Subarachnoid hemorrhage induces enhanced expression of thromboxane A2 receptors in rat cerebral arteries

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Subarachnoid hemorrhage induces enhanced expression of thromboxane A2 receptors in rat cerebral arteries Saema Ansar a,b,⁎, Carl Larsen c , Aida Maddahi a , Lars Edvinsson a,b a

Division of Experimental Vascular Research, Department of Clinical Sciences, Lund University, Sweden Department of Clinical Experimental Research, Glostrup University Hospital, Glostrup, Denmark c Department of Neurosurgery, University Hospital, Glostrup, Denmark b

A R T I C LE I N FO

AB S T R A C T

Article history:

Cerebral ischemia remains the key cause of morbidity and mortality after subarachnoid

Accepted 12 December 2009

hemorrhage (SAH) with a pathogenesis that is still poorly understood. The aim of the

Available online 22 December 2009

present study was to examine the involvement of thromboxane A2 receptors (TP) in the pathophysiology of cerebral ischemia after SAH in cerebral arteries. SAH was induced in rats

Keywords:

by injecting 250 μl of blood into the prechiasmatic cistern. Two days after the SAH, cerebral

Subarachnoid hemorrhage

arteries were harvested and contractile responses to the TP receptor agonist U46619 were

Cerebral ischemia

investigated with myographs. In addition, the contractile responses were examined after

Thromboxane receptor

pretreatment with selective TP receptor antagonist GR3219b. The TP receptor RNA and protein levels were analyzed by quantitative real-time PCR and immunohistochemistry, respectively. The global and regional cerebral blood flows (CBFs) were quantified with an autoradiographic technique. SAH resulted in enhanced contractile responses to U46619 as compared to sham. The TP receptor antagonist GR3219b abolished the enhanced contractile responses to U46619 observed after SAH. The TP receptor mRNA level was elevated after SAH as compared to sham. The level of TP receptor protein on the smooth muscle cells (SMCs) was increased in SAH compared to sham. Global and regional CBFs were reduced in SAH as compared to sham. The results demonstrate that SAH results in CBF reduction and this is associated with the enhanced expression of TP receptors in the SMC of cerebral arteries and microvessels. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

Spontaneous subarachnoid hemorrhage (SAH) arising from the rupture of an intracranial aneurysm is an important cause of premature death and disability worldwide. It is a cerebrovascular disease responsible for approximately 50% of the overall stroke mortality (Bamford et al., 1990). Studies have demonstrated that 50% of the patients die within 30 days after

the SAH and two-thirds of the deaths occur within 48 hours (Schievink et al., 1995). SAH is a biphasic disease and consists of an early, short-lived phase occurring immediately following SAH and a subsequent phase that is prolonged or chronic (Delgado et al., 1985; Grasso, 2004; Jackowski et al., 1990). In a previous study, angiographic examinations of the cerebral arteries in the rat revealed a biphasic vasospasm with a maximal acute cerebral constriction at ten minutes and a late

⁎ Corresponding author. Division of Experimental Vascular Research, Department of Clinical Sciences, BMC A13, Lund University, 221 84 Lund, Sweden. Fax: +46 46 222 0616. E-mail address: [email protected] (S. Ansar). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.031

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Fig. 1 – The global CBF after induced SAH (n = 6) compared to the control (n = 6) rats. Data were obtained by an autoradiographic method and data are expressed as mean ± SEM values, ***P < 0.001.

