All animal procedures were carried out strictly within national laws and guidelines and approved by the Danish Animal Experimentation Inspectorate and the Ethical Committee for Laboratory Animal Experiments at the University of Lund.
Rat subarachnoid hemorrhage model
Subarachnoid hemorrhage was induced by a model originally devised by Svendgaard et al  and carefully described by Prunell et al . Svendgaard has in an elegant series of studies carefully analysed the correlation between amount of blood, angiographic vasoconstriction, CBF and cerebral metabolism. In a previous study using the same SAH model Delgado et al  have revealed a biphasic vasospasm in angiographic examinations of the arteries with a maximal acute vasoconstriction at ten minutes and a late maximal constriction at two days after SAH.
Male Sprague-Dawley rats (350-400 g) were anaesthetized using 5% halothane (Halocarbon Laboratories, River Edge, New Jersey) in N2O/O2 (30:70). The rat was intubated and artificially ventilated with inhalation of 0.5-1.5% halothane in N2O/O2 (70:30) during the surgical procedure. The depth of anaesthesia was carefully monitored and the respiration checked by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer, Copenhagen, Denmark). An electric temperature probe was inserted into the rectum of the rat to record the temperature, and found to be maintained at 37°C. An arterial catheter was placed in the tail artery to measure blood pressure and a catheter to monitor intracranial pressure (ICP) was placed in the subarachnoid space under the subocciptal membrane. At either side of the midline of the skull, 3 mm from the midline and 4 mm anteriorly from the bregma, holes were drilled through the skull bone down to the dura mater (without perforation) allowing the placement of two laser-Doppler flow probes to measure cortical CBF. Finally, a 27 G blunt canula with side hole was introduced 6.5 mm anterior to bregma in the midline at an angle of 30° to the vertical. With the aperture pointing to the right, the needle was lowered until the tip reached the skull base 2 to 3 mm anterior to the chiasma. After 30 minutes of equilibration 250 μl of blood was withdrawn from the tail catheter and injected intracranially via this canula at a pressure equal to the mean arterial blood pressure (MABP) (80-100 mmHg). Subsequently the rat was kept under anaesthesia for another 60 minutes to allow recovery from the cerebral insult after which catheters were removed and incisions closed. The rat was then revitalized and extubated. A subcutaneous injection of carprofen (4.0 mg/kg) (Pfizer, Denmark) was administered and the rat was hydrated subcutaneously using 40 ml isotonic sodium chloride at the end of the operation and at day one. During the period, the rat was monitored regularly, and if showing severe distress the animal was prematurely killed. In addition, a series of sham-operated rats were prepared. Two types of sham animals were studied; no fluid injection or injection of saline (250 μl) during 15 min to avoid any change in ICP . Since both procedures revealed the same outcome, they were grouped together in the statistical analysis. After two days either autoradiography measurements or harvesting of vessels were done (see below for details)
All surviving animals were neurologically examined using an established scoring system of 0 -5 (Table 1) [21, 22]. The animals were tested on the day before surgery. On day 1 and 2 after surgery each animal was tested twice. All animals were graded by personnel blinded to the experimental groups of the animals, and subjectivity in the observations was reduced by the involvement of two observers in the testing of each animal.
Measurement of the effect of the raf inhibitor on the cortical CBF and ICP
This group of animals went through the same procedure as the above-mentioned SAH animals until the injection of blood. To investigate the effect of the raf inhibitor on the cortical CBF and ICP 20 μl; 10-6 M of SB386023-b (a kind gift from Dr A A Parsons, GSK, UK) was given at the time point 0 h and 6 h after the induced SAH. The SB386023-b was injected intracisternally via the occipital membrane into the cisterna magna. The cortical CBF and ICP were measured during the entire time period 0-7 h after the SAH. After the experiment the animals were decapitated. Control sham animals received the same volume of saline.
Rat subarachnoid hemorrhage model with raf inhibition
This group of animals went through the same procedure as the above-mentioned SAH animals. In addition they were treated with 20 μl; 10-6 M of SB386023-b or the same volume of vehicle. Three groups of treated animals were examined; (i) 20 μl; 10-6 M SB386023-b was repeatedly injected intracisternally at 0, 6, 12, 24 and 36 h after the induced SAH (ii) 20 μl; 10-6 M SB386023-b was repeatedly injected intracisternally at 6, 12, 24 and 36 h after the induced SAH or (iii) 20 μl; 10-6 M SB386023-b was repeatedly injected intracisternally in the cistern magna at 12, 24 and 36 h after the induced SAH. The dose was chosen on the basis of previous detailed work on isolated arteries  and in vivo study with SAH and ERK1/2 inhibition . The dose used was chosen at near maximum inhibition and calculation of cerebrospinal fluid volume/turn over.
