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Am J Physiol Heart Circ Physiol 293: H3750-H3758, 2007. First published September 14, 2007; doi:10.1152/ajpheart.00857.2007
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Cerebrovascular ETB, 5-HT1B, and AT1 receptor upregulation correlates with reduction in regional CBF after subarachnoid hemorrhage

Saema Ansar,1,2 Petter Vikman,1 Marianne Nielsen,2 and Lars Edvinsson1,2

1Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden; and 2Department of Clinical Experimental Research, Glostrup University Hospital, Glostrup, Denmark

Submitted 23 July 2007 ; accepted in final form 10 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We hypothesize that cerebral ischemia leads to enhanced expression of endothelin (ET), 5-hydroxytryptamine (5-HT), and angiotensin II (ANG II) receptors in the vascular smooth muscle cells. Our aim is to correlate the upregulation of cerebrovascular receptors and the underlying molecular mechanisms with the reduction in regional and global cerebral blood flow (CBF) after subarachnoid hemorrhage (SAH). SAH was induced by injecting 250 µl blood into the prechiasmatic cistern in rats. The cerebral arteries were removed 0, 1, 3, 6, 12, 24, and 48 h after the SAH for functional and molecular studies. The contractile responses to ET-1, 5-carboxamidotryptamine (5-CT), and ANG II were investigated with myograph. The receptor mRNA and protein levels were analyzed by quantitative real-time PCR and immunohistochemistry, respectively. In addition, regional and global CBFs were measured by an autoradiographic method. As a result, SAH resulted in enhanced contractions to ET-1 and 5-CT. ANG II [via ANG II type 1 (AT1) receptors] induced increased contractile responses [in the presence of the ANG II type 2 (AT2) receptor antagonist PD-123319]. In parallel the ETB, 5-HT1B, and AT1 receptor, mRNA and protein levels were elevated by time. The regional and global CBF showed a successive reduction with time after SAH. In conclusion, the results demonstrate for the first time that SAH induces the upregulation of ETB, 5-HT1B, and AT1 receptors in a time-dependent manner both at functional, mRNA, and protein levels. These changes occur in parallel with a successive decrease in CBF. Thus there is a temporal correlation between the changes in receptor expression and CBF reduction, suggesting a linkage.

cerebral blood flow; cerebral ischemia

SUBARACHNOID HEMORRHAGE (SAH) occurring after the rupture of an intracranial aneurysm is associated with a high level of morbidity and mortality. The clinical syndrome is biphasic, with an immediate acute transient vasoconstrictive phase within the first hours and a delayed cerebral ischemia phase beginning 2–4 days after the SAH and peaking at approximately day 7 in humans (6). The sequence of events is more rapid in laboratory animals, such as the rat where the late phase of ischemia peaked at day 2 (7). Despite five decades of active investigation, the pathogenesis behind this is still unclear. Several theories have appeared, and some suggest that the delayed cerebral ischemia is related to endothelial cell damage and enhanced smooth muscle cell contractility, as well as increased amounts of spasmogenic substances (14, 39) or changes in inflammatory reaction products in the vascular walls (8, 31). In humans, there are increased levels of endothelin (ET)-1 (39) and 5-hydroxytryptamine (5-HT) (37, 38) in cerebral ischemia. In addition, angiotensin II (ANG II) has been shown to have an important role in the pathophysiology of cerebral ischemia (29). ET-1, 5-HT, and ANG II are all potent vasoconstrictors in cerebral arteries that mediate effects through their respective receptors: ET A (ETA), ET B (ETB) (21, 39), 5-HT receptors (37), and the ANG II type 1 (AT1) and type 2 (AT2) receptors (5, 32). They belong to the family of G protein-coupled receptors (12, 19, 21). The ETA receptor is localized on smooth muscle cells in the vascular wall and mediates proliferation and vasoconstriction, whereas the ETB receptors mainly occur on endothelial cells mediating vasodilatation (21). The AT1 and AT2 receptors behave much like the ET receptors, exhibiting almost opposing effects. The AT1 receptor mediates vasoconstriction, cell proliferation, and salt and water retention, whereas the AT2 receptor mediates vasodilatation, inhibits cell growth, and prevents thirst (33). In human cerebral arteries it has been shown that the predominant 5-HT receptor is of the 5-HT1B receptor subtype, mainly localized on the smooth muscle cells mediating vasoconstriction (24). In rodents, the 5-HT2A receptor subtype predominates while the 5-HT1B subtype upregulates in cerebral ischemia (15). Since the etiology of cerebral vasospasm is multifactorial, several receptors are involved in the development and maintenance of this prolonged pathological contraction. Thus we have earlier demonstrated the upregulation of contractile ETB, 5-HT1B, and AT1 receptors in the major cerebral arteries after SAH (2, 3, 15, 16, 35).