maximal vasoconstriction at two days after SAH (Delgado et al., 1985). Despite intense research and major advances in surgical techniques, the mortality and morbidity rates after SAH have not changed in recent years (Schievink et al., 2004). The etiology of late cerebral ischemia that occurs after SAH is multifactorial, including free haemoglobin, enhanced level of free radicals (Asano, 1999; Macdonald et al., 2004; Nozaki et al., 1990), inflammatory reactions (Dumont et al., 2003; Prunell et al., 2005), a central nervous dysfunction (Edvinsson, 2002; Shiokawa and Svendgaard, 1994), and the enhanced presence of vasoactive substances (Edvinsson, 2002). Thromboxane A2 (TXA2) is a potent vasoconstrictor and an important modulator of vascular tone in both physiological and pathophysiological conditions (Davidge, 2001). TXA2 is generated through sequential metabolism of arachidonic acid by cyclooxygenases 1 or 2 and TXA2 synthase (Coyle et al., 2002); it mediates its effects by binding to the TXA2 receptor (TP) (Huang et al., 2004). TP receptor activation results in smooth muscle cell (SMC) constriction and platelet aggregation, promotes intravascular hemostasis (Cockerham et al., 1991; Coyle et al., 2002; Radomski, 1985) and is considered to play an important role in the pathogenesis of cerebral ischemia, myocardial infarction, and atherosclerosis (Kaneko et al., 2006; Sasaki et al., 1982). Several studies have shown an increased thromboxane biosynthesis in patients with cerebral ischemia, as reflected by the urinary excretion (Koudstaal et al., 1993; Saloheimo et al., 2005; van Kooten et al., 1999). We hypothesise that SAH induces changes in cerebrovascular receptor expression and function, which might influence the development of late cerebral ischemia. Therefore, we examined the impact of TP receptors on the pathophysiology of late cerebral ischemia after SAH in cerebral arteries, microvessels, and associated brain tissue. The cerebral arteries were examined using a sensitive in vitro pharmacological method where the contractile responses to U46619 (TP receptor agonist) were measured. In addition, the mRNA and protein levels of TP receptors were investigated by quantitative realtime polymerase chain reaction (PCR) and immunohistochemistry. The global and regional CBFs after SAH were measured by an autoradiographic technique.

2.

Results

2.1.

SAH model

The mortality rate was 5%, and there was no difference in the mortality rate between the groups. The rats did not show any distressed behaviour. They were moving around, eating, and drinking and their fur was not sprawl. All surviving animals were neurologically examined using an established scoring system (Bederson et al., 1986; Menzies et al., 1992). All SAH-operated rats received a score of 1, and the sham-operated rats got a score of 0. In all operated rats, mean arterial blood pressure (101 ± 3 mm Hg), partial pCO2 (39 ± 3 mm Hg), partial pO2 (111 ± 4 mm Hg) values, and temperature were within acceptable limits during the operation (37 ± 1.2 mm Hg). No statistical difference was seen in physiological parameters between the groups sham and SAH. As a result of injecting the blood, the cortical blood flow dropped over both hemispheres to 10 ± 5% of resting flow (there was no difference between the two Laser Doppler probe data) and the intracranial pressure (ICP) increased from 9 ± 2 to 126 ± 9 mm Hg. The laser Doppler blood flow and the elevated ICP returned to the basal values within 1 hour of postoperative monitoring.

2.2. Regional cerebral blood flow to evaluate the overall consequences of SAH There was a significant global decrease in cerebral blood flow measured at 48 h in the SAH (n = 6) group as compared to the control group (n = 6) from 143 ± 8 to 58 ± 2 ml/100 g/min (Fig. 1). The SAH animals showed a reduction in the regional CBF in 16 of the 18 brain regions examined compared to the control operated rats (Table 1).

Table 1 – Regional cerebral blood flow 48 hours after subarachnoid hemorrhage.

Frontal cortex Parietal cortex Occipital cortex Caudate putamen Septum Hippocampus Thalamus Hypothalamus Corpus callosum Sensorimotor cortex Superior colliculus Inferior colliculus Cerebellar cortex Dentate nucleus Facial nucleus Cochlear nucleus Vestibular nucleus Trigeminal nucleus

Sham (n = 6)

SAH (n = 6)

161 ± 54⁎ 172 ± 27⁎ 145 ± 33⁎ 77 ± 24 81 ± 4a 129 ± 15 139 ± 50 144 ± 30 134 ± 27 223 ± 57⁎ 187 ± 35⁎ 168 ± 45⁎ 124 ± 24⁎ 147 ± 25⁎ 130 ± 33⁎ 158 ± 28⁎ 145 ± 23⁎ 107 ± 24⁎

69 ± 24⁎ 65 ± 29⁎ 53 ± 30⁎ 34 ± 15 51 ± 21 59 ± 30⁎ 47 ± 30b 64 ± 28 65 ± 25 63 ± 50⁎ 66 ± 29⁎ 61 ± 38⁎ 50 ± 29⁎ 71 ± 25⁎ 54 ± 26⁎ 67 ± 33⁎ 60 ± 25⁎⁎ 49 ± 21⁎

Values are expressed in milliliters per minute per 100 g and given as mean ± SEM. ⁎Significant difference, P ≤ 0.05. ** Significant difference from SAH, P ≤ 0.01.

