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

Abstract

Cerebral ischemia remains the key cause of morbidity and mortality after subarachnoid hemorrhage (SAH) with a pathogenesis that is still poorly understood. The aim of the 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 by injecting 250 μl of blood into the prechiasmatic cistern. Two days after the SAH, cerebral arteries were harvested and contractile responses to the TP receptor agonist U46619 were investigated with myographs. In addition, the contractile responses were examined after 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.

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 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 and K.D., 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 real-time polymerase chain reaction (PCR) and immunohistochemistry. The global and regional CBFs after SAH were measured by an autoradiographic technique.