We suggest that cerebral ischemia leads to ET, 5-HT, and ANG II receptor upregulation in the vascular smooth muscle cells via a mechanism that involves transcription and translation. We hypothesize that the molecular events in cerebral arteries correlate closely with the changes associated with the late cerebral ischemia as monitored with quantitative measurement of regional CBF. This was addressed by closely following the time course for the upregulation of the receptors involved in cerebral arteries and the regional and global CBF after SAH. We positively link these events to the pathophysiology of cerebral ischemia following SAH. Treatment with an inhibitor of the MAPK ERK1/2 abolished both upregulation and flow reduction (2). There was a correlation between the upregulation of cerebrovascular receptors and the reduction in CBF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All animal procedures were carried out strictly within national laws and guidelines and were approved by the University Animal Experimentation Inspectorate.

Rat SAH Model

SAH was induced by a model carefully described by Prunell et al. (26). Male Sprague-Dawley rats (350–400 g) were anesthetized using 5% halothane (Halocarbon, River Edge, New Jersey) in 30% N2O-70% O2. The rat was intubated and artificially ventilated with inhalation of 0.5–1.5% halothane in 30% N2O-70% O2 during the surgical procedure. The depth of anesthesia was carefully monitored, and the respiration was checked by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer, Copenhagen, Denmark). An electric temperature probe was inserted into the rectum to record the temperature, which was maintained at 37°C. An arterial catheter to measure blood pressure was placed in the tail artery, and a catheter to monitor intracranial pressure (ICP) was placed in the subarachnoid space under the subocciptal membrane. At either side of the skull, 3 mm from the midline and 4 mm anterior of the bregma, holes were drilled through the scull bone down to the dura mater (without perforation), allowing the placement of two laser-Doppler flow probes to measure cortical CBF. Finally, a 27-gauge blunt canula with side hole was introduced stereotactically 6.5 mm anterior to the 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 scull base 2 to 3 mm anterior to the chiasma. After 30 min of equilibration, 250 µl of blood were withdrawn from the tail catheter and injected intracranially at a pressure equal to the mean arterial blood pressure (MABP; 80–100 mmHg). Subsequently, the rat was kept under anesthesia for another 60 min 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) was administered, and the rat was hydrated subcutaneously using 40 ml isotonic sodium chloride at the end of the operation. During the period of observation, the rat was monitored regularly, and if showing severe distress, the animal was prematurely euthanized. In addition, a series of sham-operated rats were prepared. They went through exactly the same procedure as described above with the exception that no blood was injected intracisternally (2, 3). Two types of sham-operated rats were studied: no fluid injection or injection of saline (250 µl) during 15 min to avoid any change in ICP. Both procedures revealed the same outcome. All surviving animals were neurologically examined using an established scoring system (1, 22).

Harvest of Cerebral Arteries

After 0, 1, 3, 6, 12, 24, and 48 h of observation, the SAH and the control/sham-operated rats were anesthetized with CO2 and decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution (see composition in In Vitro Pharmacology). Under a dissection microscope, the middle cerebral arteries (MCAs), the basilar artery (BA), and the circle of Willis were carefully dissected free from the brain and cleared of connective tissue. The vessel segments were immediately mounted in myographs for in vitro pharmacology or snap frozen at –80°C and examined by quantitative real-time PCR or immunohistochemistry.

In Vitro Pharmacology

For contractile experiments, a myograph was used for recording the isometric tension in isolated cerebral arteries (18, 23). 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, Aarhus, 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) of the following composition: (in mM) 119 NaCl, 15 NaHCO3, 4.6 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.5 CaCl2, and 5.5 glucose. The buffer was continuously aerated with oxygen enriched with 5% CO2 resulting in a pH of 7.4. The vessels were stretched to an initial resting tone of 2 mN and then allowed to stabilize at this tone for 1 h. 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 a reference for the contractile capacity (18). Only vessels responding by contraction of at least 2.0 mN to K+ for BA and 0.8 mN to K+ 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, MO) and subsequently exposing the segments to carbachol (10–5 M) (Sigma). A relaxant response by >50% of the precontracted tension was considered indicative of a functional endothelium (15).

Concentration-response curves were obtained by cumulative application of 5-CT (Sigma) in the concentration range 10–12 to 10–5 M, ANG II (Sigma) in the concentration range 10–12 to 10–6 M, and ET-1 (AnaSpec, San Jose, CA) in the concentration range 10–14 to 10–7 M. Before the application of ANG II, the arteries were pretreated with the AT2 receptor antagonist PD-123319 (10–5.5 M) for 30 min (a kind gift from Dr. P. Morsing, Astra-Zeneca, Molndal, Sweden). ANG II response was investigated both with and without the AT2 receptor antagonist PD-123319.

RNA Isolation

To quantify mRNA for the ETA, ETB, AT1, AT2, and 5-HT1B receptors, RT-PCR and real-time detection monitoring the PCR products were employed.

Total cellular RNA was extracted from BA, MCA, and circle of Willis using the TRIzol RNA isolation kit (Invitrogen) following the suppliers instructions. Briefly, the arteries were homogenized in 1 ml of TRIzol (Invitrogen) by using a TissueLyser (VWR). Subsequently, 200 µl of chloroform were added, and the samples were incubated in room temperature for 3 min, followed by centrifugation at 15,000 g for 15 min at 4°C. The supernatant was collected and the organic phase discarded. Chloroform (200 µl) was again added to remove all traces of phenol, and the samples were centrifuged at 15,000 g for 15 min at 4°C. The aqueous supernatant was again collected, and to precipitate the RNA, an equal amount of isopropanol was added and the samples were incubated overnight at –20°C.

