SAH-induced Vasospasm Research Articles

17β-Estradiol Activates Adenosine A 2a Receptor After Subarachnoid Hemorrhage

Our previous study showed that 17beta-estradiol (E2) and an adenosine A(2A) receptor (AR-A(2A)) agonist could attenuate subarachnoid hemorrhage (SAH)-induced cerebral vasospasm via preventing the augmentation of iNOS expression and preserving the normal eNOS expression. This study tests the hypothesis that E2 attenuates SAH-induced vasospasm and apoptosis by activating adenosine AR-A(2A) and extracellular signal-regulated kinase 1 and 2 (ERK1/2), and by altering antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). The two-hemorrhage SAH model in rat was used. Animals were treated with E2 with or without a nonselective estrogen receptor (ER) antagonist (ICI182,780). The cross sectional areas of the basilar artery and terminal dUTP nick-end labeling (TUNEL) were used to determine the degree of vasospasm and apoptosis, respectively. The expressions of Bcl-2, Bax, AR-A(2A), and ERK1/2 in the cerebral cortex, hippocampus, and dentate gyrus were investigated. E2 significantly attenuated vasospasm. Seven days after the first SAH, TUNEL scores were significantly increased, and protein levels of AR-A(2A), ERK1/2, and Bcl-2 were significantly decreased in the dentate gyrus only but not in the cortex and hippocampus. These changes were reversed by E2 while ICI182,780 abrogated the antiapoptotic and anti-spastic effects of E2. The expression of Bax did not change in the dentate gyrus after SAH with or without treatment. The down-regulated AR-A(2A) and ERK may play a role in vasospasm and apoptosis after SAH. The beneficial effect of E2 in the attenuating SAH-induced vasospasm and apoptosis may be due to an increased expression of AR-A(2A) and ERK via ER-dependent mechanisms. These data may support further investigation of E2 in the treatment of SAH in humans.

In vitro evidence of the role of hemoglobin during vasospasm on the modifications of the expression of PKCα and ζ

The cellular events leading to cerebral vasospasm after subarachnoid hemorrhage (SAH) are poorly understood, although the family of protein kinase C (PKC) is already known to play crucial roles in this pathology. Hemoglobin (Hb) is one of the major causes of the cerebral vasospasm that follows SAH. In the present study we investigated whether Hb can in vitro regulate PKC expression in endothelial as opposed to smooth-muscle cells. The levels of expression of PKCalpha and PKCzeta were quantitatively determined by means of computer-assisted fluorescence microscopy in the A7r5 smooth-muscle rat cells and human umbilical endothelial cells (HUVECs). Hb significantly modified both calcium-dependent PKCalpha and calcium-independent PKCzeta expression in HUVECs and A7r5 smooth-muscle rat cells. Our data showed that, in vitro, Hb promptly and markedly modified the levels of expression of both calcium-dependent PKCalpha and calcium-independent PKCzeta. We are currently investigating the effects of specific PKC antagonists associated or not with calcium channel blockers on the expression of PKC and the in vivo severity of SAH-induced vasospasm. Our results encourage the prophylactic use of specific PKC isoform antagonists associated with calcium channel blockers early after SAH to prevent cerebral vasospasm.

High-dose Bosentan in the Prevention and Treatment of Subarachnoid Hemorrhage-induced Cerebral Vasospasm: An Open-label Feasibility Study