Autoradiographic measurements of regional CBF
Regional and global cerebral blood flow was measured by a model originally described by Sakurada  and modified by Gjedde .
In brief, after 48 hours of observation rats in the various groups (sham, SAH, SAH + vehicle and SAH treated with the raf inhibitor) were anaesthetized using 5% halothane in N2O/O2 (30:70). The animal was intubated and artificially ventilated with inhalation of 0.5 -1.5% halothane in N2O/O2 (70:30) during the surgical procedure. The anaesthesia and the respiration were monitored by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer AS, Denmark). A catheter to measure MABP was placed in the right femoral artery and a catheter for blood sampling was placed in the left femoral artery. This catheter was connected to a constant velocity withdrawal pump (Harvard apparatus 22, USA) for mechanical integration of tracer concentration. In addition, a catheter was inserted in one femoral vein for injection of heparin and for infusion of the radioactive tracer. The MABP was continuously monitored with a Powerlab Unit (ADInstruments, UK). A temperature probe was inserted into the rectum of the rat to record the temperature, which was regularly maintained at 37°C. The hematocrit was measured by a hematocrit centrifuge (Beckman Microfuge 11, USA). After 30 minutes of equilibration a bolus injection of 50 uCi of 14C-iodoantipyrine 4[N-methyl-14C] (Perkin-Elmer, Boston, USA) was given i.v. Arterial blood (122 μl) was withdrawn over 20 seconds. Immediately after this the animal was decapitated, the brain removed and immersed in isopentane chilled to - 50°C. The arterial blood sample was transferred to liquid scintillation counting vials containing 1 ml mixture of Soluene-350 and Isopropanol (1:1). The β-radioactivity scintillation counting was performed on the samples with a program that included quench correction (Packard 2000 CA, Denmark). The 14C activity in the tissue was determined after sectioning the brain in 20 μm sections at -20°C in a cryostat (Wild Leitz A/S, Glostrup, Denmark). The sections were exposed to x-ray films (Kodak, Denmark) together with 14C methylmethacrylate standards (Amersham Life Science, England) and exposed the films for 20 days. Densities of the autoradiograms were measured with a Macintosh computer equipped with an analog CF 4/1 camera (Kaiser, Germany) and a transparency flat viewer (Color-Control 5000, Weilheim, Germany). The 14C content was determined in several brain regions (see Table 2). The CBF was calculated from the brain tissue 14C activity determined by autoradiography using Gjedde et al.'s equation .
Harvest of cerebral arteries
After 48 hours of observation sham, SAH treated with SB386023-b or SAH + vehicle operated rats (see above SAH model) were anaesthetized with CO2 and decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution. Under a dissection microscope, the middle cerebral artery (MCA), the basilar artery (BA) and circle of Willis were dissected out. The MCA and BA were immediately mounted in myographs for in vitro pharmacology or snap frozen at -80°C and examined by real-time PCR or immunohistochemistry.
In vitro pharmacology myograph experiments
For contractile experiments a sensitive myograph was used for recording the isometric tension in isolated cerebral arteries [53, 54]. The vessels were cut into 1 mm long cylindrical segments and mounted on two 40 μm in diameter stainless steel wires in a Myograph (Danish Myo Technology A/S, Denmark). One wire was connected to a force displacement transducer attached to an analog-digital converter unit (ADInstruments, Oxford, UK). The other wire was connected to a micrometer screw, allowing fine adjustments of vascular tone by varying the distance between the wires. Measurements were recorded on a computer by use of a PowerLab unit (ADInstruments). The segments were immersed in a temperature controlled buffer solution (37°C) . The vessels were stretched to an initial resting tone of 2 mN and then allowed to stabilize at this tone for 1 hour. The contractile capacity was determined by exposing the vessels to an isotonic solution containing 63.5 mM of K+, obtained by partial change of NaCl for KCl in the above buffer. The contraction induced by K+ was used as reference for the contractile capacity . Only vessels responding by contraction of at least 2.0 mN to potassium for BA and 0.8 mN to potassium for MCA were included in the study. The presence of the endothelium was checked by precontracting the vessel using 5-HT (10 -6.5 M) (Sigma, St Louis, USA) and subsequently exposing the segments to carbachol (10-5 M) (Sigma, St Louis, USA). A relaxant response of the precontracted tension was considered indicative of a functional endothelium .