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Table 2 Sigmoidal curve

U46619 BA Sham SAH SAH inhibited SAH inhibited U46619 MCA Sham SAH SAH inhibited SAH inhibited

+

N

K mean ± SEM

Emax (%) ± SEM

pEC50(1) ± SEM

with GR3219b 10− 8 M with GR3219b 10− 6 M

14 9 6 6

3.88 ± 0.23 4.12 ± 0.44 3.49 ± 0.31 3.21 ± 0.25

104 ± 6⁎ 133 ± 8 90 ± 10⁎ 86 ± 8⁎

7.71 ± 0.15 7.31 ± 0.08 7.73 ± 0.36 6.46 ± 0.08⁎

with GR3219b 10− 8 M with GR3219b 10− 6 M

14 9 6 6

2.38 ± 0.15 2.48 ± 0.51 1.98 ± 0.33 1.67 ± 0.31

113 ± 5⁎ 152 ± 16 118 ± 6⁎ 106 ± 15⁎⁎

7.58 ± 0.11 7.32 ± 0.13 7.26 ± 0.06 7.26 ± 0.25

Responses were characterized by Emax values, expressed as percent of 63 mM K+-induced contraction, and pEC50 values (negative logarithm of the molar concentration that produces half maximum contraction). Values are represented as mean ± SEM and n represents the number of animal. ⁎Significant difference from SAH, P ≤ 0.05. ⁎⁎Significant difference from SAH, P ≤ 0.01.

2.3.

Functional in vitro pharmacology

K+-induced contractions did not differ significantly between the cerebral arteries from the different groups (Table 2). Emax and pEC50 values for respective group are presented in Table 2.

2.3.1.

Contractile response to U46619

In the MCA and BA from SAH rats (n = 9), U46619 showed an elevated Emax compared to sham- (n = 14) operated rats. GR3219b (10− 6 M) abolished the elevated Emax and, in addition, caused a rightward shift of the concentration response curve to U46619. At the concentration 10− 8 M, the selective TP receptor antagonist GR3219b abolished the enhanced Emax but did not alter the pEC50 (Fig. 2 and Table 2).

2.4.

Quantitative mRNA expression

The standard curves for each primer pair had almost similar slopes, demonstrating that EF-1, β-actin, and TP receptor 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 the samples. Since the results from the different brain arteries examined MCA, BA, and circle of Willis (n = 7–10) were identical, they were grouped together in the statistical analysis. The results showed that SAH enhanced the expression of TP receptor mRNA levels significantly as compared to control (Fig. 3).

2.5.

Immunohistochemistry

The selective and specificity of the TP receptor immunoreactivity was checked by visualizing the smooth muscle cell localization of the TP receptor 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 TP receptor protein was expressed on the smooth muscle cells in the BA; this signal was significantly increased in SAH (166 ± 15%) as compared to sham (100 ± 14%)

Fig. 2 – Concentration–response curves elicited by cumulative application of U46619 in rat cerebral arteries. (A) U46619, BA. (B) U46619, MCA, .Data are expressed as mean ± SEM, n = 6–14, *P ≤ 0.05, **P ≤ 0.01. *Significant difference between SAH and SAH treated with GR3219b (10− 6 M).

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3.

Fig. 3 – The mRNA levels of TP receptor after experimentally induced SAH in rats. Data were obtained by real-time PCR and are expressed as mean ± SEM values relative to EF-1 mRNA levels, n = 7–10, *P ≤ 0.05.

(Figs. 4A and B). The up-regulation was not confined only to the large cerebral arteries but notable also in the parenchyma microvessels. There was no or minor TP receptor expression in endothelial cells in the control brain (sham). However, there was a somewhat tendency to increase in TP receptor expression in the brain tissue after SAH (130 ± 12%) as compared to sham (100 ± 3%) (Figs. 5A and B).

Discussion

We have demonstrated that SAH results in global and regional CBF reduction and this is associated with enhanced expression of TP receptors in the smooth muscle cells of cerebral arteries and microvessels. The results revealed that experimental SAH induces an enhanced contractile response to U46619, which was blocked by the TP receptor blocker GR3219b, demonstrating that the response was mediated via TP receptors. The selective TP antagonist GR3219b blocked both the enhanced Emax and caused a rightward shift of the TXA2 curve. The results from real-time PCR revealed enhanced levels of TP receptor mRNA; in addition, immunohistochemistry showed an enhanced expression of TP receptors in SAH as compared to sham-operated rats. The results lend support for the hypothesis of an increase in cerebrovascular TP receptor expression and this may participate in the increase of vascular tone after SAH. The method for inducing SAH has been well characterized with regard to correlation between amount of blood, angiographic vasoconstriction, CBF, metabolic changes, cerebral arterial tone, ICP, and peripheral circulation. This has been shown to induce a late phase reduction in CBF and metabolism after 48 hours (Prunell et al., 2002, 2003, 2004). In the 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 (Prunell et al., 2002). It has been discussed for decades if the SAH blood is the main determinant of the late cerebral ischemia. We have examined this at some depth;

