Signal transduction after MCA occlusion

Distal MCAO resulted in an abrupt decrease in CBF over the dorsolateral cortex; flow was reduced to 15 ± 3% of the baseline flow in the ischemic region. After 2 hours the block was removed. The flow subsequently returned to baseline. The animal were carefully monitored during the following 48 hours and then sacrificed. We calculated the neurology score (MCAO: 4.0 ± 0.5, P < 0.05 versus sham operated animals) (Table 1), collected tissue for immunostaining and Western blot, and determination of the infarct volume (24.8 ± 1.4% of total cerebrum) and the degree of edema (Fig. 1 and 2). The physiological parameters did not differ between the groups (Table 2). There was an increase in body temperature after MCAO in all groups which are in agreement with previous studies [10]; this did not differ between the different groups in our study.

Neurology score	Control (n = 7)	U0126 – 0 h (n = 7)	U0126 – 6 h (n = 6)	U0126 – 12 h (n = 6)
0 h	4 ± 0.0	4± 0.0	4 ± 0.0	4 ± 0.0
24 h	3.8 ± 3.4	3 ± 0.6	3.5 ± 0.5	3.8 ± 0.3
48 h	4 ± 0.5	2 ± 0.7	3.3 ± 0.7	3.8 ± 0.3

Comparison between the U0126 treated groups and vehicle treated rats (control). Data are expressed as mean ± S.D and n = number of rats.

Physiological Parameters	Control (n = 7)	U0126, 0 h (n = 7)	U0126, 6 h (n = 6)	U0126,12 h (n = 6)
pO2 (mmHg)	97.2 ± 2	108.3 ± 3.2	107.6 ± 7	120.4 ± 10
pCO2(mmHg)	52.6 ± 3	50.6 ± 1.9	49.6 ± 4.5	48.5 ± 0.6
pH	7.3 ± 0.01	7.3 ± 0.01	7.3 ± 0.02	7.5 ± 0.01
Plasma glucose (mmol/L)	12.1 ± 0.4	10.2 ± 0.6	11.1 ± 0.3	10.7 ± 0.4
MAP (mmHg)	91.1 ± 2.2	92.6 ± 1.8	91.8 ± 2.5	101.4 ± 4.7
Temperature during operation (°C)	37.1 ± 0.2	37.2 ± 0.1	37.2 ± 0.1	37.3 ± 0.2
Temperature before reperfusion (°C)	39.3 ± 0.1	39.2 ± 0.1	39.3 ± 0.2	38.9 ± 0.2
Temperature after reperfusion (°C)	38.1 ± 0.1	38.6 ± 0.3	38.1 ± 0.1	38.6 ± 0.2
Weight loss 48 h after occlusion (%)	13.4 ± 1.3	8.0 ± 1.8	16.1 ± 3.2	10.3 ± 2.4

Values are the mean % of control ± s.e.m., P > 0.05 between the groups, n = number of rats.

We subsequently assessed if MCAO leads to activation of pERK1/2 and pElk-1 in the smooth muscle cells of the MCA, the associated microvessels, and in brain tissue. There was weak staining of both in vehicle control (Fig. 3). The results showed that pERK1/2 and pElk-1 were markedly activated at 48 hours after the MCAO + vehicle (Fig. 3). The pERK1/2 and pElk-1 immunoreactivity were localized in the cytoplasm of the smooth muscle cells as verified by co-localization experiments with smooth muscle specific actin (Fig. 5). As can be seen in the illustration there was a significant enhanced expression of pERK1/2 (185 ± 11 %) and pElk-1 (174 ± 6 %) in the MCA leading to the infarct and in associated microvessels (130 ± 10 % and 130 ± 6 %, respectively) (Fig. 4). However, there was no significant change in their expression in associated brain tissue (106 ± 3 % and 117 ± 13 %, respectively) or in other regions of the brain. There was a weak expression of pElk-1 in cell bodies within the brain tissue around the MCA.