To evaluate the safety of high-dosages of the endothelin ET(A/B )receptor antagonist bosentan in SAH patients at high-vasospasm risk. Ten Fisher group-3 SAH patients, enrolled within 96 h of ictus, received bosentan in a dose-escalation manner (20, 30, 40 mg/kg/day orally every 4 hours on treatment days 1, 2, and 3 respectively, to a maximum dose of 4000 mg/day), followed by maintenance of the maximum tolerated dose until 14 days post-SAH or vasospasm resolution. Further management followed standard protocols: nimodipine in all patients; daily transcranial Doppler (TCD); "triple-H"/endovascular treatment, as indicated. Two of the ten patients never developed any clinical or TCD signs of vasospasm; the other eight patients exhibited some elevation of TCD velocities during the vasospasm watch period. Four of the eight patients remained asymptomatic; of them, one had only mild elevation on peak systolic velocities, thought to represent hyperemia. The other three were further assessed with CT-angiography; this revealed moderate vasospasm (asymptomatic) in only one patient. The remaining four patients developed symptomatic vasospasm requiring endovascular treatment; two developed cerebral infarction; both had started bosentan relatively later than the other subjects. The most common adverse drug effects were flushing and transient liver enzyme elevations, reversible in all. Two patients had ALT/AST elevations >3x normal limit, requiring bosentan-dose reduction or discontinuation (one case each). High-dose bosentan (up to 40 mg/kg/day) appears to be safe in SAH patients at high risk of developing vasospasm. Further studies are required to properly investigate the efficacy of this regimen in the prevention and treatment of SAH-induced vasospasm.

Antivasospastic and antiinflammatory effects of caspase inhibitor in experimental subarachnoid hemorrhage

Inflammation in the subarachnoid space and apoptosis of arterial endothelial cells have been implicated in the development of delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). The authors investigated mechanisms of possible antivasospastic effects of N-benzyl-oxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), a caspase inhibitor that can inhibit both inflammatory and apoptotic systems, in animal models of SAH. Rabbits were assigned to three groups of eight animals each and were subjected to SAH by injection of blood into the cisterna magna. The experiments were performed in the following groups: SA only, SAH + vehicle, and SAH + Z-VAD-FMK. The Z-VAD-FMK (1 mg) or vehicle (5% dimethyl sulfoxide) was intrathecally administered before SAH induction. Diameters of the basilar artery (BA) were measured on angiograms obtained before and 2 days after SAH. The BA diameter on Day 2 was expressed as a percentage of that before SAH. Interleukin (IL)-1 in the cerebrospinal fluid (CSF) was examined using Western blotting, and brains were immunohistochemically examined for caspase-1 and IL-1beta. In a separate experiment, 20 rats were subjected to SAH and their brains were immunohistochemically assessed for caspase-1, IL-1beta, and macrophages. RESULTS. In rabbits, Z-VAD-FMK significantly attenuated cerebral vasospasm (the BA diameter on Day 2 in SAH-only, SAH + vehicle, and SAH + Z-VAD-FMK groups was 66.6 +/- 3.2%, 66.3 +/- 3.7%, and 82.6 +/- 4.9% of baseline, respectively), and suppressed IL-1beta release into the CSF and also suppressed immunoreactivities of caspase-1 and IL-1P in macrophages infiltrating into the subarachnoid space. Immunoreactivities for caspase-1 and IL-1P were observed in immunohistochemically proven infiltrating macrophages in rats. These results indicate that caspase activation may be involved in the development of SAH-induced vasospasm through inflammatory reaction.

Attenuation of experimental subarachnoid hemorrhage–induced cerebral vasospasm by the adenosine A2A receptor agonist CGS 21680

Impaired endothelium-dependent relaxation is present in vasospastic cerebral vessels after subarachnoid hemorrhage (SAH) and may result from deficient production of endothelial nitric oxide synthase (eNOS) or increased production and/or activity of inducible NOS (iNOS). Accumulating evidence demonstrates that adenosine A2A receptors increase the production of NO by human and porcine arterial endothelial cells, which in turn leads to vasodilation. This study was designed to examine the effects of an adenosine A2A receptor agonist, (2(4-[2-carboxyethyl]phenyl)ethylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680), in the prevention of SAH-induced vasospasm. . Experimental SAH was induced in Sprague-Dawley rats by injecting 0.3 ml of autologous blood into the cisterna magna of each animal. Intraperitoneal injections of CGS 21680 or vehicle were administered 5 minutes and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging measurements of cross-sectional areas of the basilar artery (BA) 48 hours after SAH. Expression of eNOS and iNOS in the BA was also evaluated. Prior to perfusion-fixation, there were no significant differences among animals in the control and treated groups in any physiological parameter that was recorded. The CGS 21680 treatment significantly attenuated SAH-induced vasospasm. Induction of iNOS mRNA and protein in the BA by the SAH was significantly diminished by administration of CGS 21680. The SAH-induced suppression of eNOS mRNA and protein was also relieved by the CGS 21680 treatment. This is the first evidence that adenosine A2A receptor agonism is effective in preventing SAH-induced vasospasm without significant complications. The beneficial effect of adenosine A2A receptor agonists may be, at least in part, related to the prevention of augmented expression of iNOS and the preservation of normal eNOS expression following SAH. Adenosine A2A receptor agonism holds promise in the treatment of cerebral vasospasm following SAH and merits further investigation.