Subsequently, the RNA was centrifuged at 15,000 g for 20 min at 4°C. The supernatant was discarded, and the resulting pellet was washed with 75% ethanol, air dried, and redissolved in diethylpyrocarbonate-treated water. Total RNA was determined using a GeneQuant Pro spectrophotometer measuring absorbance at 260-to-280 ratio (Amersham Pharmacia Biotech, Uppsala, Sweden).

Real-Time PCR

Reverse transcription of total RNA to cDNA was carried out using the Gene Amp RNA kit (Perkin-Elmer Applied Biosystems) 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 (a reagent control without added cDNA) 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 housekeeping gene elongation factor-1 (EF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were designed by using the Primer Express 2.0 software (PE Applied Biosystems) and synthesized by TAG Copenhagen A/S (Copenhagen, Denmark).

Receptor primers had the following sequences: ETA receptor forward: 5'-GTCGAGAGGTGGCAAAGACC-3'; ETA receptor reverse: 5'-ACAGGGCGAAGATGACAACC-3'; ETB receptor forward: 5'-GATACGACAACTTCCGCTCCA-3'; ETB receptor reverse: 5'-GTCCACGATGAGGACAATGAG-3'; 5-HT1B receptor forward: 5'-TCCGGGTCTCCTGTGTACGT-3'; 5-HT1B receptor reverse: 5'-GGCGTCTGAGACTCGCACTT-3'; AT1 receptor forward: 5'-GGATGGTTCTCAGAGAGAGTACAT-3'; AT1 receptor reverse: 5'-CCTGCCCTCTTGTACCTGTTG-3'; AT2 receptor forward: 5'-TCTGTTAGTGGGATGCATGTAATCA-3'; and AT2 receptor forward reverse: 5'-TGTGGGCCTCCAAACCATT-3'.

The housekeeping gene EF-1, GAPDH, and β-actin were used as a reference, since they are continuously expressed to a constant amount in cells.

The housekeeping primers were designed as follows: EF-1 forward: 5'-GCAAGCCCATGTGTGTTGAA-3'; EF-1 reverse: 5'-TGATGACACCCACAGCAACTG-3'; GAPDH forward: 5'-GGCCTTCCGTGTTCCTACC-3'; GAPDH reverse: 5'-CGGCATGTCAGATCCACAAC-3'; β-actin forward: 5'-GTAGCCATCCAGGCTGTGTTG-3'; and β-actin reverse: 5'-TGCCAGTGGTACGACCAGAG-3'.

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 s and 60°C for 1 min. Each sample was examined in duplicate. To verify that each primer pair only generated one PCR product at the expected size that 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 cDNAs of EF-1, GAPDH, β-actin, AT1, AT2, ETA, ETB, and 5-HT1B receptors were amplified with a similar efficacy during real-time PCR, a standard curve was made in which the CT values were plotted against cDNA concentration on the basis of the following equation: CT = [log(1 + E)]–1 log(concentration), where CT is the number of PCR cycles performed in one sample at a specific point of time and E is the amplification efficiency with an optimal value of one. Standard curves for ETA, ETB, AT1, AT2, 5-HT1B, β-actin, GAPDH, and EF-1 were performed by dilution of cDNA sample (1:10, 1:100, and 1:1,000) (data not shown).

Immunohistochemistry

The MCA and BA were dissected out and then placed onto Tissue TEK (Gibco) and frozen. They were then sectioned into 10-µm-thick slices. The primary antibodies used were rabbit anti-human ETB (IBL, 16207), diluted 1:400; goat anti-mouse 5-HT1B (sc-1461, Santa Cruz Biotechnologies), diluted 1:100; AT1 (sc-1173, Santa Cruz Biotechnologies), diluted 1:100; mouse anti-rat CD31 (MCA1746, Serotec), diluted 1:200; and mouse anti-rat smooth muscle actin (MCA1905T, Serotec), diluted 1:100. All dilutions were done in PBS with 10% fetal calf serum. The secondary antibodies used were donkey anti-mouse Cy5 conjugated (715-175-150, Jackson ImmunoResearch), 1:100; and donkey anti-rabbit Cy3 conjugated (711-165-152, Jackson ImmunoResearch), 1:100 in PBS with 10% fetal calf serum. The antibodies were detected at the appropriate wavelength in a confocal microscopy (Zeiss). As control, only secondary antibodies were used.

Autoradiographic Measurements of Regional CBF

Local CBF was measured by a model originally described by Sakurada et al. (30) and modified by Gjedde et al. (13).