Concentration-response curves were obtained by cumulative application of 5-CT (Sigma, St. Louis, USA) in the concentration range 10 -12 to 10 -5 M, ET-1 (AnaSpec, San Jose, USA) in the concentration range 10 -14 to 10 -7 M, SB386023-b (a kind gift from Dr A A Parsons, GSK, UK) in the concentration range 10 -12 to 10 -6 M and Ang II (Sigma, St. Louis, USA) in the concentration range 10 -12 to 10 -6 M. Before application of Ang II the arteries were pretreated with the AT2 receptor antagonist PD123319 (10 -5.5 M) for 30 minutes (Sigma, St. Louis, USA). The concentration-response curves for SB386023-b were investigated both with and without precontraction with 5-HT (10 -6.5 M) (Sigma, St Louis, USA).
To quantify mRNA for the ETA, ETB, AT1, AT2 and 5-HT1B receptors, RT-PCR and real-time detection monitoring the PCR products was employed.
Total cellular RNA was extracted from BA, MCA and circle of Willis using the Trizol RNA isolation kit (Invitrogen, USA) following the suppliers instructions. Briefly, the arteries were homogenized in 1 ml of Trizol (Invitrogen, Sweden) by using a TissueLyser (VWR, Sweden). Subsequently 200 μl of chloroform was added and the samples were incubated in room temperature for 3 min, followed by centrifugation at 15000 g for 15 min at 4°C. The supernatant was collected and the organic phase discarded. 200 μl of chloroform was again added to remove all traces of phenol and the samples were centrifuged at 15000 g for 15 at 4°C. The aqueous supernatant was again collected and to precipitate the RNA equal amount of isopropanol was added and the samples incubated overnight at -20°C.
Subsequently, the RNA was centrifuged at 15000 g for 20 min at 4°C. The supernatant was discarded and the resulting pellet was washed with 75% ethanol, air dried and re-dissolved in diethylpyrocarbonate treated water. Total RNA was determined using a GeneQuant Pro spectrophotometer measuring absorbance at 260/280 (Amersham Pharmacia Biotech, Uppsala, Sweden).
Reverse transcription of total RNA to cDNA was carried out using the Gene Amp RNA kit (Perkin-Elmer Applied Biosystems, USA) in a Perkin-Elmer 2400 PCR machine at 42°C for 90 min and then 72°C for 10 min. The real-time quantitative PCR was performed with the GeneAmp SYBR Green PCR kit (PE Applied Biosystems) in a Perkin-Elmer real-time PCR machine (GeneAmp 5700 sequence detection system). The above synthesized cDNA was used as a template in a 25 μl reaction volume and a no template was included in all experiments. The system automatically monitors the binding of a fluorescent dye to double-strand DNA by real-time detection of the fluorescence during each cycle of PCR amplification. Specific primers for the rat ETA, ETB, AT1, AT2 and 5-HT1B receptor and house keeping gene elongation factor-1 (EF-1) were designed by using the Primer Express 2.0 software (PE Applied Biosystems) and synthesized by TAG Copenhagen A/S (Copenhagen, Denmark). For the primer sequence, refer to our previous studies .
The housekeeping gene EF-1 is used as a reference, since it is continuously expressed to a constant amount in cells.
The PCR reaction was carried out as follows: 50°C for 2 min, 95°C for 10 min and the following 40 PCR cycles with 95°C for 15 sec and 60°C for one min. Each sample was examined in duplicates. To verify that each primer-pair only generated one PCR product at the expected size a dissociation analysis was performed after each real-time PCR run. A blank control (without template) was used in all experiments. To prove that the cDNA of EF-1 and the ET, AT and 5-HT1B receptors were amplified with a similar efficacy during real-time PCR, a standard curve were made.
Tissue Lysis and Protein Content Determination
After dissection of the circle of Willis arteries, the vessels were collected and placed on ice, homogenized in lysis-buffer with protease- and phosphatase inhibitors. After 20 min incubation in lysis buffer on ice, homogenates were centrifuged at 4500 g for 10 min at 4°C and supernatant collected. Total protein concentration was determined using a BioRad DC kit (Hercules, CA, USA) and measuring absorbance at 750 nm on a Genesys 10 spectrophotometer (Thermo, Waltham, MA, U.S.A.). Lysates were used immediately or stored at -80°C.
Western Blot Analysis
Proteins of interest were evaluated in circle of Willis arteries from the various groups.