Attenuation of experimental subarachnoid hemorrhage–induced increases in circulating intercellular adhesion molecule–1 and cerebral vasospasm by the endothelin-converting enzyme inhibitor CGS 26303

Adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, are important mediators of inflammation, and their levels are elevated in the serum of patients following aneurysmal subarachnoid hemorrhage (SAH). The investigators previously found that CGS 26303 is effective in preventing and reversing arterial narrowing in a rabbit model of SAH. The purpose of the present study was to examine whether levels of adhesion molecules are altered after treatment with CGS 26303 in this animal model. New Zealand White rabbits were each injected with 3 ml of autologous blood in the cisterna magna, and intravenous treatment with CGS 26303 (30 mg/kg) was initiated 1 hour later. The compound was subsequently administered at 12, 24, and 36 hours post-SAH. Blood samples were collected at 48 hours post-SAH to measure ICAM-1, VCAM-1, and E-selectin levels. After the rabbits had been killed by perfusion-fixation, the basilar arteries (BAs) were removed and sliced, and their cross-sectional areas were measured. Treatment with CGS 26303 attenuated arterial narrowing after SAH. Morphologically, corrugation of the internal elastic lamina of BAs was prominently observed in the SAH only and vehicle-treated SAH groups, but not in the CGS 26303-treated SAH group or in healthy controls. There were no significant differences in the levels of VCAM-1 among the four groups. The levels of E-selectin were increased in all animals subjected to SAH (those in the SAH only, SAH plus vehicle, and SAH plus CGS 26303 groups) compared with healthy controls (no SAH); however, the levels of ICAM-1 in the SAH only and SAH plus vehicle groups were significantly elevated (p < 0.001), and treatment with CGS 26303 reduced ICAM-1 to control levels following SAH. These results show that ICAM-1 may play a role in mediating SAH-induced vasospasm and that a reduction of ICAM-1 levels after SAH may partly contribute to the antispastic effect of CGS 26303.

Regional cerebral 18FDG uptake during subarachnoid hemorrhage induced vasospasm

Objectives: The aim was to elucidate whether aneurysmal subarachnoid hemorrhage (SAH)-induced vasospasm induces changes of regional glucose uptake in surgically treated, asymptomatic cases.Methods: 18FDG uptake (standardized uptake value, SUV) was analysed with PET in eight surgically treated aneurismal patients with a mean middle cerebral artery flow velocity >120 cm/seconds measured with transcranial Doppler ultrasound. Data were compared with a healthy control group using Statistical Parametric Mapping (SPM99b).Results: Six of the eight patients had no focal neurological signs. The inhomogeneous bilateral increase in SUV (p<0.0001) was asymmetrical, with an almost 70% larger volume on the operated side. Reduced glucose uptake was found in the frontal and temporobasal regions of the two patients with neurological deficits (p<0.0001); the affected volume was 40% larger on the operated side.Discussion: SAH-induced vasospasm results in widespread increase of glucose uptake—probably reflecting increased glycolysis. This was earlier than neurological focal signs appear. Decreased glucose uptake can be detected in severe cases of vasospasm reflected by neurological deficit. Although the changes are more prominent where surgery had taken place our results suggest that not only the surgery, but also subarachnoid blood might have resulted in our findings.