In brief, rats in the various groups (0, 24, and 48 h after SAH) were anesthetized using 5% halothane in 30% N2O-70% O2. The animal was intubated and artificially ventilated with inhalation of 0.5 –1.5% halothane in 30% N2O-70% O2 during the surgical procedure. The anesthesia and the respiration were monitored by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer AS). 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) 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). 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). After 30 min of equilibration, a bolus injection of 50 µCi of 14C-iodoantipyrine 4[N-methyl-14C] (Perkin-Elmer, Boston, MA) was given intravenously. Arterial blood (122 µl) was withdrawn over 20 s. Immediately after this, the animal was decapitated, and the brain was removed and immersed in isopentane (J. T. Baker, Deventer, Netherlands) chilled to –50°C. The β-radioactivity scintillation counting was performed on the arterial blood samples with a program that included quench correction (Packard 2000 CA). 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) together with 14C methylmethacrylate standards (Amersham Life Science) 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 GoGoGoTable 4). The CBF was calculated from the brain tissue 14C activity determined by autoradiography using the equation of Gjedde et al. (13).


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  		Table 1. Contractile effects of ET-1 in MCAs and BAs after SAH

 

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  		Table 2. Contractile effects of 5-CT in MCAs and BAs after SAH

 

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  		Table 3. Contractile effects of ANG II in MCA after SAH

 

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  		Table 4. Regional cerebral blood flow 48 h post-SAH

 
Calculations and Statistics

Data are expressed as means ± SE, and n refers to the number of rats. Statistical analyses were performed with Kruskal-Wallis nonparametric test with Dunn's post hoc test, where P < 0.05 was considered significant. The correlation analyses were performed with Spearman's rank correlation coefficient.

In vitro pharmacology. Contractile responses in each segment are expressed as percentages 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 describe the high-affinity phase and Emax 2 and pEC50 2 describe the low-affinity phase.

Real-time PCR. Data were analyzed with the comparative cycle threshold (CT) method (16). 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 housekeeping genes mRNA in the sample by the formula Formula, where X0 is the original amount of target mRNA, R0 is the original amount of housekeeping gene mRNA, CTR is the CT value for housekeeping gene and CTX is the CT value for the target. GAPDH, β-actin, and EF-1 were used as housekeeping genes. Each sample was examined in triplicate, and the mean was used. Results are presented as means ± SE compared with 0 h, which was set to 100%.

Immunohistochemistry. The images were analyzed using the ImageJ software (http://rsb.info.nih.gov/ij/). The fluorescence in four to six different areas in each artery was measured, and a mean value was calculated. These values are presented as percent fluorescence in the SAH groups compared with the control group (0 h) or sham-operated group, where the control group is set to 100%.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SAH Model

The mortality rate of the animal model of SAH was 4%, and there was no difference in the mortality rate between the groups. The rats did not show any distressed behavior. All surviving animals were neurologically examined using an established scoring system (1, 22). All SAH animals received a score of 1 and the sham-operated groups a score of 0. In all operated rats, mean arterial blood pressure (105 ± 3 mmHg), PCO2 (39 ± 2 mmHg), PO2 (108 ± 4 mmHg), hematocrit (40 ± 1 mmHg) values, and temperature were within acceptable limits during the operation. As a result of the blood being injected, the cortical blood flow dropped over both hemispheres to 18 ± 3% of resting flow (there was no difference between the two laser-Doppler probe data), and the ICP increased from 9 ± 1 to 120 ± 11 mmHg. The laser-Doppler blood flow and the elevated ICP returned to the basal values within 1 h of postoperative monitoring.

In Vitro Pharmacology

K+ (63.5 mM) evoked contraction of the smooth muscle cells and was used as an internal control. K+-induced contractions did not differ significantly between the vessels from the different groups (Tables 1–3; P = 1.0). Emax and pEC50 values for respective group are presented in Tables 1–3.

Contractile response to ET-1. The contractile response to ET-1 was concentration dependent. The contractile response to ET-1 was successively increased by time after SAH, reaching a maximum at 48 h (Fig. 1A and Table 1). Both MCA and BA from SAH at 48 h showed a leftward shift of the curve to ET-1. This indicates an enhanced contractile response to ET-1 compared with that in the control rats where a sigmoidal curve was obtained. The concentration-response relation to ET-1 in the BA is illustrated in Fig. 1A.


Figure 1
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  		Fig. 1. Concentration (Conc) response curves elicited by cumulative application of endothelin (ET)-1, 5-carboxamidotryptamine (5-CT), and ANG II in rat cerebral arteries after subarachnoid hemorrhage (SAH). ET-1 [basilar artery (BA); A], 5-CT (BA; B), ANG II in presence of ANG II type 2 (AT2) receptor blocker PD-123319 [middle cerebral artery (MCA); C], and ANG II (MCA; D) are shown. Data are expressed as means ± SE; n = 4–6 rats/group.

 
Contractile response to 5-CT. 5-CT gave raise to a biphasic concentration-dependent contraction both in MCA and BA, indicating the presence of two receptors, 5-HT1B and 5-HT2A, in a manner previously characterized in detail (17). The contractile response to 5-CT successively increased with time, with a maximum at 48 h (Fig. 1B and Table 2), the BA is illustrated in the Fig. 1B.

Contractile response to ANG II. In MCA, ANG II (via AT1) induced a concentration-dependent response that increased with time in contractile response (in the presence of the AT2 receptor antagonist PD-123319). The contractile response to ANG II was successively increased with time, with a maximum at 24 h (Fig. 1C and Table 3). ANG II did not induce an increased contractility in the BA after SAH. In the absence of the AT2 receptor antagonist PD-123319, there was no increased contractile response to ANG II with time (Fig. 1D).