Inhibition of signal-transduction

Administration of the MEK1-specific inhibitor U0126 (30 mg/kg, intraperitoneal), which blocks the enzymatic activity of MEK1 [11] reduced both the infarct volume and the neurology score when given in conjunction with the start of the reperfusion (0 hour) or at 6 hours after the MCAO; the reductions were significant for infarct volume (11.8 ± 2 % and 14.6 ± 3 %; respectively of cerebrum, P < 0.05; Fig. 1 and 2A), and neurology score (2 ± 0.7 and 3.3 ± 0.7; respectively, P < 0.05) but not for the edema (Table 1) (Fig. 2B). The administration of U0126 with start at 12 hours after the initiation of reperfusion did not result in a significantly diminished infarct volume (20.3 ± 1 % of cerebrum; Fig. 2A) or the neurology score (3.8 ± 0.3; Table 1).

We subsequently assessed if MEK1 inhibition altered the cerebrovascular activation of pERK1/2 and pElk-1 in the vascular smooth muscle cells after MCAO. The results showed that systemic treatment with U0126 abolished the increase in pERK1/2 and pElk-1 activation after MCAO when the treatment was given in conjunction with reperfusion (0 hour) or with start 6 hours, and for pERK1/2 12 hours after the start of the reperfusion (Fig. 3 and 4). In the contralateral hemisphere there was only weak pERK1/2 and pElk-1 activity at baseline in the vasculature, and this was not affected by MCAO (Fig. 3); the levels of activity were comparable to those of the vehicle control. There was no significant change in brain tissue for pERK1/2 and pELK-1 activity (Fig. 3).

Cerebrovascular receptor expression

To investigate whether the MAPK activity leads to changes in receptor protein transcription, we analyzed the expression of vascular receptors using immunostaining and image analysis with confocal microscopy. We observed for the first time a significant increase in the expression of endothelin ET_B, angiotensin AT₁, and 5-hydroxytryptamine 5-HT_1B receptors in smooth muscle cells not only in the ischemic MCA but also in smooth muscle cells of cerebral microvessels associated with the ischemic region (Fig. 6 and 7). There were no changes in receptor expression in contralateral vessels. Importantly, the endothelin ET_A receptor expression was unchanged after MCAO and following treatment at all locations (Fig. 6 and 7). Double immunostaining for endothelin ET_B, angiotensin AT₁, and 5-hydroxytryptamine 5-HT_1B receptors versus smooth muscle actin, expressed in the smooth muscle cells, revealed clear co-localization. All three receptors co-localized with the smooth muscle cells, in addition, endothelin ET_B receptor protein was located in the endothelial cells of the cerebral vessels (Fig. 8); previous studies with the endothelial marker CD31 has verified this in cerebral arteries [12]. MCAO did however not show enhanced (or altered) expression of the endothelial ET_B receptors.

Systemic treatment with the MEK1 inhibitor abolished the increase in receptor expression in both vascular regions when treatment was initiated either at reperfusion (0 hour) or 6 hours afterwards but not when starting 12 hours after reperfusion (Fig. 6); this correlates well with the reduction in infarct volume at the same time points (Fig. 1 and 2A).

Western blot

The Western blot experiments showed a significant increase in ET_B protein level after MCAO as compare to the vehicle group (122 ± 9 %; p < 0.05). Treatment with the MEK inhibitor (U0126) given in conjunction with reperfusion (0 hour) prevented the increase in the ET_B receptor protein (101 ± 5%) (Fig. 9).

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 ET_B (Abcam, ab1921) 1:400, rabbit antihuman AT₁ (Santa Cruz, sc-1173), goat antihuman 5-HT_1B (Santa Cruz, sc1460) and goat antihuman ET_A (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 ET_B, angiotensin AT₁, and 5-hydroxytryptamine 5-HT_1B 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.