Endothelial cells from human cerebral aneurysm and arteriovenous malformation release ET-1 in response to vessel rupture

Cerebral aneurysms and arteriovenous malformations (AVM) are a common cause of stroke and cerebral hemorrage. Both are often discovered when they rupture, causing subarachnoid hemorrhage (SAH). SAH-induced vasospasm is mediated by enhanced vasoconstriction due to endothelin-1 (ET-1). We investigated whether endothelial cells (ECs) obtained from aneurysm and AVM express phenotypic and genotypic alterations contributing to the development of vasospasm after SAH. We isolated ECs from human AVM and aneurysm and then confirmed their EC origin by polymerase chain reaction and immunocytochemistry with endothelial markers. Experiments were also carried out with human cerebral microvascular and umbilical vein ECs (HCECs and HUVECs respectively) for comparison. We tested EC proliferation ability and microtubule formation in Matrigel at different cell passages. Five aneurysm (3 ruptured, 2 unruptured) and 3 AVM (2 ruptured, 1 unruptured) ECs were tested for ET-1 release in the culture medium. Aneurysm and AVM ECs expressed von Willebrand factor, Adrenomedullin, and exhibited a progressive reduction of proliferation and in vitro angiogenic ability after the V passage. Significantly higher levels of ET-1 have been detected in ECs from ruptured aneurysms and AVMs. We report the first successful isolation and characterization of primary EC lines from human cerebral vascular lesions. Augmented release of ET-1 is correlated with the rupture of the abnormal vessel confirming its role in vasospasm after SAH. Furthermore, ECs obtained from these vascular malformations can be used as an experimental model to study SAH-induced vasoconstriction.

SYSTEMIC ADMINISTRATION OF PHOSPHODIESTERASE V INHIBITOR, SILDENAFIL CITRATE, FOR ATTENUATION OF CEREBRAL VASOSPASM AFTER EXPERIMENTAL SUBARACHNOID HEMORRHAGE

One of the phosphodiesterase isoenzymes, Type V (PDE V), specifically hydrolyzes cyclic guanosine monophosphate to cause vasoconstriction. This study analyses the effect of PDE V inhibition with sildenafil citrate (SC) on cerebral vasospasm and its effect on apoptotic changes of the vascular endothelium. Twenty-four rabbits were divided into four groups. The first group was composed of sham-surgery animals. The second group was the subarachnoid hemorrhage (SAH) group, in which cerebral vasospasm was induced. In the third group, sham-surgery rabbits were treated with SC. In the fourth group, animals were treated with SC after SAH. SC was administered for 48 hours, 0.7 mg/kg, three times per day in Groups 3 and 4. Basilar artery lumen circumferences were measured in all groups by computerized image analysis. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method was used to evaluate the rate of apoptosis between SAH and SC-treated SAH groups. Results were compared by analysis of variance and paired t tests, and P values less than 0.05 were considered significant. Basilar artery circumferences between groups were significantly different (P < 0.001). SC (0.7 mg/kg, three times per d) significantly dilated the basilar arteries in both the sham-surgery group (2370 +/- 233 microm; P = 0.039) and the SAH group (2142 +/- 195 microm; P = 0.006) after 48 hours of treatment. The TUNEL method for apoptosis revealed that actual numbers of the apoptotic endothelial cells per cross section after SAH in the control (no treatment) (73 +/- 2) and SC-treated (0.7 mg/kg) groups(76 +/- 3) were not significantly different (P > 0.05). The vasodilatory effect of SC was observed to be significant on normal cerebral vessels and after SAH-induced vasospasm. SC did not prevent apoptosis of the endothelium in our study, which suggests that prevention of apoptosis is not necessary in the treatment of cerebral vasospasm.