Fig. 4 – (A) Sections from the basilar artery showing TP receptor immunoreactivity in the smooth muscle cell layer: (a) TP, sham; (b) TP, SAH; (c) negative control. Data were obtained with confocal microscopy. (B) Fluorescence intensity for the TP receptor in the basilar artery. Results are presented as the mean percentage relative to control ± SEM; n = 9. **P < 0.01.

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Fig. 5 – (A) Sections from the brain tissue showing TP receptor immunoreactivity in the brain tissue: (a) TP, sham; (b) TP, SAH. Data were obtained with confocal microscopy. (B) Fluorescence intensity for the TP receptor in the brain tissue. Results are presented as the mean percentage relative to control ± SEM, n = 5–6.

both the amount of blood and the rise in ICP contribute almost equally (Ansar and Edvinsson, 2009). Metabolism of arachidonic acid produces prostaglandin 2 (PG2) and PGH2, and thromboxane synthase catalyzes the reaction that converts PGH2 to TXA2, while PGIS catalyzes the reaction that converts PGH2 to prostacyclin (PGI2) (Kaneko et al., 2006). Under normal pathophysiological conditions, there is a balance between production of potent vasoconstrictor TXA2 and potent vasodilator PGI2. However, in vascular pathology conditions, such as SAH, there may be an imbalance, where the vasoconstrictor TXA2 becomes more predominant (Chan et al., 1984). It is important to note that although TXA2 is a vasoconstrictor, its immediate precursor, PGH2, also binds to the TP receptor to cause a vasoconstrictive response. TP receptors play important roles in development of cerebral ischemia and mediate vascular proliferation and contraction (De Clerck and Janssen, 1990). Kaneko et al. (2006) have, in a clinical study, demonstrated that the TP receptor gene is associated with increased susceptibility to cerebral infarction. Many studies have implicated a role for TXA2 in the pathophysiology of cerebral ischemia. Elevated levels of TXA2 in CSF have been reported in man following SAH (Pickard et al., 1994). In addition, an enhanced TX biosynthesis has been found in patients with intracerebral hemorrhage (van Kooten et al., 1999) and ischemic stroke (Koudstaal et al., 1993). A poor stroke outcome tended to be associated with increased TX production in the acute phase (van Kooten et al., 1999). The increased platelet activation may be a reflection of vascular risk factors, diffuse atherosclerotic lesions, or the extent of vascular damage due to stroke. However, thromboxane

receptor inhibition has been relatively ineffective in models of SAH. The effect of TP receptor blockade after transient cerebral ischemia in dog showed that the TP receptor antagonist reduced the cerebral ischemia (Satoh et al., 1994). The TXA2 synthetase inhibitor, ozagrel, has been shown to prevent cerebral vasospasm in a single-hemorrhage canine model (Komatsu et al., 1986); however, in contrast, another study demonstrated that ozagrel did not reverse the spastic artery during the chronic stage of cerebral vasospasm (Toshima et al., 1997). Thus, although TXA2 may contribute to cerebral ischemia, studies using inhibitors of prostaglandin and thromboxane synthesis have had little effect on experimental and human vasospasm (Nosko et al., 1988; Suzuki et al., 1985). Late cerebral ischemia is multifactorial, and importantly, there are several vasoactive mediators altering cerebral ischemia. These systems do interact with each other and do not act in isolation. This view is supported by previous studies with our SAH model. Thus, we found upregulation of contractile ETB, 5-HT1B, and AT1 receptors in the cerebral arteries (Ansar et al., 2007). Hence, several receptor systems are upregulated in late cerebral ischemia. This multitude of changes could explain the lack of effect of the many substances tested in clinical trials; inhibiting one system will not remedy other receptor systems involved (Wilkins, 1980, 1986). Treatments aimed at a common signaling pathway could be more beneficial. We have recently demonstrated that the extracellular-regulated kinase (ERK) 1/2 inhibitor SB386023-b prevented the upregulation of ETB and 5-HT1B in cerebral arteries after SAH. In addition, the reduction in global and regional CBF was prevented by the

