametric Kruskal-Wallis test with Dunn's post hoc test for quantitative immunohistochemistry and one-way ANOVA analysis of variance with Dunnett's test for infarct and edema brain evaluation. For Western blot comparisons between two groups were performed using two-tailed unpaired Student's t-test and at least 3 different samples or independent experiments were analyzed in each group. P-values less than 0.05 were considered significant, n = number of rats.