Controlled release of lipopolysaccharide in the subarachnoid space of rabbits induces chronic vasospasm in the absence of blood

Objective Leukocyte-endothelial cell interactions appear to play a role in the development of vasospasm after SAH. Using a purely inflammatory protein, LPS, we evaluated the effect of inflammation on the development of chronic vasospasm in the absence of blood and compared it to SAH-induced vasospasm in rabbits. Methods Lipopolysaccharide was incorporated into EVAc polymers to produce 20% LPS/EVAc polymers (wt/wt). Rabbits (n = 23) were randomized to 4 experimental groups: (1) empty polymer (n = 6), (2) SAH (n = 5), (3) 0.7 mg/kg polymeric LPS dose (n = 6), and (4) 1.4 mg/kg polymeric LPS dose (n = 6). Blood and polymers were inserted into the cisterna magna. The rabbits were killed 3 days postoperatively, and the basilar arteries were harvested for morphometric analysis. Clinical response and lumen patencies were analyzed using ANOVA and a post hoc Newman-Keuls Multiple Comparisons test. Results Significant narrowing of the basilar artery was observed by insertion of 20% LPS/EVAc polymers into the subarachnoid space at a polymeric dose of 1.4 mg/kg (actual dose, 66 μg kg −1 d −1) (75.4% ± 4.2%; P < .01) and by SAH (80.3% ± 8.1%; P < .01) as compared with the empty polymer group. A trend toward narrowing was observed in the 0.7 mg/kg polymeric LPS dose group (actual dose, 33 μg kg −1 d −1) (85.2% ± 2.6%; P > .05). Symptoms associated with SAH were noted in 50% of the rabbits in the 0.7 mg/kg LPS group and in 100% of rabbits in the 1.4 mg/kg LPS group. Conclusion Controlled release of LPS into the subarachnoid space of rabbits produced chronic vasospasm in a dose-dependent manner. At a polymeric dose of 1.4 mg/kg, LPS-induced vasospasm was equivalent to that induced by SAH. This suggests that LPS and SAH may induce vasospasm through similar mechanisms and provides further evidence that inflammation plays a central role in the etiology of chronic vasospasm.

17β-Estradiol Inhibits Subarachnoid Hemorrhage–Induced Inducible Nitric Oxide Synthase Gene Expression by Interfering With the Nuclear Factor κB Transactivation

Previously, we showed that 17beta-estradiol (E(2)) treatment prevented the subarachnoid hemorrhage (SAH)-induced cerebral vasospasm in male rats. The aim of this study was designed to further delineate the molecular mechanisms underlying E(2)-induced inhibition of inducible nitric oxide synthase (iNOS) upregulation and relief of vasospasm caused by SAH. The 2-hemorrhage SAH model was induced by 2 autologous injections of blood into the cisterna magna of adult male rats. The rats were then subcutaneously implanted of a Silastic tube containing corn oil with or without 17beta-estradiol benzoate and received daily intraperitoneal injections of various doses of ICI 182,780, a nonselective estrogen receptor (ER) antagonist, for 7 days after the first hemorrhage. Basilar arteries were then removed for protein extraction, RNA isolation, and gel mobility assay. The protein levels of iNOS, p65, and ER were examined by Western blot analysis, and that iNOS mRNA expression was evaluated by reverse-transcription polymerase chain reaction. E(2) prevented the SAH-induced vasospasm and increases of the levels of iNOS protein and mRNA in basilar artery through an ER-dependent mechanism. Treatment of the SAH rat with E(2) did not affect the nuclear translocation of p65 subunit of nuclear factor kappaB, but caused an increase of the association of p65/ER, and reversed the SAH-induced increase of the p65 binding on iNOS promoter. E(2) inhibits the SAH-induced increase of iNOS by increasing the association of p65/ER, which in turn inhibits the binding of p65 to iNOS DNA. Our data suggest the potential applications of E(2) in the treatment of SAH patient.