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ERK1/2 inhibitor (Beg et al., 2006). The possible intracellular mediator of the TP receptor upregulation could very well be mitogen-activated protein kinase (MAPK). In the present study, the increase in TP receptor mRNA suggests a transcriptional event. In organ culture, we have examined the involvement of the intracellular MAPK pathways in the upregulation of TP receptors in cerebral arteries. Inhibition of the MAPK pathway with the specific MEK1/2 inhibitor U0126 in cerebral arteries decreased the functional response of TP receptor upregulation (unpublished data). In conclusion, the present study revealed that SAH induces the upregulation of TP receptors in cerebrovascular smooth muscle cells and it is associated with a reduction in regional and global CBFs. Thus; there is a correlation between the changes in receptor expression and reduction in CBF.

4.

Experimental procedures

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.

4.1.

Rat subarachnoid hemorrhage model

Subarachnoid hemorrhage was induced by a model originally devised by Prunell et al. (2002) and carefully described by Prunell et al. (2003). Prunell et al. have, in an elegant series of studies, carefully analyzed the correlation between amount of blood, angiographic vasoconstriction, CBF, and cerebral metabolism. In a previous study using the same SAH model, Delgado et al. (1985) revealed a biphasic vasospasm in angiographic examinations of the arteries with a maximal acute vasoconstriction at 10 minutes and a late maximal constriction at 2 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, 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 suboccipital membrane. At either side of the skull, 3 mm from the midline and 4 mm anteriorly from the bregma, holes were drilled through the skull bone down to dura mater (without perforation) allowing the placement of two laser-Doppler flow probes to measure cortical CBF. Finally, a 27-gauge blunt cannula 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 intracranial via this

cannula at a pressure equal to the mean arterial blood pressure (MABP) (80–100 mm Hg). 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 of isotonic sodium chloride at the end of the operation and at day 1. 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 were studied: no fluid injection or injection of saline (250 μl) during 15 minutes to avoid any change in ICP. Since both procedures revealed the same outcome, they were grouped together in the statistical analysis (Ansar and Edvinsson, 2009). All surviving animals were neurologically examined using an established scoring system (Table 3) (Bederson et al., 1986; Menzies et al., 1992). After 2 days, either autoradiographic measurements or harvesting of vessels was done (see below for details).

4.2.

Autoradiographic measurements of regional CBF

Regional and global cerebral blood flows were measured by a model originally described by Sakurada et al. (1978) and modified by Gjedde et al. (1980). In brief, after 48 hours of observation, rats in the various groups (sham and SAH) 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

Table 3 – Neurological score after SAH. Score 0 1 2 3 4 5

Interpretation No visible deficits Contralateral forelimb flexion, when hold by tail Decreased grip of contralateral forelimb Spontaneous movement in all directions, but contralateral circling if pulled by tail Spontaneous contralateral circling Death

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bolus injection of 50 μCi of 14C-iodoantipyrine 4[N-methyl-14C] (Perkin-Elmer, Boston, USA) was given i.v. Arterial blood (122 μl) was withdrawn for 20 seconds. Immediately after this the animal was decapitated, the brain removed and immersed in isopentane (J.T. Baker, Deventer, Netherlands) chilled to − 50 °C. The arterial blood sample was transferred to liquid scintillation counting vials containing 1 ml of a mixture of Soluene-350 (Perkin-Elmer, Boston, USA) and isopropanol (J. T. Baker, Deventer, Netherlands) (1:1). After 2 hours at 60 °C, 0.2 ml of 30% hydrogen peroxide was added to the vials, and the samples were maintained at room temperature for 15– 30 minutes. Thereafter, the samples were kept at 60 °C for 30 minutes and 10–15 ml of Hionic-Fluor (Perkin-Elmer, Foster, CA, USA) was added. 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 1). The CBF was calculated from the brain tissue 14C activity determined by autoradiography using the equation of Gjedde et al. (1980): f bl =

CBr ðTÞ RT 0 Ca ðtÞdt

EðTÞ

where fbl is the blood flow per unit mass, CBr(T) is the isotope content, E(T) is the next extraction fraction of the isotope in the time from t = 0 to t = T, t = the variable time, T = the experimental time, and Ca(t) is the arterial blood concentration of the isotope at time t.

4.3.

Harvest of cerebral arteries

After 48 hours of observation, sham- and SAH-operated rats were anaesthetized with CO2 and decapitated. The brains were quickly removed and chilled in ice-cold bicarbonate buffer solution (see composition below). Under a dissection microscope, the middle cerebral artery (MCA), the basilar artery (BA), circle of Willis, and some brain tissue were carefully dissected free from the brain and cleared of connective tissue. 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.