Lysates were dissolved in Tris-glycine SDS sample buffer and boiled for 5 min. Equal amounts of protein (50 μg/lane) were loaded on a 8% Tris-glycine gel (Invitrogen A/S, Taastrup, Denmark) and separated by SDS-PAGE. Molecular weight markers (New England BioLabs, Ipswich, MA, USA) were loaded on each gel for protein band identification. After separation, proteins were transferred to a nitrocelullose membrane (BioRad, Hercules, CA, USA). Subsequently the membrane was blocked with 6.5% non-fat milk in Tween-TBS (T-TBS) overnight 4°C. Membranes were then incubated with the primary antibody of interest: pERK1/2 (1:5000 dilution; Promega, Madison, WI, U.S.A.) or β-actin (1:1000 dilution; Sigma, Saint Louis, USA) for 1 h at 37°C, followed by 3 × 5 min wash with T-TBS. Subsequently the membranes were incubated with the appropriate secondary antibody: goat anti-rabbit IgG-horseradish peroxidase or goat anti-mouse IgG-horseradish peroxidase (1:5000; Pierce, Rockford, IL, U.S.A) for 1 h at room temperature, followed by 5 × 5 min wash with T-TBS. Levels of β-actin were used to confirm equal loading of the lanes. The membranes were developed using the Supersignal Dura kit (Pierce, Rockford, IL, U.S.A.) and visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer (Stamford, CT, U.S.A.).
For immunohistochemistry the indirect immunofluorescence method was used. The BA, with surrounding brain tissue were dissected out and frozen in ice cold isopentane. They were then sectioned into 10 μm thick slices in a cryostat. The cerebral artery crysections were fixed for 10 minutes in ice cold acetone and thereafter rehydrated in phosphate buffer solution (PBS) containing 0.25% Triton X-100 for 15 minutes. The tissue was then permeabilized and blocked for 1 hour in blocking solution containing PBS, 0.25% Triton X-100, 1% BSA and 5% normal donkey serum. The sections were incubated over night at 4°C with the following primary antibodies: rabbit antihuman ETB (IBL, 16207), diluted 1:400, goat anti mouse 5-HT1B (Santa Cruz Biotechnologies, sc-1461), diluted 1:100, AT1 (Santa Cruz Biotechnologies), diluted 1:100, mouse anti rat CD31 (Serotec, MCA1746), diluted 1:200, rabbit antiphospho ERK 1/2 MAPK (Cellsignalling #4376) diluted 1:50. and mouse anti rat smooth muscle actin (Serotec, MCA1905T) diluted 1:100. All dilutions were done in PBS containing 0.2% Triton X- 100, BSA 1% and 2% normal donkey serum. Sections were subsequently washed with PBS and incubated with secondary antibody for 1 hour at room temperature. The secondary antibody used were donkeyantimouse Cy™5 conjugated (JacksonImmunoResearch, 715-175-150), donkeyantirabbit Cy™3 conjugated (JacksonImmunoResearch, 711-165-152) diluted 1:200 in PBS containing 0.2% TritonX- 100 and BSA 1%. The sections were washed subsequently with PBS and mounted with permafloure mounting medium (Beckman coulter, PN IM0752). The same procedure was used for the negative controls but primary antibodies were omitted. The immunoreactivity of the antibodies were visualized and photographed with a Nikon Eclipse E800 microscope fitted with fluorescence optics at the appropriated wavelength.
Calculations and statistics
Data are expressed as mean ± standard error of the mean (s.e.m.), and n refers to the number of rats. Statistical analyses were performed with Kruskal-Wallis non-parametric test with Dunn's post-hoc test, where P < 0.05 was considered significant.
In vitro Pharmacology
Contractile responses in each segment are expressed as percentage of the 63.5 mM K+ induced contraction. Emax value represents the maximum contractile response elicited by an agonist and the pEC50 the negative logarithm of the drug concentration that elicited half the maximum response. For biphasic responses, Emax(1) and pEC50(1) describes the high affinity phase and Emax(2) and pEC50(2) describes the low affinity phase.
Data were analysed with the comparative cycle threshold (CT) method . The CT values of EF-1 mRNA were used as a reference to quantify the relative amount of ETA, ETB, AT1, AT2 and 5-HT1B mRNA. The relative amount of mRNA was calculated with the CT values of ETA, ETB, AT1, AT2 and 5-HT1B receptor mRNA in relation to the CT values of EF-1 mRNA in the sample by the formula X
0 = 2
, where X
0 is the original amount of target mRNA, R
0 is the original amount of EF-1 mRNA, CtR is the C
value for EF-1 and C
X is the C
value for the target.
Cerebrovascular protein lysates from the different groups were compared. Cerebral arteries from 2 animals were pooled for each group of experiment and each experiment was repeated 3 times. Quantitation of band density was performed with the electrophoresis computer analysis program Fujifilm Science Laboratory Image Gauge 4.0. The immunoblot optical density values were determined with repeated measurement and presented as percentage activity in the treated groups compared with the sham in which the sham group was set to 100%.
The images were analysed using the ImageJsoftware http://rsb.info.nih.gov/ij. The fluorescence in 4-6 different areas in each artery was measured and a mean value was calculated. These values are presented as percentage fluorescence in the SAH groups compared to the sham group, where the sham group is set to 100%.