Endothelin-converting enzyme inhibitors for the treatment of subarachnoid hemorrhage-induced vasospasm

A burgeoning body of evidence suggests that endothelin-1 (ET-1), the most potent endogenous vasoconstrictor yet identified, may be critical in the pathophysiology of various cardiovascular diseases. The ET system may also be implicated in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). Clinical studies have shown that the levels of ET-1 are increased in the cerebrospinal fluid (CSF) of patients following SAH, suggesting that ET-1-mediated vasoconstriction plays a major role in the development of vasospasm after SAH. The potential involvement of ETs in SAH-induced vasospasm has triggered considerable interest in developing therapeutic strategies that inhibit the biologic effects of ET. One promising approach to block the biosynthesis of ETs is suppressing the proteolytic conversion of the precursor peptide (big ET-1) to its vasoactive form (ET-1) using metalloprotease as endothelin-converting enzyme (ECE) inhibitor. To date, three types of ECE-1 inhibitors have been synthesized: dual ECE-1/neutral endopeptidase 24.11 (NEP) inhibitors, triple ECE-1/NEP/angiotensin-converting enzyme (ACE) inhibitors and selective ECE-1 inhibitors. The therapeutic effects of ECE-1 inhibitors on the prevention and reversal of SAH-induced vasospasm in animal studies are reviewed and discussed.

Ion channels and calcium signaling in cerebral arteries following subarachnoid hemorrhage

Entry of Ca2+ through voltage-dependent calcium channels (VDCCs) is critical to the regulation of intracellular free calcium concentration ([Ca2+]i) in vascular smooth muscle and thus the control of cerebral artery diameter. Increased VDCC activity in cerebral artery myocytes may contribute to decreased cerebral blood flow and the accompanying neurological deficits associated with subarachnoid hemorrhage (SAH). This review will focus on the impact of SAH on VDCCs and K+-selective ion channels, two important classes of ion channels located in the plasma membrane of cerebral artery myocytes. SAH may act through a variety of direct and indirect mechanisms to increase the activity of VDCCs promoting cerebral artery constriction and reduced cerebral blood flow. Further, SAH may lead to suppression of K+ channel activity to cause membrane potential depolarization to enhance VDCC activity. The ability of VDCC blockers or K+ channel activators to alleviate SAH-induced vasospasm will also be examined.

Safety and technical efficacy of over-the-wire balloons for the treatment of subarachnoid hemorrhage–induced cerebral vasospasm

Over the past decade, low-pressure, flow-directed balloons have been replaced by over-the-wire balloons in the treatment of vasospasm induced by subarachnoid hemorrhage (SAH). The authors assess the procedural safety and technical efficacy of these newer devices. Seventy-five patients who underwent 85 balloon angioplasty procedures for the treatment of SAH-induced vasospasm were identified from a prospective quality-assurance database. Medical records and angiographic reports were reviewed for evidence of procedural complications and technical efficacy. No vessel rupture or perforation occurred, but thromboembolic complications were noted in four (4.7%) of the 85 procedures. Balloon angioplasty was frequently attempted and successfully accomplished in the distal internal carotid (100%), proximal middle cerebral (94%), vertebral (73%), and basilar (88%) arteries. Severe narrowing was present in 89 proximal anterior cerebral arteries. Angioplasty was attempted in 41 of these vessels and was successful in only 14 (34%). In 19 of the 27 unsuccessful attempts, the balloon could not be advanced over the wire due to severe vasospasm or unfavorable vessel angle. Follow-up angiography in a subset of patients demonstrated that severe recurrent vasospasm occurred in 15 (13%) of 116 vessels studied after angioplasty. Over-the-wire balloons involve a low risk for vessel rupture. The anterior cerebral artery remains difficult to access and successfully treat with current devices. Further improvements in balloon design, such as smaller inflated diameters and better tracking, are necessary. Finally, thromboembolic complications remain an important concern, and severe vasospasm may recur after balloon angioplasty.