Real-Time PCR

The standard curves for each primer pair had almost similar slopes, demonstrating that EF-1, β-actin, GAPDH, ETA, ETB, AT1, AT2, and 5-HT1B cDNA were amplified with the same efficiency (data not shown). In each PCR experiment, a no-template control was included, and there were no signs of contaminating nucleic acids in those samples. Gene expression was normalized to the expression of three different housekeeping genes: β-actin, GAPDH, and EF-1. All the housekeeping genes gave the same results after normalization. Since the results from the three types of brain arteries were identical, the MCA, BA, and circle of Willis (n = 5–9) were added together for the statistical analysis. The ETB, 5-HT1B, and AT1 receptor mRNA levels were elevated by time after SAH. The mRNA receptor levels for ETB showed a quick response to SAH with an initial transient peak at about 1 h and a maximum at the time point 24 h after SAH (Fig. 2A). There was a significant increase in AT1 receptor mRNA at 24 h (Fig. 2B). The 5-HT1B receptor mRNA level showed an initial peak at 12 h with a maximum response at 24 h after SAH (Fig. 2C). The level of ETA (P = 0.54) and AT2 (P = 0.51) receptor mRNA levels remained unchanged in SAH compared with the control group (data not shown).


Figure 2
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  		Fig. 2. mRNA levels of ETB (A), ANG II type 1 (AT1; B), and 5-hydroxytryptamine 1B (5-HT1B; C) receptors after 0–48 h after SAH in rat cerebral arteries (MCA + BA + circle of Willis). ETB, AT1, and 5-HT1B receptor mRNA levels were elevated by time. Data were obtained by real-time PCR and are expressed as means ± SE values relative to elongation factor-1 mRNA levels; n = 7–10 rats/group. *P < 0.05.

 
Immunohistochemistry

Selective antibodies toward the ETB, 5-HT1B, and AT1 receptors visualized their smooth muscle cell localization using confocal microscopy. Double immunohistochemistry staining versus smooth muscle actin, expressed in the smooth muscle cells, and CD31, expressed in the endothelial cells, were performed to verify the localization. The immunohistochemistry experiments showed that the ETB, 5-HT1B, and AT1 receptor protein levels were elevated by time with a maximum at 48 h for the receptor subtypes (Fig. 3, A and B). The ETB receptor protein level was elevated already at 3 h after SAH, whereas both the AT1 and 5-HT1B receptor protein levels increased at 6 h after SAH. The enhanced expression was localized to the smooth muscle cells.


Figure 3
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  		Fig. 3. A: sections from the BA showing ETB, 5-HT1B, and AT1 immunoreactivity in the smooth muscle cell layer 0–48 h after SAH. There are increased expressions of the ETB, 5-HT1B, and AT1 receptor protein levels with time. Data were obtained with confocal microscopy. B: activation of the different protein levels in the BA measured with immunohistochemistry. Values are expressed as percentage of control and given as means ± SE. *P < 0.05.

 
Regional Cerebral Blood Flow

There was a significant global decrease in CBF in the SAH group compared with the control from 134 ± 7 to 53 ± 3 ml·min–1·100 g–1 at 48 h after SAH. At the time point 24 h after SAH, the global CBF was 82 ± 4 ml·min–1·100 g–1 (Fig. 4). In addition, there was significantly lower blood flow at 48 h compared with 24 h. All the regions showed similar results, and there was no significant difference between the regions in their response to the SAH (Table 4). Furthermore, as can be seen in the Table 4, the CBF was successively reduced with the time after SAH.


Figure 4
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  		Fig. 4. Global cerebral blood flow (CBF) after induced SAH in rats. There is a reduction in the global CBF compared with that in control rats. Data were obtained by an autoradiographic method, and data are expressed as means ± SE; n = 6 rats in each group. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Correlation Between Functional Data, mRNA and Protein Levels, and CBF

SAH induces a significant upregulation of ETB, 5-HT1B, and AT1 receptors in a time-dependent manner both at functional, mRNA, and protein levels. There is a difference in the time profile of the upregulation of the different receptors. The contractile ETB receptor appeared within the first 12–24 h after SAH, whereas both the AT1 and 5-HT1B receptors appeared between 24 to 48 h. The receptor mRNA expression revealed a maximum at 12–24 h for all receptor subtypes; however, the ETB receptor mRNA level showed an initial peak already at 1 h. The receptor protein expression revealed maximum at 24–48 h after SAH. The functional contractile responses were parallel with the protein expression. These changes are parallel with a successive decrease in CBF (Fig. 5). Thus there is a clear temporal correlation between the changes in receptor expression and reduction in CBF. This is illustrated for the ETB receptor (Fig. 5), but it is similar for AT1 and 5-HT1B receptors (data not shown). A recalculation of the data in our previous study (2) revealed that a specific inhibitor of raf resulted in the normalization of both protein and function of ETB and 5-HT1B receptors at the same time as the return of the CBF from 67 ± 4 to 125 ± 7 ml·min–1·100 g–1.