4.4.

In vitro pharmacology

For contractile experiments, a myograph was used for recording the isometric tension in isolated cerebral arteries (Hogestatt et al., 1983; Mulvany and Halpern, 1977). The vessels were cut into 1-mm-long cylindrical segments and mounted on two 40-μm-diameter stainless steel wires in a

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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 (mM): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. 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 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 (Hogestatt et al., 1983). 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 (Hansen-Schwartz et al., 2003). Concentration–response curves were obtained by cumulative application of U46619 (TP receptor agonist) (Sigma, St. Louis, USA) in the concentration range 10− 12 to 10− 6 M. The concentration–response curves for U46619 were investigated both with and without TP receptor antagonist. In the experiments with TP receptor antagonist, the arteries were pretreated with the TP receptor antagonist GR3219b in different concentrations (10− 8 to 10− 6 M) (Sigma, St. Louis, USA) 30 minutes before the concentration curves were performed.

4.5.

Immunohistochemistry

For immunohistochemistry, the indirect immunofluorescence method was used. The BA, microvessels, and cortex 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 phosphatebuffered solution (PBS) containing 0.25% Triton X-100 for 15 minutes. The tissue was then permeabilized and blocked for 1 hour in a blocking solution containing PBS, 0.25% Triton X-100, 1% BSA, and 5% normal donkey serum. The sections were incubated overnight at 4 °C with the following primary antibodies: TP receptor antibody, diluted 1:150 (Santa Cruz Biotechnologies), mouse anti-rat CD31 (Serotec, MCA1746), diluted 1:200, and mouse anti-rat smooth muscle actin (Serotec, MCA1905T) diluted 1:100. All dilutions were done in PBS containing 0.2 % Triton X- 100, 1% BSA, 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 was donkey antirabbit CY ™² conjugated (Jackson ImmunoResearch, 711-165152) diluted 1:200 in PBS containing 0.2% Triton X-100 and 1%

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BSA. The sections were washed subsequently with PBS and mounted with PermaFlour 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.

4.6.

RNA isolation

To quantify mRNA for the TP 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 supplier's 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 minutes, followed by centrifugation at 15,000 × g for 15 minutes at 4 °C. The supernatant was collected, and the organic phase was discarded. Two hundred microliters of chloroform was again added to remove all traces of phenol, and the samples were centrifuged at 15,000 × g for 15 minutes 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 minutes 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 260/280 (Amersham Pharmacia Biotech, Uppsala, Sweden).

4.7.

Real-time PCR

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 minutes and then at 72 °C for 10 minutes. 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 TP receptor and housekeeping 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). Receptor primers had the following sequences: TP receptor forward: 5′-ATCTCCCATCTTGCCATAGTCC-3′ TP receptor reverse: 5′-CCGATGATCCTTGGAGCCTAAAG-3′ The housekeeping gene EF-1 is used as a reference, since it is continuously expressed to a constant amount in cells.

The EF-1 primers were designed as follows: EF-1 forward: 5′-GCA AGC CCA TGT GTG TTG AA-3′ EF-1 reverse: 5′-TGA TGA CAC CCA CAG CAA CTG-3′ The PCR was carried out as follows: 50 °C for 2 minutes, 95 °C for 10 minutes, and the following 40 PCR cycles at 95 °C for 15 seconds and at 60 °C for 1 minute. 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 TP receptors were amplified with a similar efficacy during real-time PCR, a standard curve were made in which the CT values were plotted against cDNA concentration on the basis of the following equation: CT = (log(1 + E))−1log (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 1. Standard curves for TXA2 and EF-1 were performed by the dilution of cDNA sample (1:10, 1:100, and 1:1000) (data not shown).

4.8.

Calculations and statistics

Data are expressed as mean ± standard error of the mean (SEM), and n refers to the number of rats. Statistical analyses were performed using the nonparametric Mann–Whitney test, where P < 0.05 was considered significant.

4.8.1.

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.

4.8.2.

Immunohistochemistry

The images were analyzed blindly using the ImageJ software (http://rsb.info.nih.gov/ij/). The fluorescence in 4–6 different areas in each artery segment (3 sections per point) was measured. The mean value of this 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%.

Acknowledgments The study was supported by the Swedish Research Council (grant no. 5958), the Heart and Lung Foundation (Sweden), the Danish Research Council, and the Lundbeck Foundation (Denmark).

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