The effect of an adenosine A1 receptor agonist in the treatment of experimental subarachnoid hemorrhage-induced cerebrovasospasm

Adenosine is a potent vasodilator and an important modulator of cardiovascular function. It has been postulated that nitric oxide (NO) is involved in adenosine-induced vasodilation. This study was designed to examine the effect of an adenosine A1 agonist, N6-cyclopentyladenosine (CPA), in the prevention of subarachnoid haemorrhage (SAH)-induced vasospasm. Method. Experimental SAH was induced in Sprague-Dawley rats by injecting 0.3 mL autogenous blood into the cisterna magna. Intraperitoneal injections of CPA (0.003 mg/kg), or vehicle were administered 5 min and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging the cross sectional areas of the basilar artery 2 days after SAH. Expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in basilar artery were evaluated. Findings. There were no significant differences among the control and treated groups in physiological parameters recorded before sacrifice. When compared with animals in the control group, cross-sectional area of basilar arteries areas in the SAH only, SAH plus vehicle and SAH plus CPA groups were reduced by 19% (p < 0.01), 22% (p < 0.01), and 9% (p = 0.133), respectively. The cross-sectional areas of the CPA-treated group differed significantly from those of the SAH only and SAH plus vehicle group (p < 0.05). Induction of iNOS-mRNA and protein in basilar artery by SAH was not significantly diminished by CPA. The SAH-induced suppression of eNOS-mRNA and protein were relieved by CPA treatment. Conclusions. This is the first evidence to show an adenosine A1 receptor agonist is effective in partially preventing SAH-induced vasospasm without significant cardiovascular complications. The mechanisms of adenosine A1 receptor agonists in attenuating SAH-induced vasospasm may be, in part, related to preserve the normal eNOS expression after SAH. Inability in reversing the increased iNOS expression after SAH may lead to the incomplete anti-spastic effect of CPA.

17beta-estradiol inhibits endothelin-1 production and attenuates cerebral vasospasm after experimental subarachnoid hemorrhage.

Though cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) has been recognized for over half a century, it remains a major complication in patients with SAH. Clinical studies have shown that elevated levels of endothelin-1 (ET-1) are present in the cerebrospinal fluid of patients with SAH, suggesting that ET-1-mediated vasoconstriction contributes to vascular constriction after SAH. Administration of estrogen promotes vasodilation in humans and in experimental animals, in part by decreasing the production of ET-1. This study evaluated the influence of 17beta-estradiol (E2) on the production of ET-1 and cerebrovasospasm in an experimental SAH 2-hemorrhage model in rat. A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats just before SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Plasma samples collected before death were assayed for ET-1. The protective effect of E2 in attenuating vasospasm achieved statistical significance when compared with the SAH only or SAH plus vehicle groups (P < 0.01). Concentrations of ET-1 were higher in the SAH only and SAH plus vehicle groups than in controls (P < 0.001). Serum levels of ET-1 in the SAH plus E2 and E2 only groups were significantly lower than those in the SAH only and SAH plus vehicle groups (P < 0.001). There was no significant difference between ET-1 levels in the healthy control and SAH plus E2 groups. A significant correlation was found between the cross-sectional areas of basilar artery and ET-1 levels (P < 0.001). The beneficial effect of E2 in attenuating SAH-induced vasospasm may be due in part to decreasing ET-1 production after SAH. The role of E2 in the treatment of cerebral vasospasm after SAH is promising and is worthy of further investigation.

Oxyhemoglobin (oxyhb) induces R‐type calcium channel expression in cerebral arteries