Figure 5
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  		Fig. 5. Correlation between functional data, protein levels, mRNA levels, and CBF for the ETB receptor. The functional contractile responses are parallel with the protein expression, and these changes are parallel with a successive decrease in CBF. Thus there is a temporal correlation between the changes in receptor expression and reduction in CBF.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to directly correlate the functional and protein expression of cerebrovascular smooth muscle receptors with the global and regional CBF after SAH. The receptor mRNA clearly preceded this event. Cerebral vasoconstriction is commonly seen after SAH due to the rupture of an intracranial aneurysm (40–70%) and sometimes after head trauma (5–10%) and intracranial surgery. A considerable number of the affected subjects die as a cause of cerebral ischemia. The first angiographic association was provided by Ecker and Riemenschneider (9) in 1951, and, subsequently, in 1966 Kågstrom et al. (20) demonstrated the delayed progressive vasoconstriction several days after the bleed (4, 36). This pattern of events has been confirmed in experimental studies (25). The relationship between the amount of subarachnoid blood and vasospasm is supported in both clinical and experimental studies (10, 11). Svendgaard and colleagues (7, 26, 27) have in an elegant series of studies carefully analyzed the correlation between the amount of blood, angiographic vasoconstriction, CBF, and cerebral metabolism. By intracisternal administration of 250 µl of blood, a rodent model was created that replicates the major parts of the events that occur in human SAH (28). By using this model, we have demonstrated that the late phase of cerebral ischemia is associated with de novo transcription of genes for contractile G protein-coupled receptors, for cytokines, and for inflammatory mediators (35); however, one missing piece of evidence is the relationship between late cerebral ischemia and cerebrovascular gene regulation. To address this crucial question, we have examined the detailed time course of SAH-induced upregulation of smooth muscle ETB, 5-HT1B, and AT1 receptors at both functional and molecular levels.

There is a difference in the time profile of the upregulation of the different receptors. The receptor mRNA expression revealed a maximum at 12–24 h for all receptor subtypes; however, the ETB receptor mRNA level showed an initial peak already at 1 h. The protein level for the ETB receptor increased at an earlier time point compared with the 5-HT1B and AT1. However, all three receptors showed a maximum at 48 h after SAH. The functional responses appeared later compared with mRNA and protein levels. The contractile ETB receptor appeared within the first 12–24 h after SAH, whereas both the AT1 and 5-HT1B receptors appeared between 24 to 48 h. The functional, mRNA, and protein levels for AT1 receptors showed parallel results with a significant upregulation of the receptor at 24 h. Thus we have demonstrated a tight correlation between transcriptional activation of the ETB, 5-HT1B, and AT1 receptors genes and the contractile responses toward their respective receptor agonists. However, the mRNA expression was always earlier than protein and function. In addition, the ETB receptor had an earlier upregulation compared with the other receptors, which suggest a differentiated system of activation and, therefore, again, different signal transduction pathways for the upregulation of the respective receptors. Importantly, the contractile responses and the molecular changes were associated with a parallel reduction in CBF. This is in agreement with our hypothesis that an increase in cerebrovascular receptor expression results in an increase in vascular tone after SAH, which in turn causes late cerebral vasoconstriction and reduced cerebral blood flow with subsequent development of cerebral ischemia and neuronal death.

We have previously studied in detail the molecular mechanisms involved in the upregulation of receptors in brain vessels. We demonstrated that the upregulation of ETB receptors after organ culture is mediated via increased transcription and subsequent translation of receptor mRNA. The transcriptional blocker actinomycin D prevented the upregulated ETB receptor observed after organ culture in cerebral arteries both for function and mRNA, whereas the translational inhibitor cyclohexamide prevented only functional responses. In the present study we have examined both the mRNA, protein expression, and the functional responses to the respective receptor activation. Because they were all modified in SAH, it clearly suggests that there is an enhanced expression of the receptors in the cerebral vessels.

In a previous study with the same methodology, angiographic examinations of the cerebral arteries revealed a biphasic vasospasm with a maximal acute cerebral constriction at 10 min and a late maximal vasoconstriction at 2 days after the SAH (7). Here we have demonstrated that the maximum reduction in regional and global CBF appears at 48 h after SAH. In addition, the enhanced contractility toward the endogenous agonists appeared with a maximum at 48 h, except from AT1 receptors. This suggests that the upregulation of cerebrovascular receptors and the reduction in CBF all correlate with the late vasoconstriction phase that occurs 48 h after SAH. In fact, plotting the functional data and molecular data in a correlation graph showed a significant correlation between function and protein expression. The response also correlates with the regional CBF in the present study. Further evidence for such a tight coupling between these events comes from our treatment studies; intracisternal administration of a MAPK ERK1/2 inhibitor (2) or a PKC inhibitor (3) resulted in loss of vascular ETB and 5-HT1B receptor upregulation and prevention of CBF reduction after SAH.

We have demonstrated that SAH is not linked to a single specific receptor subtype, and this may therefore explain the partial therapeutic effects observed in many trials. In clinical studies, 5-HT antagonists have proven ineffective in relieving clinical vasospasm (37); however, this does not exclude the involvement of 5-HT receptors, since the antagonists used were nonspecific, but suggests a multifactor pathogenesis. Clinical trials with the selective ETA antagonist clazosentan have been performed. The results demonstrate that clazosentan reduces the severity of vasospasm following aneurysmal SAH; however, there was no positive effect in the outcome of the patients (34). This supports our view that the inhibition of only one receptor system will not remedy other receptor systems involved. Instead, the mechanism responsible for the receptor upregulation might be a more promising target (2, 3).

In conclusion, we have revealed that SAH induces an upregulation of ETB, 5-HT1B, and AT1 receptors, which occur in a time-dependent manner both at functional, mRNA, and protein levels. The protein expression changes are parallel with a successive decrease in CBF. Thus there is a close temporal correlate between the changes in receptor expression and the reduction in CBF.


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This study was supported by the Swedish Research Council Grant 5958, the Heart and Lung foundation, King Oscar V. and Queen Victoria Foundation (Sweden), and the Danish Research Council and Lundbeck Foundation (Denmark).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Ansar, Dept. of Clinical Sciences, Div. of Experimental Vascular Research, BMC A13, Lund Univ., 221 84 Lund, Sweden (e-mail: saema.ansar{at}med.lu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17: 472–476, 1986.[Abstract/Free Full Text]
  2. Beg SA, Hansen-Schwartz JA, Vikman PJ, Xu CB, Edvinsson LI. ERK1/2 inhibition attenuates cerebral blood flow reduction and abolishes ETB and 5-HT1B receptor upregulation after subarachnoid hemorrhage in rat. J Cereb Blood Flow Metab 26: 846–856, 2006.[CrossRef][Web of Science][Medline]
  3. Beg SS, Hansen-Schwartz JA, Vikman PJ, Xu CB, Edvinsson LI. Protein kinase C inhibition prevents upregulation of vascular ETB and 5-HT1B receptors and reverses cerebral blood flow reduction after subarachnoid haemorrhage in rats. J Cereb Blood Flow Metab 27: 21–32, 2007.[CrossRef][Web of Science][Medline]
  4. Bergvall U, Galera R. Time relationship between subarachnoid haemorrhage, arterial spasm, changes in cerebral circulation and posthaemorrhagic hydrocephalus. Acta Radiol Diagn (Stockh) 9: 229–237, 1969.[Medline]
  5. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PB. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 165: 196–203, 1989.[CrossRef][Web of Science][Medline]
  6. Cook DA. Mechanisms of cerebral vasospasm in subarachnoid haemorrhage. Pharmacol Ther 66: 259–284, 1995.[CrossRef][Web of Science][Medline]
  7. Delgado TJ, Brismar J, Svendgaard NA. Subarachnoid haemorrhage in the rat: angiography and fluorescence microscopy of the major cerebral arteries. Stroke 16: 595–602, 1985.[Abstract/Free Full Text]
  8. Dumont AS, Dumont RJ, Chow MM, Lin CL, Calisaneller T, Ley KF, Kassell NF, Lee KS. Cerebral vasospasm after subarachnoid hemorrhage: putative role of inflammation. Neurosurgery 53: 123–133, 2003.[CrossRef][Web of Science][Medline]
  9. Ecker A, Riemenschneider PA. Arteriographic demonstration of spasm of the intracranial arteries, with special reference to saccular arterial aneurysms. J Neurosurg 8: 660–667, 1951.[Web of Science][Medline]
  10. Espinosa F, Weir B, Boisvert D, Overton T, Castor W. Chronic cerebral vasospasm after large subarachnoid hemorrhage in monkeys. J Neurosurg 57: 224–232, 1982.[Web of Science][Medline]
  11. Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 6: 1–9, 1980.[Web of Science][Medline]
  12. Furuta H, Guo DF, Inagami T. Molecular cloning and sequencing of the gene encoding human angiotensin II type 1 receptor. Biochem Biophys Res Commun 183: 8–13, 1992.[CrossRef][Web of Science][Medline]
  13. Gjedde A, Hansen AJ, Siemkowicz E. Rapid simultaneous determination of regional blood flow and blood-brain glucose transfer in brain of rat. Acta Physiol Scand 108: 321–330, 1980.[Web of Science][Medline]
  14. Grasso G. An overview of new pharmacological treatments for cerebrovascular dysfunction after experimental subarachnoid hemorrhage. Brain Res Brain Res Rev 44: 49–63, 2004.[CrossRef][Medline]
  15. Hansen-Schwartz J, Hoel NL, Xu CB, Svendgaard NA, Edvinsson L. Subarachnoid hemorrhage-induced upregulation of the 5-HT1B receptor in cerebral arteries in rats. J Neurosurg 99: 115–120, 2003.[Web of Science][Medline]
  16. Hansen-Schwartz J, Hoel NL, Zhou M, Xu CB, Svendgaard NA, Edvinsson L. Subarachnoid hemorrhage enhances endothelin receptor expression and function in rat cerebral arteries. Neurosurgery 52: 1188–1194, 2003.[Web of Science][Medline]
  17. Hoel NL, Hansen-Schwartz J, Edvinsson L. Selective up-regulation of 5-HT1B/1D receptors during organ culture of cerebral arteries. Neuroreport 12: 1605–1608, 2001.[CrossRef][Web of Science][Medline]
  18. Hogestatt ED, Andersson KE, Edvinsson L. Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels. Acta Physiol Scand 117: 49–61, 1983.[Web of Science][Medline]
  19. Iwai N, Inagami T. Identification of two subtypes in the rat type I angiotensin II receptor. FEBS Lett 298: 257–260, 1992.[CrossRef][Web of Science][Medline]
  20. Kågström EGT, Hanson J, Galera R. Changes in cerebral blood flow after subarachnoid haemorrhage. Excerpta Med Int Cong Series 110: 629–633, 1966.
  21. Masaki T, Vane JR, Vanhoutte PM. International Union of Pharmacology nomenclature of endothelin receptors. Pharmacol Rev 46: 137–142, 1994.[Web of Science][Medline]
  22. Menzies SA, Hoff JT, Betz AL. Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model. Neurosurgery 31: 100–106, 1992.[Web of Science][Medline]
  23. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41: 19–26, 1977.[Free Full Text]
  24. Nilsson T, Longmore J, Shaw D, Olesen IJ, Edvinsson L. Contractile 5-HT1B receptors in human cerebral arteries: pharmacological characterization and localization with immunocytochemistry. Br J Pharmacol 128: 1133–1140, 1999.[CrossRef][Web of Science][Medline]
  25. Peerless SJ, Fox AJ, Komatsu K, Hunter IG. Angiographic study of vasospasm following subarachnoid hemorrhage in monkeys. Stroke 13: 473–479, 1982.[Abstract/Free Full Text]
  26. Prunell GF, Mathiesen T, Diemer NH, Svendgaard NA. Experimental subarachnoid hemorrhage: subarachnoid blood volume, mortality rate, neuronal death, cerebral blood flow, and perfusion pressure in three different rat models. Neurosurgery 52: 165–175, 2003.[CrossRef][Web of Science][Medline]
  27. Prunell GF, Mathiesen T, Svendgaard NA. Experimental subarachnoid hemorrhage: cerebral blood flow and brain metabolism during the acute phase in three different models in the rat. Neurosurgery 54: 426–436, 2004.[Web of Science][Medline]
  28. Prunell GF, Mathiesen T, Svendgaard NA. A new experimental model in rats for study of the pathophysiology of subarachnoid hemorrhage. Neuroreport 13: 2553–2556, 2002.[CrossRef][Web of Science][Medline]
  29. Saavedra JM, Benicky J, Zhou J. Mechanisms of the anti-ischemic effect of angiotensin II AT1 receptor antagonists in the brain. Cell Mol Neurobiol 26: 1099–1111, 2006.[Web of Science][Medline]
  30. Sakurada O, Kennedy C, Jehle J, Brown JD, Carbin GL, Sokoloff L. Measurement of local cerebral blood flow with iodo[14C]antipyrine. Am J Physiol Heart Circ Physiol 234: H59–H66, 1978.[Abstract/Free Full Text]
  31. Takizawa T, Tada T, Kitazawa K, Tanaka Y, Hongo K, Kameko M, Uemura KI. Inflammatory cytokine cascade released by leukocytes in cerebrospinal fluid after subarachnoid hemorrhage. Neurol Res 23: 724–730, 2001.[CrossRef][Web of Science][Medline]
  32. Timmermans PB, Chiu AT, Herblin WF, Wong PC, Smith RD. Angiotensin II receptor subtypes. Am J Hypertens 5: 406–410, 1992.[Web of Science][Medline]
  33. Touyz RM, Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res 35: 1001–1015, 2002.[Web of Science][Medline]
  34. Vajkoczy P, Meyer B, Weidauer S, Raabe A, Thome C, Ringel F, Breu V, Schmiedek P. Clazosentan (AXV-034343), a selective endothelin A receptor antagonist, in the prevention of cerebral vasospasm following severe aneurysmal subarachnoid hemorrhage: results of a randomized, double-blind, placebo-controlled, multicenter phase IIa study. J Neurosurg 103: 9–17, 2005.[CrossRef][Web of Science][Medline]
  35. Vikman P, Beg S, Khurana T, Hansen-Schwartz J, Edvinsson L. Gene expression and molecular changes in cerebral arteries following subarachnoid hemorrhage in the rat. J Neurosurg 105: 438–444, 2006.[Web of Science][Medline]
  36. Weir B, Grace M, Hansen J, Rothberg C. Time course of vasospasm in man. J Neurosurg 48: 173–178, 1978.[Web of Science][Medline]
  37. Zervas NT. Vasospasm: an update. Clin Neurosurg 26: 643–656, 1979.[Medline]
  38. Zervas NT, Lavyne MH, Negoro M. Neurotransmitters and the normal and ischemic cerebral circulation. N Engl J Med 293: 812–816, 1975.[Web of Science][Medline]
  39. Zimmermann M, Seifert V. Endothelin and subarachnoid hemorrhage: an overview. Neurosurgery 43: 863–875, 1998.[CrossRef][Web of Science][Medline]



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