Cerebral vasospasm after subarachnoid hemorrhage (SAH) represents a major cause of mortality and morbidity following aneurysm rupture. Our laboratory has recently shown the emergence of R-type Ca2+ channels is associated with increased cerebral artery constriction in a rabbit SAH model (Ishiguro et al., Cir. Res. 2005). Here, we have examined whether the blood component, oxyhb, can mimic the ability of SAH to induce expression of R-type Ca2+ channels in the cerebral vasculature. Rabbit cerebral arteries were organ cultured for 1–5 days in the presence or absence of oxyhb. In the absence of oxyhb, constriction to 60 mM K+ is completely abolished by diltiazem, a L-type VDCC antagonist. However, following 5days of oxyhb treatment, K+-induced constriction became partially resistant to diltiazem. Further, the combination of SNX-482 (R-type VDCC antagonist) and diltiazem fully dilated oxyhb-exposed arteries. RT-PCR revealed that after 5 days of oxyhb, arteries expressed R-type Ca2+ channels (Cav2.3) in addition to L-type Ca2+ channels (Cav1.2). In contrast, arteries cultured in the absence of oxyhb expressed only L-type Ca2+ channels. These results demonstrate that oxyhb exposure for 5 days induces the expression of Cav2.3 in cerebral arteries. We propose that oxyhb contributes to SAH-induced vasospasm via the emergence of R-type Ca2+ channels in cerebral arteries. Expression of R-type Ca2+ channels may help to explain why L-type antagonists are relatively ineffective at reversing SAH-induced vasospasm. The work was supported by NIH (P20 RR16435, R01 HL078983) and Totman Medical Research Trust.

BilirubinOxidation Products (BOXes) and Their Role in Cerebral Vasospasm after Subarachnoid Hemorrhage

Many factors have been postulated to cause delayed subarachnoid hemorrhage (SAH)-induced vasospasm, including hemoglobin, nitric oxide, endothelin, and free radicals. We propose that free radicals (because of the high levels that are produced in the blood clots surrounding blood vessels after SAH) act on bilirubin, biliverdin, and possibly heme to produce BOXes (Bilirubin OXidized Products). Bilirubin oxidation products act on vascular smooth muscle cells to produce chronic vasoconstriction and vasospasm combined with a vasculopathy because of smooth muscle cell injury. This review summarizes recent evidence that BOXes play a role in SAH-induced vasospasm. The data supporting a role for BOXes includes (1) identification of molecules in cerebrospinal fluid (CSF) of patients with vasospasm after SAH that have structures consistent with BOXes; (2) BOXes are vasoactive in vitro and mimic the biochemical actions of CSF of patients with vasospasm; (3) BOXes are vasoactive in vivo, constricting rat cerebral vessels; and (4) there is a correlation between clinical occurrence of vasospasm and BOXes concentration in our preliminary study of patients with SAH. Since oxidation of bilirubin, biliverdin, and perhaps heme is proposed to produce BOXes that contribute to vasospasm, either blocking bilirubin formation, inactivating bilirubin or BOXes, or removing all of the blood clot before vasospasm are potential treatment targets.

The effect of 17β-estradiol in attenuating experimental subarachnoid hemorrhage–induced cerebral vasospasm

Sex differences in the outcome of aneurysmal subarachnoid hemorrhage (SAH) are controversial, and the potential influence of estradiol on vasodilation is unclear. In the present study the authors evaluate the effect and possible mechanism of 17beta-estradiol (E2) on SAH-induced vasospasm in a two-hemorrhage rodent model of SAH. A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats. Serum levels of E2 were measured on Days 0, 1, 2, 3, 4, and 7 postimplantation. The degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery (BA) 7 days after the first SAH. Expressions of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the BA were also evaluated. Serum levels of E2 in the E2-treated rats were at physiological levels (56-92 pg/ml) and were significantly higher than those in the control and vehicle-treated groups. Treatment with E2 significantly (p < 0.01) attenuated SAH-induced vasospasm. Induction of iNOS messenger (m)RNA and protein in the BA by SAH was significantly diminished by the E2 treatment but not by vehicle treatment. The SAH-induced suppression of eNOS mRNA and protein was relieved by E2 treatment. These results suggest that continuous treatment with E2 at physiological levels prevents cerebral vasospasm following SAH. The beneficial effect of E2 may be in part related to the prevention of augmentation of iNOS expression and the preservation of normal eNOS expression after SAH. Treatment with E2 holds therapeutic promise in the treatment of cerebral vasospasm following SAH and merits further investigation.

Co‐expression/co‐location of S100 proteins (S100B, S100A1 and S100A2) and protein kinase C (PKC‐β, ‐η and ‐ζ) in a rat model of cerebral basilar artery vasospasm

The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm.