endothelin-1 and Subarachnoid-Hemorrhage

Aneurysmal subarachnoid hemorrhage (SAH) is an acute cerebrovascular emergency resulting from the rupture of a brain aneurysm. Despite only accounting for 5% of all strokes, SAH imposes a significant health burden on society due to its relatively young age at onset. Those who survive the initial bleed are often afflicted with severe disabilities thought to result from delayed cerebral ischemia (DCI). Consequently, elucidating the underlying mechanistic pathways implicated in DCI development following SAH remains a priority. Neuroinflammation has recently been implicated as a promising new theory for the development of SAH complications. However, despite this interest, clinical trials have failed to provide consistent evidence for the use of anti-inflammatory agents in SAH patients. This may be explained by the complexity of SAH as a plethora of inflammatory pathways have been shown to be activated in the disease. By determining how these pathways may overlap and interact, we hope to better understand the developmental processes of SAH complications and how to prevent them. The goal of this review is to provide insight into the available evidence regarding the molecular pathways involved in the development of inflammation following SAH and how SAH complications may arise as a result of these inflammatory pathways.

Topics: Brain Ischemia; Endothelin-1; Humans; Inflammation; Intracranial Aneurysm; Metabolic Networks and Pathways; Mitogen-Activated Protein Kinases; Oxyhemoglobins; Platelet Activating Factor; Reactive Oxygen Species; Serotonin; Subarachnoid Hemorrhage; Thrombin

Despite intensive research efforts, by our own team and many others, the molecules responsible for acute neurological damage following subarachnoid hemorrhage (SAH) and contributing to delayed ischemic neurological deficit (DIND) have not yet been elucidated. While there are a number of candidate mechanisms, including nitric oxide (NO) scavenging, endothelin-1, protein kinase C (PKC) activation, and rho kinase activation, to name but a few, that have been investigated using animal models and human trials, we are, it seems, no closer to discovering the true nature of this complex and enigmatic pathology. Efforts in our laboratory have focused on the chemical milieu present in hemorrhagic cerebrospinal fluid (CSF) following SAH and the interaction of the environment with the molecules generated by SAH and subsequent events, including NO scavenging, immune response, and clot breakdown. We have identified and characterized a group of molecules formed by the oxidative degradation of bilirubin (a clot breakdown product) and known as BOXes (bilirubin oxidation products). We present a synopsis of the characterization of BOXes as found in human SAH patients' CSF and the multiple signaling pathways by which BOXes act. In summary, BOXes are likely to play an essential role in the etiology of acute brain injury following SAH, as well as DIND.

Topics: Animals; Bilirubin; Brain Injuries; Brain Ischemia; Endothelin-1; Humans; Models, Biological; Muscle, Smooth, Vascular; Nitric Oxide; Oxidation-Reduction; Protein Kinase C; rho-Associated Kinases; Signal Transduction; Subarachnoid Hemorrhage

Delayed ischemic neurological deficits (DINDs) contribute to poor outcomes after aneurysmal subarachnoid hemorrhage (SAH). Endothelin-1 is an important mediator involved in the development of vasospasm.. We performed a systematic review and meta-analysis of randomized controlled trials assessing the use of endothelin-receptor antagonists (ETRAs) in patients with SAH.. Three studies met eligibility criteria, enrolling 867 patients. ETRAs significantly reduced the occurrence of DINDs (OR 0.68 [0.49 to 0.95]) and radiographic vasospasm (OR 0.31 [0.19 to 0.49]), but did not have any impact on mortality (OR 1.09 [0.69 to 1.72]) or poor neurological outcomes (OR 0.87 [0.63 to 1.20]). Any benefit of ETRAs may have been partially offset by adverse effects, including hypotension(OR 2.39 [1.37 to 4.17]) and pulmonary complications (OR 2.12 [1.51 to 2.98]).. Although ETRAs reduce radiographic vasospasm and DINDs, there is currently no evidence that they improve outcomes.

Topics: Brain Infarction; Cerebral Arteries; Endothelin Receptor Antagonists; Endothelin-1; Humans; Outcome Assessment, Health Care; Radiography; Randomized Controlled Trials as Topic; Receptors, Endothelin; Subarachnoid Hemorrhage; Treatment Outcome; Vasospasm, Intracranial

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acute Disease; Angioplasty, Balloon; Calcium Channel Blockers; Cell Membrane; Combined Modality Therapy; Endothelin-1; Hemoglobins; Humans; Muscle, Smooth, Vascular; Nicardipine; Nitric Oxide; Oxidative Stress; Subarachnoid Hemorrhage; Vasodilator Agents; Vasospasm, Intracranial; Verapamil

Cerebral vasospasm lingers as the leading preventable cause of death and disability in patients who experience aneurysmal subarachnoid hemorrhage. Despite the potentially devastating consequences of cerebral vasospasm, the mechanisms behind it are incompletely understood. Nitric oxide, endothelin-1, bilirubin oxidation products and inflammation appear to figure prominently in its pathogenesis. Therapies directed at many of these mechanisms are currently under investigation and hold significant promise for an ultimate solution to this substantial problem.

Topics: Anti-Inflammatory Agents; Bilirubin; Calcium Channel Blockers; Cerebral Angiography; Encephalitis; Endothelin-1; Fibrinolytic Agents; Heme Oxygenase (Decyclizing); Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracranial Aneurysm; Magnesium; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

Vasospasm is one of the leading causes of morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH). Radiographic vasospasm usually develops between 5 and 15 days after the initial hemorrhage, and is associated with clinically apparent delayed ischemic neurological deficits (DID) in one-third of patients. The pathophysiology of this reversible vasculopathy is not fully understood but appears to involve structural changes and biochemical alterations at the levels of the vascular endothelium and smooth muscle cells. Blood in the subarachnoid space is believed to trigger these changes. In addition, cerebral perfusion may be concurrently impaired by hypovolemia and impaired cerebral autoregulatory function. The combined effects of these processes can lead to reduction in cerebral blood flow so severe as to cause ischemia leading to infarction. Diagnosis is made by some combination of clinical, cerebral angiographic, and transcranial doppler ultrasonographic factors. Nimodipine, a calcium channel antagonist, is so far the only available therapy with proven benefit for reducing the impact of DID. Aggressive therapy combining hemodynamic augmentation, transluminal balloon angioplasty, and intra-arterial infusion of vasodilator drugs is, to varying degrees, usually implemented. A panoply of drugs, with different mechanisms of action, has been studied in SAH related vasospasm. Currently, the most promising are magnesium sulfate, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, nitric oxide donors and endothelin-1 antagonists. This paper reviews established and emerging therapies for vasospasm.

Topics: Angioplasty, Balloon; Calcium Channel Blockers; Cerebral Angiography; Endothelin-1; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Neuroprotective Agents; Nimodipine; Nitric Oxide Donors; Pregnatrienes; Subarachnoid Hemorrhage; Thrombolytic Therapy; Ultrasonography, Doppler, Transcranial; Vasodilator Agents; Vasospasm, Intracranial

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.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Humans; Metalloendopeptidases; Neprilysin; Subarachnoid Hemorrhage; Vasoconstriction; Vasospasm, Intracranial

Genetic modification of cerebral vessels represents a promising and novel approach for prevention and/or treatment of various cerebral vascular disorders, including cerebral vasospasm. In this review, we focus on the current understanding of the use of gene transfer to the cerebral arteries for prevention and/or treatment of cerebral vasospasm following subarachnoid hemorrhage (SAH). We also discuss the recent developments in vascular therapeutics, involving the autologous use of progenitor cells for repair of damaged vessels, as well as a cell-based gene delivery approach for the prevention and treatment of cerebral vasospasm.

Topics: Animals; Cerebral Arteries; Endothelin-1; Gene Transfer Techniques; Genetic Therapy; Humans; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Regeneration; Stem Cells; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Endothelin (ET)-mediated vasoconstriction has been implicated in the pathophysiology of various disorders, e.g. hypertension, chronic heart failure, acute renal failure, pulmonary hypertension, and subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. The potential involvement of ETs in cerebral vasospasm following SAH has triggered considerable interest in designing therapeutic strategies to inhibit biological effects of ET. Major approaches include: (a) reducing the levels of circulating ET- 1 by the the specific anti- ET- 1 antibodies, (b) antagonizing the ET receptors, and (c) suppressing the biosynthesis of ET-1. To date, numerous antagonists of ET(A) and/or ET(B) receptors have been discovered, and some are under clinical evaluation. Inhibitors of endothelin-converting enzymes (ECEs), which catalyze the biosynthesis of ET-1, have also been synthesized. Two types of ECE-1 inhibitors have been evaluated in various animal disease models: dual ECE-1/neutral endopeptidase 24.11 (NEP) inhibitors and selective ECE-1 inhibitors. In this article, the effects of ET receptor antagonists and ECE-1 inhibitors on the prevention and reversal of SAH-induced cerebral vasospasm in preclinical animal models are reviewed.

Topics: Adrenergic Agents; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Humans; Metalloendopeptidases; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Molecular biologic investigations in the past decades have begun to unravel the intracellular mechanisms involved in vasomotor regulation of cerebral blood vessels and their failure in delayed cerebral vasospasm produced by aneurysmal subarachnoid hemorrhage. Progress in deciphering macrovascular regulatory mechanisms and their failure in delayed cerebral vasospasm induced by aneurysmal subarachnoid hemorrhage have revealed that there are at least two important vasoactive substances-nitric oxide and endothelin-1-that play important roles in the clinical manifestations of subarachnoid-hemorrhage-induced cerebral vasospasm. Nitric oxide is a cell-membrane-permeable free radical gas that accounts for the phenomenon of vasodilatation by a variety of vasodilator agents. Endothelin-1, a 21 amino acid peptide, is one of the most potent constricting factors. Cerebral vasospasm is thought to represent a disturbance in the cerebral vasomotor equilibrium for which these two physiologically antagonistic compounds are at least partly responsible. Advances in our understanding of the molecular responses of the cerebral vasculature to subarachnoid hemorrhage should lead to more comprehensive management as knowledge becomes translated into development of effective pharmacologic agents to reverse or prevent cerebral vasospasm following aneurysmal subarachnoid hemorrhage.

Topics: Endothelin-1; Humans; Intracranial Aneurysm; Nitric Oxide; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

Topics: Animals; Endothelin-1; Male; Rabbits; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Increasing evidence implicates endothelin (ET)-1 in the pathophysiological development of cerebral vasospasm. This study examined the ability of TBC 11251 (TBC), a new ETA receptor antagonist, to prevent vasospasm in a rabbit model of subarachnoid hemorrhage (SAH).. Eighty-five New Zealand White rabbits were assigned to 1 of 10 groups. SAH was induced by injecting autologous blood into the cisterna magna. The treatment groups were as follows: 1) control (no SAH), 2) SAH alone, 3) SAH plus vehicle every 12 hours (BID), 4) SAH plus 5 mg/kg TBC BID, 5) SAH plus 10 mg/kg TBC BID, 6) SAH plus 20 mg/kg TBC BID, 7) SAH plus vehicle at 24 and 36 hours after SAH (24/36), 8) SAH plus 5 mg/kg TBC 24/36, 9) SAH plus 10 mg/kg TBC 24/36, and 10) SAH plus 20 mg/kg TBC 24/36. Animals were killed 48 hours after SAH, by perfusion-fixation, and then basilar arteries were histologically prepared and their cross-sectional areas were measured.. The mean basilar artery cross-sectional area was constricted from 0.332 mm2 in the control group to 0.131 mm2 in the SAH alone group, 0.132 in the vehicle 24/36 group, and 0.125 in the vehicle BID group. All groups treated with TBC showed an increase in cross-sectional luminal basilar artery area, relative to the vehicle-treated groups. The 5 mg/kg TBC BID group exhibited a mean basilar artery area of 0.217 mm2, and the 10 mg/kg TBC BID group showed a mean basilar artery area of 0.240 mm2; both groups were statistically improved, compared with the vehicle-treated groups (P < 0.05). No side effects were seen, and there were no differences in the mean arterial pressures between drug- and vehicle-treated groups.. These findings demonstrate that systemic administration of the ETA receptor antagonist TBC significantly attenuates cerebral vasospasm after SAH, thus providing additional support for the role of ET-1 in vasospasm.

Topics: Animals; Basilar Artery; Calcium Signaling; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endothelin Receptor Antagonists; Endothelin-1; Infusions, Intravenous; Ischemic Attack, Transient; Isoxazoles; Male; Rabbits; Receptor, Endothelin A; Subarachnoid Hemorrhage; Thiophenes

The endothelin family of peptides are extremely potent endogenous vasoconstrictor and pressor agents. Of the 3 isoforms, endothelin-1 is the major isoform produced by the vascular endothelium and is, therefore, likely to be of most importance for regulation of vascular function. Two endothelin receptor subtypes have so far been cloned in mammalian species; ET A, and ET B. Both receptor subtypes are found on smooth muscle cells and mediate the vasoconstrictor and pressor actions of endothelin. The ET B receptor is also found on vascular endothelial cells and mediates endothelin-dependent vasodilatation through release of nitric oxide and prostacyclin. Since their discovery in 1988, the endothelins have been the subject of intense research on their physiological function and potential pathophysiological role in cardiovascular disease. There is now good evidence that endothelin regulates vascular tone and blood pressure, and studies to support the development of endothelin receptor antagonists in conditions associated with chronic vasoconstriction, such as hypertension and heart failure, as well as in vasospastic disorders, such as subarachnoid haemorrhage and Raynaud's disease. There are now a number of selective ET A and combined ET A/B receptor antagonists available for preclinical studies. However, it is still not clear which of these will prove to be of most therapeutic value. Some of these agents are currently being assessed in early phase clinical trials. Endothelin receptor antagonists represent a novel therapeutic approach to a fundamental and newly discovered endogenous vasoconstrictor mechanism. The results of the current clinical trials are awaited with considerable interest.

Topics: Amino Acid Sequence; Animals; Cardiovascular Diseases; Coronary Disease; Endothelin Receptor Antagonists; Endothelin-1; Heart Failure; Humans; Hypertension; Migraine Disorders; Raynaud Disease; Subarachnoid Hemorrhage

The purpose of this study is to establish the diagnostic sensitivity of Endothelin-1 for risk stratification and screening of clinical vasospasm after subarachnoid hemorrhage.This is a multicentre, observational study, correlating daily blood Endothelin-1 with clinical variables. Binary logistic regression used to examine if Endothelin-1 levels could be used to predict clinical vasospasm. Bivariate modelling used to explore associations between patient characteristics and vasospasm. A Receiver Operating Curve used to explore cut-off values for Endothelin-1. Sensitivity and specificity was used to validate the cut-point found in the pilot study. A total of 96 patients were enrolled over two years. Median Endothelin-1 was higher for patients who experienced clinical vasospasm except for day-5, where median endothelin for patients without vasospasm was higher (3.6 IQR = 5.3), compared to patients with vasospasm (3.3 IQR = 8.5) although differences were not significant. The Receiver Operating Curve analysis confirmed that day-5 Endothelin-1 was not a good indicator of vasospasm, with an area under the curve of 0.506 (95% CI: 0.350-0.663,

Topics: Double-Blind Method; Endothelin-1; Humans; Pilot Projects; Subarachnoid Hemorrhage; Vasospasm, Intracranial

To investigate the effects and possible mechanisms of puerarin on the vascular active factors correlated to cerebral vasospasm (CVS) after aneurysm subarachnoid hemorrhage (aSAH).. Fifty-four patients with aSAH were randomly assigned to the puerarin group (30 cases) and the control group (24 cases) by lot. On the basis of routine treatment, the patients in the puerarin group were intravenously dripped with 0.5 g puerarin by adding in 250 mL glucose injection once daily. The injection was given starting from the 3rd day of the disease course, for 14 successive days. The plasma levels of nitric oxide (NO), endothelin-1 (ET-1), thromboxane B, (TXB2), 6-Keto-prostaglandin F1alpha (6-K-PGF1alpha) were compared between the two groups pre- and post-therapy. The incidence of cerebral vasospasm (CVS) was observed using transcranial Doppler (TCD). The Glasgow outcome scale (GOS) were compared at discharge between the two groups.. Compared with the control group, the plasma levels of NO, ET-1, and 6-K-PGF1alpha increased in the puerarin group (P < 0. 05), the TXB2 level decreased (P < 0.05), the incidence of CVS decreased (P < 0.05), the mean MCA velocity increased (P < 0.05), and the GOS at discharge increased (P < 0.05).. Puerarin is an effective agent for the prophylaxis and treatment of the CVS in patients after aSAH. Moreover, it can improve the prognosis. The mechanism might be correlated with improving the levels of the vascular active factors, i.e., increasing the plasma levels of NO and PGl2, decreasing TXA, in plasma, increasing the cerebral blood flow, and improving cerebral perfusion.

Topics: 6-Ketoprostaglandin F1 alpha; Adult; Aged; Endothelin-1; Female; Humans; Isoflavones; Male; Middle Aged; Nitric Oxide; Prognosis; Subarachnoid Hemorrhage; Thromboxane B2; Vasospasm, Intracranial

Plasma and cerebrospinal fluid (CSF) concentrations of endothelin-1 (ET-1) were measured in patients with subarachnoid hemorrhage (SAH) after aneurysmal rupture and compared with levels of ET-1 in volunteers. We analyze the relationship between levels of ET-1 in both CSF and plasma with the risk of developing cerebral vasospasm (CVS).. Cerebrospinal fluid and blood samples were collected from 30 selected patients after SAH and from 10 healthy volunteers who were used as control. All samples were stored at -70 degrees C and the levels of ET-1 in CSF and blood were measured by using enzyme-linked immunosorbent assay and Western blot. All patients were submitted to angiography to confirm vasospasm.. From the 30 patients admitted at different days of SAH, 18 (60%) developed clinical CVS and 10 (33%) presented angiographic CVS. The levels of ET-1 in the CSF were significantly higher (P = .0001) in patients (1.618 +/- 1.05 fmol/mL) than in controls (0.365 +/- 0.328 fmol/mL). There was statistical difference (P < .05) in CSF levels of ET-1 between each group of the Hunt-Hess scale and controls. The mean plasma concentration of ET-1 was similar (P > .05) in the control group (1.531 +/- 0.753 fmol/mL) and in patients with SAH (1.920 +/- 1.15 fmol/mL).. These findings indicate that a significant rise in ET-1 levels in the CSF, but not in the plasma, occurs in patients who develop CVS after SAH. Our observation suggests that ET-1 might be involved in the pathogenesis of SAH-associated CVS.

Topics: Cerebral Angiography; Cerebral Arteries; Endothelin-1; Humans; Predictive Value of Tests; Reference Values; Risk Factors; Subarachnoid Hemorrhage; Subarachnoid Space; Up-Regulation; Vasospasm, Intracranial

Endothelin 1 (ET-1) is a potent vasoconstrictor that may play a role in cerebral vasospasm following subarachnoid hemorrhage (SAH). However, data regarding its pathogenic role in the development of vasospasm are controversial. We planned a prospective, observational clinical study to investigate the temporal relationship between increased ET-1 production and cerebral vasospasm or other neurological sequelae after SAH.. ET-1 levels in cerebrospinal fluid (CSF) were measured in 20 SAH patients from admission (within 24 hours from the bleeding) until day 7. Patients received a daily transcranial Doppler study and a neurological evaluation. On day 7, angiography was performed to verify the degree and extent of vasospasm. Patients were then classified as having (1) clinical vasospasm, (2) angiographic vasospasm, (3) no vasospasm, or (4) poor neurological condition without significant vasospasm (low Glasgow Coma Scale score [GCS]).. On admission, ET-1 levels were increased in the low-GCS group compared with the other groups (P=0.04). On day 4 ET-1 levels were not significantly different among groups, whereas on day 7 ET-1 levels were significantly increased in both the clinical vasospasm and low-GCS groups compared with the angiographic vasospasm and no vasospasm groups (P<0.005). Moreover, when the low-GCS group was excluded, there was a significant relationship between vasospasm grade and CSF ET-1 levels (R(2)=0.73).. CSF ET-1 levels were markedly elevated in patients with clinical manifestations of vasospasm (day 7) and with a poor neurological condition not related to vasospasm. However, ET-1 levels were low in clinical vasospasm patients before clinical symptoms were evident (day 4) and remained low in angiographic vasospasm patients throughout the study period. Thus, our data suggest that CSF ET-1 levels are increased in conditions of severe neuronal damage regardless whether this was due to vasospasm or to the primary hemorrhagic event. In addition, CSF ET-1 levels paralleled the neurological deterioration but were not predictive of vasospasm.

Topics: Cerebral Angiography; Disease Progression; Endothelin-1; Female; Humans; Incidence; Male; Middle Aged; Predictive Value of Tests; Severity of Illness Index; Subarachnoid Hemorrhage; Ultrasonography, Doppler, Transcranial; Vasospasm, Intracranial

This study aimed to study the association between rs12976445 polymorphism and the incidence of IA re-bleeding. Genotype and allele frequency analysis was performed to study the association between rs12976445 polymorphism and the risk of IA re-bleeding. Western blot, ELISA and real-time RT-PCR were conducted to measure the relative expression of miR-125a, ET1 mRNA and ET1 protein. Computational analysis and luciferase assays were utilized to investigate the association between the expression of miR-125a and ET1 mRNA. No significant differences were observed between IA patients with or without symptoms of re-bleeding. Subsequent analyses indicated that the T allele was significantly associated with the reduced risk of IA re-bleeding. In patients carrying the CC genotype, miR-125a level was up-regulated while ET1 mRNA/protein levels were reduced compared with those in patients carrying the CT or TT genotype. And ET1 mRNA was identified as a virtual target gene of miR-125a with a potential miR-125a binding site located on its 3'UTR. Accordingly, the ET mRNA/protein levels could be suppressed by the transfection of miR-125a precursors, but the transfection of ET1 siRNA exhibited no effect on the expression of miR-125a. Therefore, an increased level of miR-125a can lead to the increased risk of IA re-bleeding. Since miR-125a level is higher in CC-genotyped patients, it can be concluded that the presence of T allele in the rs12976445 polymorphism is associated with a lower risk of IA re-bleeding, and miR-125a may be used as a novel diagnostic and therapeutic target for IA rupture.

Topics: 3' Untranslated Regions; Antifibrinolytic Agents; Brain; Cell Line; Cerebral Arteries; Endothelin-1; Female; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Intracranial Aneurysm; Male; MicroRNAs; Polymorphism, Single Nucleotide; Risk; Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH), especially aneurysmal subarachnoid hemorrhage, is a serious cerebrovascular disease with high mortality and morbidity. However, there is no effective treatment in clinics. In recent years, more and more studies have shown that early brain injury (EBI) may be an important reason for poor prognosis of SAH. Explore the mechanism of early brain injury after subarachnoid hemorrhage (SAH). In this study, 20 male New Zealand white rabbits were selected and divided into the experimental group and sham operation group, with 10 rabbits in each group. The neurobehavioral scores, food intake, and cerebral perfusion parameters, cerebral blood volume (CBV), cerebral blood flow velocity (CBF), ET-1, IL-1, and IL-6, in rabbit plasma were compared. The food intake scores and neurological dysfunction scores of the experimental group at 1 h, 6 h, 24 h, and 72 h after modeling were higher than those of the sham operation group, which had a statistical significance (

Topics: Animals; Brain Injuries; Endothelin-1; Female; Humans; Interleukin-1; Interleukin-6; Male; Microcirculation; Rabbits; Rats; Subarachnoid Hemorrhage

Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH.. Human aneurysmal blood and cerebral spinal fluid were collected for evaluation. To confirm mechanism, c57/bl6 wild type and c57/bl6 IL-6 female knockout (KO) mice were utilized with groups: saline injected, SAH, SAH + IL-6 blockade, SAH IL-6 KO, SAH IL-6 KO + IL-6 administration, SAH + p-STAT3 inhibition. Dual-labeled microglia/myeloid mice were used to show myeloid diapedesis. For SAH, 50 μm blood was collected from tail puncture and administered into basal cisterns. IL-6 blockade was given at various time points. Various markers of neuroinflammation were measured with western blot and immunohistochemistry. Cerebral blood flow was also measured. Vasospasm was measured via cardiac injection of India ink/gelatin. Turning test and Garcia's modified SAH score were utilized. P < 0.05 was considered significant.. IL-6 expression peaked 3 days following SAH (p < 0.05). Human IL-6 was increased in aneurysmal blood (p < 0.05) and in cerebral spinal fluid (p < 0.01). Receptor upregulation was periventricular and perivascular. Microglia activation following SAH resulted in increased caveolin 3 and myeloid diapedesis. A significant increase in BBB markers endothelin 1 and occludin was noted following SAH, but reduced with IL-6 blockade (p < 0.01). CV occurred 5 days post-SAH, but was absent in IL-6 KO mice and mitigated with IL-6 blockade (p < 0.05). IL-6 blockade, and IL-6 KO mitigated effects of SAH on cerebral blood flow (p < 0.05). SAH mice had impaired performance on turn test and poor modified Garcia scores compared to saline and IL-6 blockade. A distinct microglia phenotype was noted day 5 in the SAH group (overlap coefficients r = 0.96 and r = 0.94) for Arg1 and iNOS, which was altered by IL-6 blockade. Day 7, a significant increase in toll-like receptor 4 and Stat3 was noted. This was mitigated by IL-6 blockade and IL-6 KO, which also reduced Caspase 3 (p < 0.05). To confirm the mechanism, we developed a p-STAT3 inhibitor that targets the IL-6 pathway and this reduced NFΚB, TLR4, and nitrotyrosine (p < 0.001). Ventricular dilation and increased Tunel positivity was noted day 9, but resolved by IL-6 blockade (p < 0.05).. Correlation between IL-6 and CV has been well documented. We show that a mechanistic connection exists via the p-STAT3 pathway, and IL-6 blockade provides benefit in reducing CV and its consequences mediated by myeloid cell origin diapedesis.

Topics: Animals; Caspase 3; Caveolin 3; Endothelin-1; Female; Gelatin; Humans; Interleukin-6; Mice; Mice, Knockout; Subarachnoid Hemorrhage; Toll-Like Receptor 4; Vasospasm, Intracranial

Cerebral vasospasm (CVS) remains a major cause of delayed cerebral ischaemia following aneurysmal subarachnoid haemorrhage (SAH), making it a life-threatening type of stroke with high morbidity and mortality. Endothelin-1 is known as key player mediating a strong vasocontractile effect. Interestingly, losartan restores the impaired vasorelaxative ET(B. Cerebral vasospasm was induced by the use of an established double-injection rat model. Sprague-Dawley rats were culled on Day 3 after the ictus, and the vasospastic basilar artery was harvested for isometric investigations of the vessel tone. Ring segments were preincubated with and without L-NAME and/or losartan.. Preincubation with L-NAME induced dose-dependent vasoconstriction via endothelin-1 in the non-SAH cohort, which was dose-dependently reduced by losartan. After SAH and dose-dependent endothelin-1 administration, maximal contraction was achieved in the control group without losartan. Furthermore, this maximal contraction was significantly decreased in the losartan group and was reversed by L-NAME.. After SAH, losartan was shown to positively influence the ET(B

Topics: Animals; Basilar Artery; Disease Models, Animal; Endothelin-1; Losartan; Male; NG-Nitroarginine Methyl Ester; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Regenerative Medicine; Subarachnoid Hemorrhage; Vasoconstriction; Vasodilation; Vasodilator Agents; Vasospasm, Intracranial

Subarachnoid hemorrhage (SAH) is associated with increased cerebral artery sensitivity to vasoconstrictors and release of the perivascular sensory vasodilator CGRP. In the current study the constrictive phenotype and the vasodilatory effects of exogenous and endogenous perivascular CGRP were characterized in detail applying myograph technology to cerebral artery segments isolated from experimental SAH and sham-operated rats. Following experimental SAH, cerebral arteries exhibited increased vasoconstriction to endothelin-1, 5-hydroxytryptamine and U46419. In addition, depolarization-induced vasoconstriction (60 mM potassium) was significantly increased, supporting a general SAH-associated vasoconstrictive phenotype. Using exogenous CGRP, we demonstrated that sensitivity of the arteries to CGRP-induced vasodilation was unchanged after SAH. However, vasodilation in response to capsaicin (100 nM), a sensory nerve activator used to release perivascular CGRP, was significantly reduced by SAH (P = 0.0079). Because CGRP-mediated dilation is an important counterbalance to increased arterial contractility, a reduction in CGRP release after SAH would exacerbate the vasospasms that occur after SAH. A similar finding was obtained with artery culture (24 h), an in vitro model of SAH-induced vascular dysfunction. The arterial segments maintained sensitivity to exogenous CGRP but showed reduced capsaicin-induced vasodilation. To test whether a metabolically stable CGRP analogue could be used to supplement the loss of perivascular CGRP release in SAH, SAX was systemically administered in our in vivo SAH model. SAX treatment, however, induced CGRP-desensitization and did not prevent the development of vasoconstriction in cerebral arteries after SAH.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Cerebral Arteries; Endothelin-1; Male; Models, Animal; Rats; Rats, Sprague-Dawley; Serotonin; Subarachnoid Hemorrhage; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

In subarachnoid hemorrhage (SAH), occurrence of cerebral vasospasm (CVS) mediated by endothelin (ET)-1 might be a result of a compartmental inflammatory response with interleukin (IL)-6 release. We aim to investigate the relationship between ET-1 and IL-6 in association of CVS.. A total of 24 New Zealand white rabbits where randomly allocated into 3 groups: SAH (N.=10), IL-6 (N.=10), and sham (N.=4). SAH was induced by a closed cranium extracranial-intracranial shunt model. In the IL-6 group, IL-6 was injected into the cisterna magna. CVS of the basilar artery was assessed by digital subtraction angiography. IL-6 and ET-1 concentrations were measured using enzyme-linked immunosorbent assay. Neuronal damage was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling.. A significant increase between baseline (day 0) and follow-up (day 3) was found in CSF IL-6 levels of animals in the SAH and IL-6 group. There was a statistically significant correlation between IL-6 and ET-1 levels in the CSF (Pearson's r=0.454, P=0.003). CVS at day 3 was more pronounced in the SAH than in the IL-6 group: 26.0 ±7.2 % and 16.7 ±5.0 % respectively. TUNEL positive apoptotic neurons in the hippocampal formation were present in the SAH group and in a lesser degree in the IL-6 group.. The results indicate that IL-6 triggered CVS after SAH is ET-1 dependent. IL-6 may be a target for new therapeutic approaches.

Topics: Animals; Endothelin-1; Interleukin-6; Rabbits; Subarachnoid Hemorrhage; Vasospasm, Intracranial

OBJECTIVE Endothelin-1, a potent vasoconstrictor, and its receptors may be involved in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), clinical vasospasm, delayed cerebral ischemia (DCI), and functional outcome following aSAH. In the present study, common endothelin single nucleotide polymorphisms (SNPs) and their relation to aSAH were evaluated. METHODS Blood samples from all patients enrolled in the Cerebral Aneurysm Renin Angiotensin System (CARAS) study were used for genetic evaluation. The CARAS study prospectively enrolled patients with aSAH at 2 academic institutions in the US from 2012 to 2015. Common endothelin SNPs were detected using 5' exonnuclease (TaqMan) genotyping assays. Analysis of associations between endothelin SNPs and aSAH and its clinical sequelae was performed. RESULTS Samples from 149 patients with aSAH and 50 controls were available for analysis. In multivariate logistic regression analysis, the TG (odds ratio [OR] 2.102, 95% confidence interval [CI] 1.048-4.218, p = 0.036) and TT genotypes (OR 7.884, 95% CI 1.003-61.995, p = 0.05) of the endothelin-1 T/G SNP (rs1800541) were significantly associated with aSAH. There was a dominant effect of the G allele (CG/GG genotypes; OR 4.617, 95% CI 1.311-16.262, p = 0.017) of the endothelin receptor A G/C SNP (rs5335) on clinical vasospasm. Endothelin SNPs were not associated with DCI or functional outcome. CONCLUSIONS Common endothelin SNPs were found to be associated with presentation with aSAH and clinical vasospasm. Further studies are required to elucidate the relevant pathophysiology and its potential implications in the treatment of patients with aSAH.

Topics: Brain Ischemia; Endothelin-1; Female; Follow-Up Studies; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Intracranial Aneurysm; Male; Middle Aged; Polymorphism, Single Nucleotide; Prospective Studies; Receptor, Endothelin A; Receptor, Endothelin B; Subarachnoid Hemorrhage; Treatment Outcome; Vasospasm, Intracranial

Under physiologic conditions, losartan showed a dose-dependent antagonistic effect to the endothelin-1 (ET-1)-mediated vasoconstriction. This reduced vasoconstriction was abolished after preincubation with an endothelin B

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensins; Animals; Basilar Artery; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Losartan; Male; Oligopeptides; Peptides, Cyclic; Piperidines; Rats; Rats, Sprague-Dawley; Receptor Cross-Talk; Subarachnoid Hemorrhage; Vasoconstriction; Vasodilation; Vasospasm, Intracranial

The aim of the present study was to assess the therapeutic effects of atorvastatin on cerebral vessel autoregulation and to explore the underlying mechanisms in a rabbit model of subarachnoid hemorrhage (SAH). A total of 48 healthy male New Zealand rabbits (weight, 2‑2.5 kg) were randomly allocated into SAH, Sham or SAH + atorvastatin groups (n=16/group). The Sham group received 20 mg/kg/d saline solution, whereas 20 mg/kg/d atorvastatin was administered to rabbits in the SAH + atorvastatin group following SAH induction. Changes in diameter, perimeter and basilar artery (BA) area were assessed and expression levels of the vasoactive molecules endothelin‑1 (ET‑1), von Willebrand factor (vWF) and thrombomodulin (TM) were measured. Neuronal apoptosis was analyzed 72 h following SAH by terminal deoxynucleotidyl-transferase‑mediated dUTP nick‑end labeling (TUNEL) staining. The mortality rate in the SAH group was 18.75, 25% in the SAH + atorvastatin treated group and 0% in the Sham group (n=16/group). The neurological score in the SAH + atorvastatin group was 1.75±0.68, which was significantly higher compared with the Sham group (0.38±0.49; P<0.05). The BA area in the SAH + atorvastatin group (89.6±9.11) was significantly lower compared with the SAH group (115.4±11.0; P<0.01). The present study demonstrated that SAH induction resulted in a significant increase in the diameter, perimeter and cross‑sectional area of the BA in the SAH + atorvastatin group. Administration of atorvastatin may significantly downregulate the expression levels of ET‑1, vWF and TM (all P<0.01) vs. sham and SAH groups. TUNEL staining demonstrated that neuronal apoptosis was remarkably reduced in the hippocampus of SAH rabbits following treatment with atorvastatin (P<0.05). Atorvastatin treatment may alleviate cerebral vasospasm and mediate structural and functional remodeling of vascular endothelial cells, in addition to promoting anti‑apoptotic signaling. These results provided supporting evidence for the use of atorvastatin as an effective and well‑tolerated treatment for SAH in various clinical settings and may protect the autoregulation of cerebral vessels.

Topics: Animals; Anticholesteremic Agents; Apoptosis; Atorvastatin; Basilar Artery; Brain; Endothelin-1; Male; Neurons; Protective Agents; Rabbits; Subarachnoid Hemorrhage; Vasospasm, Intracranial

This study aimed to demonstrate the potential of salvinorin A (SA) for cerebral vasospasm after subarachnoid hemorrhage (SAH) and investigate mechanisms of therapeutic effect using rat SAH model.. Salvinorin A was injected intraperitoneally, and the neurobehavioral changes were observed at 12 hours, 24 hours, 48 hours, and 72 hours after SAH. Basilar artery was observed by magnetic resonance imaging (MRI). The inner diameter and thickness of basilar artery were measured. The morphological changes and the apoptosis in CA1 area of hippocampus were detected. Endothelin-1 (ET-1) and nitric oxide (NO) levels were detected by ELISA kit. The protein expression of endothelial NO synthase (eNOS) and aquaporin-4 (AQP-4) was determined by Western blot for potential mechanism exploration.. Salvinorin A administration could relieve neurological deficits, decrease the neuronal apoptosis, and alleviate the morphological changes in CA1 area of hippocampus. SA alleviated CVS by increasing diameter and decreasing thickness of basilar artery, and such changes were accompanied by the decreased concentration of ET-1 and increased level of NO. Meanwhile, SA increased the expression of eNOS and decreased the expression of AQP-4 protein in the basilar artery and hippocampus.. Salvinorin A attenuated CVS and alleviated brain injury after SAH via increasing expression of eNOS and NO content, and decreasing ET-1 concentration and AQP-4 protein expression.

Topics: Animals; Aquaporin 4; Basilar Artery; Diterpenes, Clerodane; Endothelin-1; Nitric Oxide; Nitric Oxide Synthase Type III; Rats; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Perturbations in cerebral microcirculation (eg, microvasospasms) and reduced neurovascular communication determine outcome after subarachnoid hemorrhage (SAH). ET-1 (endothelin-1) and its receptors have been implicated in the pathophysiology of large artery spasms after SAH; however, their role in the development of microvascular dysfunction is currently unknown. Here, we investigated whether inhibiting ET. SAH was induced in male C57BL/6 mice by filament perforation of the middle cerebral artery. Three hours after SAH, a cranial window was prepared and the pial and parenchymal cerebral microcirculation was measured in vivo using two-photon microscopy before, during, and after administration of the ET. Clazosentan treatment had no effect on the number or severity of SAH-induced cerebral microvasospasms nor did it affect neurological outcome.. Our results indicate that ET

Topics: Animals; Dioxanes; Endothelin A Receptor Antagonists; Endothelin-1; Male; Mice; Microscopy, Fluorescence, Multiphoton; Pyridines; Pyrimidines; Receptor, Endothelin A; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Vasospasm, Intracranial

Cerebral vasospasm may lead to delayed ischemic neurological deficits following subarachnoid hemorrhage (SAH). Endothelin (ET-1) is an important factor participating in cerebral vasospasm underlying SAH. We used a specific endothelin receptor antagonist, BQ123 to assess the specific role of endothelin-1 receptor antagonist in cerebral vasospasm in a rabbit model of SAH by examining plasma ET-1 levels and the principal CT perfusion (CTP) parameters pertinent to the hemodynamic status of microcirculation following SAH.. 102 male New Zealand white rabbits were divided into control, SAH and SAH + BQ123 intervention group (BQ123 group). Rabbit SAH model was established by double hemorrhage injection of autologous blood into the cisterna magna; Aquilion ONE was used to collect cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) which were used to evaluate cerebral microcirculation hemodynamics; Elisa was used to assess plasma ET-1 levels. Data were collected on days 1, 4, 7 and 14 following SAH, respectively.. Compared with the control group, the CBF in the SAH group was significantly lower, while the MTT was significantly higher. The CBF decreased on the 4th day and reached the lowest on the 7th day. The MTT began to rise on the 4th day and peaked on the 7th day. While in the BQ123 intervention group, the CBF significantly increased while the MTT significantly decreased on the 1st and the 4th days, respectively. Compared with SAH group, plasma ET-1 levels in BQ123 group significantly increased on the earlier (1st and 4th days) but not later days (between the 7th and 14th days). In addition, the inflammatory infiltration of brain tissues in rabbits treated with BQ123 post-SAH was significantly reduced compared with SAH group.. CTP can quantify the therapeutic effect of BQ123 after SAH; Selective blockade of ET-1 endothelin receptor, BQ123 significantly improved microcirculatory perfusion along with a reduction in resultant vasogenic inflammatory responses. The effect of BQ123 on the cerebral microcirculation was lobe dependent.

Topics: Animals; Cerebrovascular Circulation; Endothelin A Receptor Antagonists; Endothelin-1; Endothelins; Hemodynamics; Male; Microcirculation; Peptides, Cyclic; Perfusion Imaging; Rabbits; Receptor, Endothelin A; Subarachnoid Hemorrhage; Tomography, X-Ray Computed; Vasospasm, Intracranial

Cerebral vasospasm (CVS) is a severe complication of subarachnoid hemorrhage (SAH), and endothelin‑1 (ET‑1) may be involved in its pathogenesis. The present study aimed to investigate the expression of ET‑1 in cerebrospinal fluid (CSF) in patients with SAH and to analyze rat arterial contractility and the expression levels of ET‑1 receptors in vitro. CSF samples were collected from 28 patients and the expression levels of ET‑1 were measured. Rat cerebral basilar arteries were isolated and incubated with hemorrhagic or clear CSF. Contractility, as well as ETA and ETB mRNA expression were measured. ET‑1 levels in CSF increased and reached a peak within the initial 5 days after SAH onset and then gradually subsided. After 12 or 24 h, the contraction of arteries incubated in hemorrhagic CSF was substantially stronger than those in clear CSF. The mRNA expression levels of endothelin receptor type A and B in arteries incubated in hemorrhagic CSF were significantly higher than those in clear CSF. ET‑1 and its receptors may be involved in the pathogenic mechanism of CVS following SAH. ET‑1 expression in CSF may be used as a marker in CVS and its receptors may provide novel therapeutic targets in CVS.

Topics: Adult; Aged; Animals; Computed Tomography Angiography; Disease Models, Animal; Endothelin-1; Female; Gene Expression; Humans; Male; Middle Aged; Rats; Receptor, Endothelin A; RNA, Messenger; Subarachnoid Hemorrhage; Time Factors; Tomography, X-Ray Computed; Vasospasm, Intracranial

BACKGROUND The aim of this study was to investigate the protective effect of ADM gene mediated by plasmid pVAX1 on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). MATERIAL AND METHODS The recombinant plasmid pVAX-ADM was successfully established, and 40 SD rats were randomly divided into normal saline, pVAX1, pVAX1-ADM low-dose, pVAX1-ADM mid-dose, and pVAX1-ADM high-dose groups. The circumference and diameter of basilar artery, diameter of middle cerebral artery and internal carotid artery, and thickness of basilar artery wall were observed. The levels of circulating endothelial cells (CEC) and levels of regional cerebral blood flow (rCBF) of the parietal cortex were detected at different time-points. The expression levels of serum ADM, ET-1, and NOS of each group and the neurological functions were compared. RESULTS The circumference and diameter of basilar artery and the diameter of the middle cerebral artery and internal carotid artery in pVAX1-ADM groups were significantly longer than those in the saline group and pVAX1 group (P<0.05), but the thickness of the basilar artery wall in pVAX1-ADM groups was significantly lower (P<0.05), and the levels of growth or decrease were both dose-dependent (P<0.05). Compared with the saline group and pVAX1 group, the expression levels of serum ADM, NOS, and rCBF in pVAX1-ADM groups were significantly higher (P<0.05), but the levels of serum ET-1 and CEC were significantly lower (P<0.05). The scores of neurobehavioral functions of pVAX1-ADM groups were significantly lower (P<0.05), and the scores were also dose-dependent (P<0.05). CONCLUSIONS The recombinant eukaryotic expression plasmid pVAX1-ADM can significantly relieve cerebral vasospasm, increase the expression of serum ADM and NOS, and decrease the expression of serum ET-1 in a rat model of CVS; it is dose-dependent and can also improve nervous system function.

Topics: Adrenomedullin; Animals; Base Sequence; Basilar Artery; Cerebrovascular Circulation; Endothelin-1; Genetic Therapy; Nitric Oxide Synthase; Plasmids; Rats, Sprague-Dawley; Recombination, Genetic; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

The therapeutic effects of atorvastatin on early brain injury (EBI), cerebral edema and its association with aquaporin 4 (AQP4) were studied in rabbits after subarachnoid hemorrhage (SAH) using western blot analysis and the dry-wet method. Seventy-two healthy male New Zealand rabbits weighing between 2.5 and 3.2 kg were randomly divided into three groups: the SAH group (n=24), sham-operated group (n=24) and the SAH + atorvastatin group (n=24). A double SAH model was employed. The sham-operated group were injected with the same dose of saline solution, the SAH + atorvastatin group received atorvastatin 20 mg/kg/day after SAH. All rabbit brain samples were taken at 72 h after the SAH model was established successfully. Brain edema was detected using the dry-wet method after experimental SAH was induced; AQP4 and caspase-3 expression was measured by western blot analysis, and neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining at 72 h after SAH. The results indicated that brain edema and injury appeared soon after SAH, while brain edema and EBI were ameliorated and increased behavior scores were noted after prophylactic use of atorvastatin. Compared with the SAH group, the level of AQP4 and the cerebral content of water was significantly decreased (P<0.01) by atorvastatin, and TUNEL staining and studying the expression of caspase-3 showed that the apoptosis of neurons was reduced markedly both in the hippocampus and brain cortex by atorvastatin. The results suggest that atorvastatin ameliorated brain edema and EBI after SAH, which was related to its inhibition of AQP4 expression. Our findings provide evidence that atorvastatin is an effective and well-tolerated approach for treating SAH in various clinical settings.

Topics: Animals; Apoptosis; Aquaporin 4; Atorvastatin; Brain; Brain Edema; Brain Injuries; Caspase 3; Endothelin-1; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Neuroprotective Agents; Rabbits; Subarachnoid Hemorrhage; Water

Endothelial dysfunctions that include decreased nitric oxide (NO) bioactivity and increased endothelin-1 (ET-1) bioactivity have been considered to be involved in the pathogenesis of cerebral vasospasm (CVS) after aneurysmal subarachnoid hemorrhage (SAH). Recent cardiovascular studies have revealed that cyclooxygenase-2 (COX-2) is involved in a disturbance in cross-talk between NO and ET-1. COX-2 expression was detected in the endothelial cells of a spastic artery after experimental SAH; however, the pathophysiological significance of COX-2 in relation to CVS remains unclear. The aim of this study was to investigate the role of COX-2 in relation to NO and ET-1 in the pathogenesis of CVS by using the COX-2 selective inhibitor, celecoxib. In the SAH group, SAH was simulated using the double-hemorrhage rabbit model. In the celecoxib group, SAH was simulated and celecoxib was administered. The basilar artery was extracted on day 5 and examined. The cross-section area of the basilar artery in the celecoxib group was significantly larger than in the SAH group. An increased expression of COX-2, ET-1, and ETA receptor (ETAR), and a decreased expression of endothelial NO synthase (eNOS) were seen in the SAH group. In the celecoxib group compared to the SAH group, expression of COX-2, ET-1, and ETAR were statistically significantly decreased, and eNOS expression was significantly increased. COX-2 might be involved in the pathogenesis of CVS due to up-regulation of ET-1 and ETAR and down-regulation of eNOS, and celecoxib may potentially serve as an agent in the prevention of CVS after SAH.

Topics: Animals; Cyclooxygenase 2; Disease Models, Animal; Endothelin-1; Nitric Oxide; Rabbits; Subarachnoid Hemorrhage; Up-Regulation; Vasospasm, Intracranial

The present research was designed to investigate whether endothelin-1 (ET-1) secretion can be induced by oxyhemoglobin and whether nuclear factor κB (NF-κB) is involved in the regulation of ET-1 transcription in cerebrovascular muscle cells. Cerebrovascular muscle cells isolated from a rabbit basilar artery were stimulated by oxyhemoglobin (OxyHb) and ET-1 production was increased significantly in the supernatant. Inhibition of NF-κB with pyrrolidine dithiocarbamate and small interfering RNA decreased the expression of ET-1. Nuclear translocation of NF-κB and the degradation of IkB-α was observed with the stimulation of OxyHb. The supernatant obtained from cerebrovascular muscle cells stimulated by OxyHb produced contractions in arterial rings and was blocked by the ET-1 receptor antagonist (BQ-123). The time course of the OxyHb-induced contractions of the basilar artery rings correlated with the time course of the OxyHb-induced ET-1 secretion. The contraction of the basilar artery rings induced by OxyHb was attenuated when the artery rings were preincubated with pyrrolidine dithiocarbamate and SN50 (20 and 10 µM, respectively). These results indicate that cerebrovascular muscle cells may be an important source of ET-1 production after subarachnoid hemorrhage. NF-κB was involved in the expression of ET-1 and the inhibition of the NF-κB pathway may be beneficial for the treatment of cerebral vasospasm.

Topics: Animals; Basilar Artery; Cell Nucleus; Endothelin-1; Gene Expression Regulation; Male; Muscle Contraction; Myoblasts, Smooth Muscle; NF-kappa B; Oxyhemoglobins; Rabbits; RNA, Messenger; Subarachnoid Hemorrhage

Aneurysmal subarachnoid hemorrhage is a type of stroke with high morbidity and mortality. Increased endothelin-1 (ET-1) levels have been associated with increased risk of cerebral vasospasm, which is associated with increased morbidity. The purpose of this study was to investigate the relationships between ET-1 genotypes and ET-1 protein levels in cerebrospinal fluid (CSF) measured 72 hr before angiographic vasospasm measurement in subjects at high risk of cerebral vasospasm. Specifically, this study evaluated the differences between variant positive and variant negative groups of nine different ET-1 single-nucleotide polymorphisms (SNPs) in relationship with the ET-1 protein exposure rate. The CSF ET-1 protein levels were quantified using enzyme-linked immunosorbent assay. One functional SNP and eight ET-1 tagging SNPs were selected because they represent genetic variability in the entire ET-1 gene. The variant negative group of SNP rs2070699 was associated with a significantly higher ET-1 exposure rate than the variant positive group (p = 0.004), while the variant positive group of the rs5370 group showed a trend toward association with a higher ET-1 exposure rate (p = 0.051). Other SNPs were not informative. This is the first study to show differences in ET-1 exposure rate 72 hr before angiography in relation to ET-1 genotypes. These exploratory findings need to be replicated in a larger study; if replicated, these differences in genotypes may be a way to inform clinicians of those patients at a higher risk of increased ET-1 protein levels, which may lead to a higher risk of angiographic vasospasm.

Topics: Endothelin-1; Female; Genotyping Techniques; Humans; Intracranial Aneurysm; Male; Middle Aged; Polymorphism, Single Nucleotide; Radiography; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) often leads to poor outcomes in SAH patients. Overexpression of endothelin-1 (ET-1) could contribute to the development of CVS. The purpose of this study was to investigate cerebral microcirculation by whole-brain perfusion CT scan and ET-1 expression following SAH.. SAH was induced in rabbits. Cerebral blood flow (CBF), cerebral blood volume (CBV), time to peak (TTP), and mean transit time (MTT) were measured with CT perfusion techniques at days 1, 4, 7, and 14 following SAH. Expression of ET-1 was determined by ELISA accordingly. Histological sections of the brain tissue were also examined.. Whole-brain perfusion showed that CBV and TTP increased at day 4 and maintained elevated rate until day 14. MTT increased at day 4, peaked at day 7, and then decreased at day 14. CBV of the occipital lobe was greater than that in the frontal and parietal lobes at day 4. CBF of the occipital lobe increased significantly compared to that of other lobes at day 7. ET-1 expression in the SAH group was significantly greater than that in the control at various time points. Moreover, ET-1 levels were positively correlated with MTT value.. CTP detects changes in cerebral microcirculation following SAH. Microcirculation of each lobe was different and could be quantified to identify high-risk areas of cerebral ischemia. ET-1 expression was significantly increased and was correlated with MTT as well, suggesting that ET-1 influences cerebral microcirculation following SAH.

Topics: Animals; Blood Flow Velocity; Blood Volume; Cerebrovascular Circulation; Disease Models, Animal; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Microcirculation; Rabbits; Subarachnoid Hemorrhage; Tomography, X-Ray Computed; Vasospasm, Intracranial

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

Topics: Animals; Brain Ischemia; Butadienes; Cerebral Arteries; Dioxanes; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Up-Regulation

Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ETA and ETB receptors.. Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ETA and ETB receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.. Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ETA receptors and new expression of ETB receptors was apparent; 5) reduction in the enhanced response to endothelin after ETB blockade in the low range and after ETA blockade in the high range of endothelin concentrations; 6) after combined ETA and ETB blockade a complete inhibition of endothelin contraction was observed.. Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ETA and ETB receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.

Topics: Aged; Animals; Cerebral Arteries; Cerebrospinal Fluid; Endothelin Receptor Antagonists; Endothelin-1; Endothelins; Endothelium, Vascular; Female; Humans; Male; Middle Aged; Rats; Receptors, Endothelin; Subarachnoid Hemorrhage; Vasoconstriction

Cerebral vasospasm (CVS) and early brain injury are major causes of morbidity and mortality following subarachnoid hemorrhage (SAH). We investigated the efficiency of human tissue kallikrein (HTK) to prevent CVS in a rabbit model of SAH.. Forty-eight Japanese white rabbits were randomly divided into four groups (n = 12 each): control (sham-operated), SAH, SAH + phosphate-buffered saline (PBS, vehicle), and SAH + HTK. Basilar artery (BA) diameters were measured by three-dimensional computed tomography angiography at three time points. Endothelin-1 (ET-1) and nitric oxide (NO) levels in the cerebrospinal fluid (CSF) were assayed 24 h before and 5 and 7 days after SAH. After the last measurement, the animals were killed, and endothelial cell apoptosis was assessed. Bax and Bcl-2 levels in the BA were measured by western blotting.. HTK was found to significantly reduce CVS following SAH in rabbits. Inverse changes were observed in ET-1 and NO levels in the CSF collected from the SAH group. HTK increased levels of NO, which has a vasodilatory effect, but did not affect levels of ET-1, which has a vasoconstrictive effect. CTA revealed that HTK treatment significantly increased BA diameter. Moreover, HTK treatment reduced the number of apoptotic cells following SAH, presumably by increasing and decreasing Bcl-2 and Bax expression, respectively.. HTK ameliorated CVS and inhibited apoptosis in the BA in a rabbit model of SAH.

Topics: Animals; Basilar Artery; Coagulants; Disease Models, Animal; Endothelin-1; Humans; Image Processing, Computer-Assisted; In Situ Nick-End Labeling; Kallikreins; Neurologic Examination; Nitric Oxide; Rabbits; Radiography; Statistics, Nonparametric; Subarachnoid Hemorrhage; Tomography Scanners, X-Ray Computed; Vasospasm, Intracranial

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET₁₈₋₃₄ (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5 nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET₁₈₋₃₄ were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.

Topics: Aspartic Acid Endopeptidases; Benzofurans; Bradykinin; Endothelin-1; Endothelin-Converting Enzymes; Enzyme Inhibitors; Humans; Hydrocephalus; Mass Spectrometry; Metalloendopeptidases; Organophosphonates; Subarachnoid Hemorrhage

Cerebral vasospasm complicating subarachnoid hemorrhage causes ischemic stroke and worsens the neurological outcome. The potential role of endothelin-1 in vasospasm pathogenesis may provide therapeutic opportunities. A recent meta-analysis however, did not support the use of endothelin antagonists. Apart from clinical assessment, transcranial Doppler and interval angiography, there are no sensitive screening markers for evolving vasospasm. We investigate the ability of serial measurement of endothelin-1 to predict the development of vasospasm following subarachnoid hemorrhage.. Endothelin-1 levels in cerebrospinal fluid and blood were measured daily in 20 patients admitted to the ICU with subarachnoid hemorrhage from days 1 to 10 following the inception bleed. In addition to clinical assessment, patients had daily transcranial Doppler. Digital subtraction angiography was performed on the suspicion of vasospasm based upon clinical or transcranial Doppler assessment. Neuron-specific enolase and SB100 were measured in blood as comparative biomarkers of neurological injury.. Mean plasma endothelin-1 on day 5, was 4.2 mcg/L (CI 3.1-5.8) in patients with vasospasm compared to 2.5 mcg/L (CI 1.5-4.0) in those without vasospasm (P = 0.047). There were no time-related differences in cerebrospinal fluid endothelin-1, plasma NSE, or SB100 for patients with and without vasospasm.. In patients with subarachnoid hemorrhage and vasospasm, endothelin-1 is significantly higher in plasma than in CSF on day 5. Neither NSE nor SB100 is associated with the development of vasospasm. Measurement of serial plasma endothelin-1 concentration is a potential screening marker of vasospasm.

Topics: Adult; Aged; Biomarkers; Endothelin-1; Female; Humans; Middle Aged; Predictive Value of Tests; Radiography; Subarachnoid Hemorrhage; Ultrasonography; Vasospasm, Intracranial

The vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). Previous results showed that CGS 26303, an endothelin converting enzyme (ECE) inhibitor, effectively prevented and reversed arterial narrowing in animal models of SAH. In the present study, we assessed the effect of CGS 26303 on neurological deficits in SAH rats. The involvement of vasoactive pathways downstream of ET-1 signaling in SAH was also investigated.. Sprague-Dawley rats were divided into five groups (n = 6/group): (1) normal control, (2) SAH, (3) SAH+vehicle, (4) SAH+CGS 26303 (prevention), and (5) SAH+CGS 26303 (reversal). SAH was induced by injecting autologous blood into cisterna magna. CGS 26303 (10 mg/kg) was injected intravenously at 1 and 24 hr after the initiation of SAH in the prevention and reversal protocols, respectively. Behavioral changes were assessed at 48 hr after SAH. Protein expression was analyzed by Western blots.. Deficits in motor function were obvious in the SAH rats, and CGS 26303 significantly improved the rate of paraplegia. Expressions of rho-kinase-II and membrane-bound protein kinase C- δ and rhoA were significantly increased, while those of soluble guanylyl cyclase α 1 and β 1 as well as protein kinase G were significantly decreased in the basilar artery of SAH rats. Treatment with CGS 26303 nearly normalized these effects.. These results demonstrate that the rhoA/rho-kinase and sGC/cGMP/PKG pathways play pivotal roles in cerebral vasospasm after SAH. It also shows that ECE inhibition is an effective strategy for the treatment of this disease.

Topics: Animals; Basilar Artery; Behavior, Animal; Cyclic AMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Endothelin-1; Guanylate Cyclase; Male; Organ Specificity; Organophosphonates; Protein Kinase C-delta; Protein Kinase Inhibitors; Protein Transport; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; rho-Associated Kinases; Signal Transduction; Soluble Guanylyl Cyclase; Subarachnoid Hemorrhage; Tetrazoles; Vasospasm, Intracranial

Stellate ganglion block (SGB) is a blockade of sympathetic ganglia innervating the head and neck, and is known to function through vasodilation of the target region. However, the effectiveness of SGB in relieving cerebral vasospasm (CVS) through dilation of intracerebral vessels has not been evaluated. The aim of the present study is to investigate the therapeutic effects of SGB in a rat model of subarachnoid hemorrhage (SAH) complicated by delayed CVS, and explore the underlying mechanisms. The SAH model was established by double injection of autologous arterial blood into the cisterna magna. We simulated SGB by transection of the cervical sympathetic trunk (TCST), and measured changes in the diameter, perimeter and cross-sectional area of the basilar artery (BA) and middle cerebral artery (MCA) to evaluate its vasodilatory effect. To investigate the underlying mechanisms, we determined the expression level of vasoactive molecules endothelin-1 (ET-1) and calcitonin gene-related peptide (CGRP) in the plasma, and apoptotic modulators Bcl-2 and Bax in the hippocampus. We found a significant increase in the diameter, perimeter and cross-sectional area of the BA and right MCA in SAH rats subjected to TCST. Application of SGB significantly reduced the expression of ET-1 while increasing that of CGRP in SAH rats. We also found a significant increase in the expression of Bcl-2 and decrease in the expression of Bax in the hippocampus of SAH rats subjected to TCST, when compared to untreated SAH rats. The mechanism of action of SGB is likely mediated through alterations in the ratio of ET-1 and CGRP, and Bax and Bcl-2. These results suggest that SGB can alleviate the severity of delayed CVS by inducing dilation of intracerebral blood vessels, and promoting anti-apoptotic signaling. Our findings provide evidence supporting the use of SGB as an effective and well-tolerated approach to the treatment of CVS in various clinical settings.

Topics: Animals; Autonomic Nerve Block; Basilar Artery; bcl-2-Associated X Protein; Calcitonin Gene-Related Peptide; Disease Models, Animal; Endothelin-1; Hippocampus; Male; Middle Cerebral Artery; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Stellate Ganglion; Subarachnoid Hemorrhage; Vasodilation; Vasospasm, Intracranial

Although relaxin causes vasodilatation in systemic arteries, little is known about its role in cerebral arteries. We investigated the expression and role of relaxin in basilar arteries after subarachnoid hemorrhage (SAH) in rabbits.. Microarray analysis with rabbit basilar artery RNA was performed. Messenger RNA expression of relaxin-1 and relaxin/insulin-like family peptide receptor 1 (RXFP1) was investigated with quantitative RT-PCR. RXFP1 expression in the basilar artery was investigated with immunohistochemistry. Relaxin concentrations in cerebrospinal fluid (CSF) and serum were investigated with an enzyme-linked immunosorbent assay. Using human brain vascular smooth muscle cells (HBVSMC) preincubated with relaxin, myosin light chain phosphorylation (MLC) was investigated with immunoblotting after endothelin-1 stimulation.. After SAH, RXFP1 mRNA and protein were significantly downregulated on day 3, whereas relaxin-1 mRNA was significantly upregulated on day 7. The relaxin concentration in CSF was significantly elevated on days 5 and 7. Pretreatment with relaxin reduced sustained MLC phosphorylation induced by endothelin-1 in HBVSMC.. Upregulation of relaxin and downregulation of RXFP1 after SAH may participate in development of cerebral vasospasm. Downregulation of RXFP1 may induce a functional decrease in relaxin activity during vasospasm. Understanding the role of relaxin may provide further insight into the mechanisms of cerebral vasospasm.

Topics: Animals; Basilar Artery; Cells, Cultured; Disease Models, Animal; Endothelin-1; Humans; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosin Light Chains; Oligonucleotide Array Sequence Analysis; Phosphorylation; Rabbits; Receptors, Peptide; Relaxin; RNA, Messenger; Subarachnoid Hemorrhage; Time Factors; Up-Regulation; Vasospasm, Intracranial

Endothelin-1 (ET-1) is a potent vasoconstrictor, and astrocytic ET-1 is reported to play a role in the pathogenesis of cerebral ischemic injury and cytotoxic edema. However, it is still unknown whether astrocytic ET-1 also contributes to vasogenic edema and vasospasm during subarachnoid hemorrhage (SAH). In the present study, transgenic mice with astrocytic endothelin-1 over-expression (GET-1 mice) were used to investigate the pathophysiological role of ET-1 in SAH pathogenesis.. The GET-1 mice experienced a higher mortality rate and significantly more severe neurological deficits, blood-brain barrier breakdown and vasogenic edema compared to the non-transgenic (Ntg) mice following SAH. Oral administration of vasopressin V1a receptor antagonist, SR 49059, significantly reduced the cerebral water content in the GET-1 mice. Furthermore, the GET-1 mice showed significantly more pronounced middle cerebral arterial (MCA) constriction after SAH. Immunocytochemical analysis showed that the calcium-activated potassium channels and the phospho-eNOS were significantly downregulated, whereas PKC-α expression was significantly upregulated in the MCA of the GET-1 mice when compared to Ntg mice after SAH. Administration of ABT-627 (ETA receptor antagonist) significantly down-regulated PKC-α expression in the MCA of the GET-1 mice following SAH.. The present study suggests that astrocytic ET-1 involves in SAH-induced cerebral injury, edema and vasospasm, through ETA receptor and PKC-mediated potassium channel dysfunction. Administration of ABT-627 (ETA receptor antagonist) and SR 49059 (vasopressin V1a receptor antagonist) resulted in amelioration of edema and vasospasm in mice following SAH. These data provide a strong rationale to investigate SR 49059 and ABT-627 as therapeutic drugs for the treatment of SAH patients.

Topics: Animals; Astrocytes; Disease Models, Animal; Endothelin-1; Immunohistochemistry; Mice; Mice, Transgenic; Receptor, Endothelin A; Subarachnoid Hemorrhage; Up-Regulation; Vasospasm, Intracranial

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease that affects approximately 30,000 people a year in the United States. Delayed cerebral ischemia (DCI) and cerebral vasospasm (CV) are common complications after aSAH. In addition, aSAH patients have a high risk of poor long-term outcomes. Endothelin-1 (ET-1), a potent vasoconstrictor, or its two types of receptors, ET receptor A (ETA) and ET receptor B (ETB), may play a role in the pathogenesis of DCI and CV. Genetic variations within the ET-1, ETA, or ETB genes may also account for variance observed in the outcomes of aSAH patients. The purpose of this study was to describe the distribution of the Lys198Asn polymorphism, a known functional SNP in the ET-1 gene, and tagging SNPs of the ET-1, ETA, and ETB genes in individuals recovering from aSAH. This study also investigated the relationships among the ET polymorphisms, DCI, and global functional outcomes measured at 3 and 6 months after aSAH. Participants included individuals aged 18-75 years with a diagnosis of aSAH. There was a trend found between the variant allele of an ET-1 SNP (rs6912834) and angiographic vasospasm. There were also associations found between two ETB SNPs (rs9574124 and rs3027111) and poor outcomes as measured by the Glasgow Outcome scale at 3 months. These findings support the role of ET-1 and ETB in recovery following aSAH.

Topics: Adolescent; Adult; Aged; Brain Ischemia; Endothelin-1; Female; Genetic Variation; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Receptor, Endothelin A; Receptor, Endothelin B; Risk Factors; Subarachnoid Hemorrhage; Vasospasm, Intracranial; Young Adult

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is characterized by prolonged severe constriction of the basilar artery, which often leads to ischemic brain damage. Locally elevated concentrations of spasmogenic substances induce persistent depolarization of myocytes in the basilar artery, leading to continuous influx of calcium (Ca) through voltage-sensitive Ca channels and myocyte contraction. Potassium (K) channel openers may have therapeutic utility to oppose membrane depolarization, dilate the arteries, and reduce ischemia. Here, we examined the involvement of vascular Kv7 K channels in the pathogenesis of cerebral vasospasm and tested whether Kv7 channel openers are effective therapeutic agents in a rat model of SAH. Patch-clamp experiments revealed that 3 different spasmogens (serotonin, endothelin, and vasopressin) suppressed Kv7 currents and depolarized freshly isolated rat basilar artery myocytes. These effects were significantly reduced in the presence of a Kv7 channel opener, retigabine. Retigabine (10 μM) also significantly blocked L-type Ca channels, reducing peak inward currents by >50%. In the presence of a selective Kv7 channel blocker, XE991, the spasmogens did not produce additive constriction responses measured using pressure myography. Kv7 channel openers (retigabine or celecoxib) significantly attenuated basilar artery spasm in rats with experimentally induced SAH. In conclusion, we identify Kv7 channels as common targets of vasoconstrictor spasmogens and as candidates for therapeutic intervention for cerebral vasospasm.

Topics: Animals; Anthracenes; Arginine Vasopressin; Basilar Artery; Carbamates; Celecoxib; Disease Models, Animal; Endothelin-1; KCNQ Potassium Channels; Male; Membrane Transport Modulators; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myography; Patch-Clamp Techniques; Phenylenediamines; Potassium Channel Blockers; Pyrazoles; Rats; Rats, Sprague-Dawley; Serotonin; Signal Transduction; Subarachnoid Hemorrhage; Sulfonamides; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Vasospasm, Intracranial

Increased vascular smooth muscle contractility has an important role in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Myofilament Ca(2+) sensitivity is a major determinant of smooth muscle contractility. We investigated changes in the Ca(2+)-sensitizing effect of endothelin-1 (ET-1) and the mechanisms underlying ET-1-induced Ca(2+) sensitization after SAH using a rabbit SAH model. After SAH, the contractile response to ET-1 was enhanced, and the ET(A) receptor expression was upregulated in the basilar artery. In α-toxin-permeabilized preparations, ET-1 induced enhanced and prolonged contraction after SAH, suggesting that ET-1-induced Ca(2+) sensitization is potentiated after SAH. Endothelin-1-induced Ca(2+) sensitization became less sensitive to inhibitors of Rho-associated coiled-coil protein kinase (ROCK) and protein kinase C (PKC) after SAH. The expression of PKCα, ROCK2, PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17) and myosin phosphatase target subunit 1 (MYPT1) was upregulated, and the level of phosphorylation of CPI-17 and MYPT1 was elevated after SAH. This study demonstrated for the first time that the Ca(2+)-sensitizing effect of ET-1 on myofilaments is potentiated after SAH. The increased expression and activity of PKCα, ROCK2, CPI-17, and MYPT1, as well as the upregulation of ET(A) receptor expression are suggested to underlie the enhanced and prolonged Ca(2+) sensitization induced by ET-1.

Topics: Animals; Basilar Artery; Calcium; Endothelin-1; Gene Expression Regulation; Male; Muscle Contraction; Muscle Proteins; Myofibrils; Myosin-Light-Chain Phosphatase; Phosphoproteins; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Kinase Inhibitors; Rabbits; Receptor, Endothelin A; rho-Associated Kinases; Subarachnoid Hemorrhage; Vasoconstriction

In the subarachnoid hemorrhage (SAH) blood mixes with cerebrospinal fluid, what starts immunoinflammatory processes - increased synthesis of proinflammatory cytokines, and formation of reactive oxygen species (ROS), resulting in pre-activation of different populations of peripheral leukocytes. Migration of leukocytes to the brain parenchyma through broken blood brain barrier may produce extra brain tissue injury besides of that resulting from SAH. We examined in adult rats the effect of interleukin-1β (IL-1β) neutralization on secretion of cytokines as well as production of ROS in the course of SAH. SAH was produced by injection of 150 μL of autologous arterial blood into cisterna magna. In 50% of animals, IL-1beta activity was inhibited by intracerebroventricular administration of anti-rat IL-1β antibodies. Ninety minutes or 24 hrs following surgery, blood samples were drawn from the extraorbital plexus and centrifuged to obtain two leukocyte subpopulations - polymorphonuclear (PMN) and mononuclear (MN). The chemiluminescence, a hallmark of ROS synthesis, was measured in PMNs. In supernatants from MNs cultures, concentrations endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were assessed. SAH caused the increase ofn PMNs chemiluminescence as well as the increase of production of ET-1 and TNF-α by MNs but had no influence on IL-6 concentration. Neutralization of IL-1β resulted in significant decrease of chemiluminescence as well as concentration of both ET-1 and TNF-α, while IL-6 concentration was increased. These revealed an important role of IL-1β in the activation of peripheral leukocytes in the course of subarachnoid hemorrhage.

Topics: Analysis of Variance; Animals; Antibodies; Disease Models, Animal; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Injections, Intraventricular; Interleukin-1beta; Leukocytes; Male; Neutrophils; Rats; Rats, Wistar; Reactive Oxygen Species; Subarachnoid Hemorrhage; Time Factors; Tumor Necrosis Factor-alpha

Under physiological conditions, vasoconstrictors and vasodilators are counterbalanced. After aneurysmal subarachnoid hemorrhage (SAH) disturbance of this equilibrium may evoke delayed cerebral vasospasm (CVS) leading to delayed cerebral ischemia (DCI). Most studies examined either the vasoconstrictor endothelin-1 (ET-1) or the vasodilative pathway of nitric oxide (NO) and did not include investigations regarding the relationship between vasospasm and ischemia. Asymmetric dimethyl-L-arginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), decreases the concentration of NO. Studies have correlated increasing concentrations of ADMA with the course and degree of CVS after SAH. We sought to determine, if ADMA and endothelin-1 (ET-1) are associated with CVS and/or DCI after SAH. CSF concentrations of ADMA and ET-1 were retrospectively determined in 30 patients after SAH and in controls. CVS was detected clinically and by arteriogaphy. DCI was monitored by follow-up CT scans. 17 patients developed arteriographic CVS and 4 patients developed DCI. ADMA but not ET-1 concentrations were correlated with occurrence and degree of CVS. However, ET-1 concentrations were correlated with WFNS grade on admission. Neither ADMA nor ET-1 correlated with DCI in this cohort. ET-1 concentrations seem to be associated with the impact of the SAH bleed. ADMA may be directly involved in the development and resolution of CVS after SAH via inhibition of NOS disturbing the balance of vasodilative and -constrictive components.

Topics: Adult; Arginine; Brain Ischemia; Endothelin-1; Female; Humans; Male; Middle Aged; Nitric Oxide; Retrospective Studies; Severity of Illness Index; Subarachnoid Hemorrhage; Vasodilation; Vasospasm, Intracranial

Increased endothelin-1 (ET-1) production and diminished nitric oxide synthase (NOS) bioavailability has been observed in aneurysmal subarachnoid hemorrhage (SAH). The authors previously found that 6-mercaptopurine (6-mp) is effective in preventing and reversing arterial narrowing in a rodent SAH model. This present study is of interest to examine the effect of 6-mp on ET-1/endothelial nitric oxide synthase (eNOS) in this animal model.. A rodent double hemorrhage SAH model was employed. Animals were randomly assigned to six groups (sham, SAH only, vehicle, 0.5, 1.0 and 2 mg kg(-1) day(-1) 6-mp treatment). Monoclonal CD45 immunostaining was utilized to evaluate monocytes and microglia. The level of pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-α(RT-PCR), and ET-1 (ELISA) was measured. The basilar arteries (BAs) were harvested and sliced, and their cross-sectional areas were determined. Radiolabeled NOS assay kit was applied to detect eNOS.. Morphologically, convolution of internal elastic lamina, endothelial cells distortion, and necrotic smooth muscle were prevalently present in the basilar artery of SAH groups, which was absent in the 1 and 2 mg kg(-1) day(-1) 6-mp plus SAH group or the healthy controls. Significant vasospasm was noted in the vehicle group (lumen patency, 54.6%, p ≤ 0.01 compared with the sham group), but it was less prominent in the 2 mg kg(-1) day(-1) 6-mp treatment group (lumen patency, 87.6%, p < 0.05). In addition, administration with 2 mg kg(-1) day(-1) 6-mp reduced cytokine levels by 11%, 47%, and 34% for IL-1, IL-6, and TNF-α, respectively, and increased ET-1 levels were found in all the animals subject to SAH (SAH only, SAH plus vehicle, SAH plus 0.5 and 1.0 mg kg(-1) day(-1) 6-mp) except in the 2 mg kg(-1) day(-1) 6-mp SAH group, when compared with the healthy controls (no SAH). Meanwhile, treatment with 6-mp did not induce the levels of expressed eNOS in BAs in the 6-mp groups (0.5, 1.0, and 2 mg kg(-1) day(-1) 6-mp plus SAH) when compared with that in the SAH groups (p > 0.1).. In summary, treatment with 6-mp decreased the release of pro-inflammatory cytokines and diminished experimental vasospasm. This study offered first evidence that 6-mp dose-dependently reduces the level of ET-1 in a NO-independent mechanism, which corresponds to its antivasospastic effect in the condition of chronic vasospasm.

Topics: Aneurysm, Ruptured; Animals; Chemotaxis; Connective Tissue; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Immunosuppressive Agents; Inflammation Mediators; Intracranial Aneurysm; Male; Mercaptopurine; Microglia; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Endothelin-1 (ET-1) is a potent vasoconstrictor implicated in the pathogenesis of vasospasm and delayed cerebral ischemia (DCI) in aneurysmal subarachnoid hemorrhage (aSAH) patients. The aim of this study was to investigate the relationship between cerebrospinal fluid (CSF) ET-1 levels and angiographic vasospasm and DCI.. Patients with aSAH were consented (n = 106). Cerebral vasospasm was determined by angiography. DCI was determined by transcranial Doppler (TCD) results and/or angiogram results with corresponding clinical deterioration. CSF ET-1 levels over 14 days after the initial insult was quantified by ELISA. ET-1 analysis included a group-based trajectory analysis and ET-1 exposure rate during 24, 48, and 72 h prior to, as well as 72 h post angiography, or clinical deterioration.. Trajectory analysis revealed two distinct groups of subjects with 56% of patients in the low ET-1 trajectory group (mean at day 1 = 0.31 pg/ml; SE = 0.04; mean at day 14 = 0.41 pg/ml; SE = 0.15) and 44% of patients in the high ET-1 trajectory group (mean at day 1 = 0.65 pg/ml; SE = 0.08; mean at day 14 = 0.61 pg/ml; SE = 0.06). Furthermore, we observed that ET-1 exposure rate 72 h before angiography and clinical spasm was a significant predictor of both angiographic vasospasm and DCI, whereas, ET-1 exposure after angiography and clinical spasm was not associated with either angiographic vasospasm or DCI.. Based on these results we conclude that ET-1 concentrations are elevated in a sub-group of patients and that the acute (72 h prior to angiography and clinical neurological deterioration), but not chronic, elevations in CSF ET-1 concentrations are indicative of the pathogenic alterations of vasospasm and DCI in aSAH patients.

Topics: Adult; Aged; Brain Ischemia; Cohort Studies; Endothelin-1; Female; Humans; Male; Middle Aged; Risk Factors; Socioeconomic Factors; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

Using vascular heat-exchange controller implemented mild hypothermia treatment, the authors established the cerebral vasospasm model in which blood was injected twice into dog's foramen magnum; and it was discussed the influence of the concentration of endothelin-1 and NO in blood plasma and cerebrospinal fluid through continuing treatment of mild hypothermia at different times in secondary brain vasospasm model after subarachnoid hemorrhage. Thirty healthy mongrel dogs were randomly divided into five groups; artificial cerebrospinal fluid group (group A), normal temperature control group (group C), mild hypothermia 8 h group (group H1), mild hypothermia 16 h group (group H2), and mild hypothermia 32 h group (group H3). The authors injected the artificial CSF into dog's foramen magnum in group A while the other four groups were injected with autologous arterial blood. The normal group's temperature maintained 38.5°C. The authors set the temperature at 33.5°C in mild hypothermia groups and this was maintained for 8, 16, and 32 h, respectively. ET-1 and NO levels in the cerebrospinal fluid and plasma were assayed in each group on days 0, 7, 14, and 21. Then the changes of the diameter of blood vessels of cerebral basilar artery and overall performance categories score in each group through application of CT angiography were recorded. In the cerebral vasospasm model which was constructed by injecting the blood to dog twice, mild hypothermia treatment, through the application of vascular heat-exchange controller, could reduce cerebral vasospasm. It was observed that the duration of the mild hypothermia is directly proportional to the longer duration of the relieving of cerebral vasospasm. The reciprocal changes observed in the levels of ET-1 and NO in cerebrospinal fluid and plasma revealed that it might be possible to reduce the cerebral vasospasm by regulating the rising amplitude of ET-1 and the decrease in NO in CSF and plasma.

Topics: Animals; Cerebral Angiography; Disease Models, Animal; Dogs; Endothelin-1; Female; Hypothermia, Induced; Male; Nitric Oxide; Subarachnoid Hemorrhage; Tomography, X-Ray Computed; Vasospasm, Intracranial

Magnesium lithospermate B (MLB), a working extract from Salvia miltiorrhiza, was effective against coronary artery disease, ischemic stroke, and chronic renal disease. This study examined the effect of MLB on endothelin-1/endothelial nitric oxide synthase (eNOS) in a subarachnoid hemorrhage (SAH) animal model.. A rodent double-hemorrhage model was employed. Animals were randomly assigned to five groups (sham, SAH only, vehicle, 10 mg/kg/day MLB treatment, and pretreatment groups). A radiolabeled NOS Assay Kit was used to detect eNOS. Serum and cerebrospinal fluid sampling for ET-1 (ELISA) was measured. The basilar arteries (BAs) were garnered and sliced, and their cross-sectional areas were determined. In addition, NOS inhibitor nitro-arginine methyl ester (L-NAME) was employed in the SAH+ MLB treatment groups.. Significant vasoconstriction was perceived in the SAH group (lumen patency: 44.6%, p < 0.01), but not in the MLB group (lumen patency: 89.3%). The ET-1 level was reduced in the MLP plus SAH group (34%, p < 0.01) when compared with the SAH groups (SAH only and vehicle). MLB dose-dependently increased the level of eNOS when compared with the vehicle plus SAH group. However, the administration of L-NAME reversed the expression of eNOS and vasoconstriction (lumen patency: 56.2%) in the MLB group.. The enhanced expression of eNOS and decreased ET-1 levels in the MLB groups may reflect its anti-spastic effect. In the study of NOS, L-NAME reversed MLB's anti-vasospastic effect. This finding lends credence to the hypothesis that MLB modulates ET-1 levels through a NOS-dependent mechanism in the pathogenesis of cerebral vasospasm following SAH.

Topics: Animals; Disease Models, Animal; Drugs, Chinese Herbal; Endothelin-1; Free Radical Scavengers; Male; Nitric Oxide; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Cerebral vasospasm determines the prognosis of subarachnoid hemorrhage (SAH). The increased vascular reactiveness has an important role in the development of cerebral vasospasm. This study analyzed the roles of the receptor-mediated signaling and the myofilament Ca(2+) sensitivity in the increased vascular reactiveness in SAH, using the basilar artery of a rabbit SAH model. Endothelin-1, thrombin, and phenylephrine induced transient increases in [Ca(2+)](i), myosin light chain phosphorylation, and contraction in the controls. All these responses were not only enhanced but also became sustained in SAH. In the sequential stimulation of thrombin receptor or alpha(1)-adrenoceptor, the second response was substantially attenuated in the controls, whereas it was maintained in SAH. The thrombin-induced contraction in SAH irreversibly persisted even after terminating the thrombin stimulation. This contraction was completely reversed by trypsin and a Galpha(q) inhibitor YM254890, thus suggesting the sustained receptor activity during the sustained contraction. YM254890 also inhibited the endothelin-1- and phenylephrine-induced sustained contraction. Furthermore, the GTPgammaS-induced transient contraction in the control alpha-toxin-permeabilized strips was converted to a sustained contraction in SAH. The results provide the first evidence that the feedback inactivation of the receptor activity and the myofilament Ca(2+) sensitivity was impaired in SAH, thus contributing to the increased vascular reactiveness.

Topics: Actin Cytoskeleton; Animals; Basilar Artery; Calcium; Cytosol; Electrophoresis, Polyacrylamide Gel; Endothelin-1; Endothelium, Vascular; Feedback, Physiological; Muscle Contraction; Muscle, Smooth, Vascular; Myosin Light Chains; Phenylephrine; Phosphorylation; Rabbits; Receptor, PAR-1; Receptors, Adrenergic, alpha-1; Subarachnoid Hemorrhage; Tachyphylaxis; Thrombin; Vasoconstrictor Agents

In human cortex it has been suggested that the tissue at risk is indicated by clusters of spreading depolarizations (SDs) with persistent depression of high-frequency electrocorticographic (ECoG) activity. We here characterized this zone in the ET-1 model in rats using direct current (DC)-ECoG recordings. Topical application of the vasoconstrictor endothelin-1 (ET-1) induces focal ischemia in a concentration-dependent manner restricted to a region exposed by a cranial window, while a healthy cortex can be studied at a second naïve window. SDs originate in the ET-1-exposed cortex and invade the surrounding tissue. Necrosis is restricted to the ET-1-exposed cortex. In this study, we discovered that persistent depression occurred in both ET-1-exposed and surrounding cortex during SD clusters. However, the ET-1-exposed cortex showed longer-lasting negative DC shifts and limited high-frequency ECoG recovery after the cluster. DC-ECoG recordings of SD clusters with persistent depression from patients with aneurysmal subarachnoid hemorrhage were then analyzed for comparison. Limited ECoG recovery was associated with significantly longer-lasting negative DC shifts in a similar manner to the experimental model. These preliminary results suggest that the ischemic zone in rat and human cortex is surrounded by a normally perfused belt with persistently reduced synaptic activity during the acute injury phase.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Cortical Spreading Depression; Electroencephalography; Endothelin-1; Humans; Male; Rats; Rats, Wistar; Subarachnoid Hemorrhage

Induced endothelin-1 (ET-1) production and decreased nitric oxide synthase (NOS) bioavailability have been found in aneurysmal subarachnoid hemorrhage (SAH). Atorvastatin is recognized to have pleiotropic effects including increasing NOS bioavailability as well as reducing inflammation and oxidative damage other than reducing dyslipidemia. This study is of interest to examine the effect of atorvastatin on ET-1/endothelial nitric oxide synthase (eNOS) in experimental SAH.. A rodent double-hemorrhage SAH model was employed. Animals were randomly assigned as sham-operated, SAH, vehicle plus SAH, 5 mg/day atorvastatin treatment plus SAH and 5 mg/day atorvastatin precondition plus SAH groups. Administration with atorvastatin (5 mg/day) was initiated 1 week before (precondition) and 24 hr later (treatment). Cerebrospinal fluid samples were collected at 72 hr after second SAH. ET-1 (ELISA) was measured. The basilar arteries (BAs) were harvested and sliced, and their cross-sectional areas were measured. Radiolabeled NOS assay kit was used to detect eNOS.. Morphologically, convoluted internal elastic lamina, distorted endothelial cells and myonecrosis of the smooth muscle were predominantly observed in the BA of SAH and vehicle-treated SAH groups, which was not detected in the atorvastatin-preconditioned SAH group or the healthy controls. Significant vasospasm was noted in the vehicle group (lumen potency 64.5%, compared with the sham group, p

Topics: Animals; Atorvastatin; Disease Models, Animal; Endothelin-1; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ischemic Preconditioning; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Pyrroles; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Treatment Outcome; Vasodilation; Vasospasm, Intracranial

Cerebral ischemia remains the key cause of disability and death in the late phase after subarachnoid hemorrhage (SAH), and its pathogenesis is still poorly understood. The purpose of this study was to examine whether the change in intracranial pressure or the extravasated blood causes the late cerebral ischemia and the upregulation of receptors or the cerebral vasoconstriction observed following SAH.. Rats were allocated to 1 of 3 experimental conditions: 1) cisternal injection of 250 microl blood (SAH Group), 2) cisternal injection of 250 microl NaCl (Saline Group), or 3) the same procedure but without fluid injection (Sham Group). Two days after the procedure, the basilar and middle cerebral arteries were harvested, and contractile responses to endothelin (ET)-1 and 5-carboxamidotryptamine (5-CT) were investigated by means of myography. In addition, real-time polymerase chain reaction was used to determine the mRNA levels for ET(A), ET(B), and 5-HT(1) receptors. Regional and global cerebral blood flow (CBF) were quantified by means of an autoradiographic technique.. Compared with the sham condition, both SAH and saline injection resulted in significantly enhanced contraction of cerebral arteries in response to ET-1 and 5-CT. Regional and global CBF were reduced both in the Saline and SAH groups compared with the Sham Group. The mRNA levels for ET(B) and 5-HT(1B) receptors were upregulated after SAH and saline injection compared with the sham procedure. The effects in all parameters were more pronounced for SAH than for saline injection.. This study revealed that both the elevation of intracranial pressure and subarachnoid blood per se contribute approximately equally to the late CBF reductions and receptor upregulation following SAH.

Topics: Animals; Autoradiography; Cerebral Arteries; Cerebrovascular Circulation; Endothelin-1; Intracranial Pressure; Male; Myography; Potassium; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA; Serotonin; Serotonin Receptor Agonists; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction

The role of endothelin-1 (ET-1) and nitric oxide (NO) as two important mediators in the development of cerebral vasospasm (CVS) after subarachnoid haemorrhage (SAH) is controversial. The objective of this study was to determine whether local levels of ET-1 and NO in cerebral arterial plasma and/or in cerebrospinal fluid (CSF) are associated with the occurrence of CVS after SAH.. CVS was induced using the one-haemorrhage rabbit model and confirmed by digital subtraction angiography of the rabbits' basilar artery on day 5. Prior to sacrifice, local CSF and basilar arterial plasma samples were obtained by a transclival approach to the basilar artery. Systemic arterial plasma samples were obtained. ET-1 levels were determined by immunometric technique (pg/ml +/- SEM) and total nitrate/nitrite level spectrophotometrically (micromol/l +/- SEM).. Angiographic CVS was documented after SAH induction (n = 12, P < 0.05). The ET-1 level in CSF was significantly elevated by 27.3% to 0.84 +/- 0.08 pg/ml in SAH animals (n = 7) in comparison to controls (0.66 +/- 0.04 pg/ml, n = 7, P < 0.05). There was no significant difference in ET-1 levels in systemic and basilar arterial plasma samples of SAH animals compared to controls. A significant lack of local NO metabolites was documented in basilar arterial plasma after SAH (36.8 +/- 3.1 micromol/l, n = 6) compared to controls (61.8 +/- 6.2 micromol/l, n = 6, P < 0.01).. This study demonstrates that an elevated ET-1 level in CSF and local lack of NO in the basilar arterial plasma samples are associated with CVS after experimental SAH.

Topics: Angiography, Digital Subtraction; Animals; Basilar Artery; Cerebrovascular Circulation; Disease Models, Animal; Endothelin-1; Female; Male; Nitric Oxide; Rabbits; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction; Vasodilation; Vasospasm, Intracranial

We designed the present experiments to investigate the involvement of endogenous nitric oxide synthase (NOS) inhibitors, dimethylarginine dimethylaminohydrolase (DDAH) as a hydrolyzing enzyme of the NOS inhibitors, NOS, arginase which shares l-arginine as a common substrate with NOS, and endothelin-1 (ET-1) in the pulmonary dysfunction after induction of experimental subarachnoid hemorrhage (SAH) in the rabbit. SAH was induced by injecting autologous blood into the cisterna magna, and controls were injected with saline. On day 2, pulmonary arteries were isolated for determinations. A significant impairment of the endothelium-dependent relaxation (EDR) caused by acetylcholine was found in 20 cases (43.5%) out of 46 SAH animals, and the same animals exhibited accompanying the significantly impaired cyclic GMP production, accumulated endogenous NOS inhibitors, attenuated DDAH activity, enhanced arginase activity and accumulated ET-1 within the vessel wall. Meanwhile, there were no differences in endothelial NOS activity per se and sodium nitroprusside-induced relaxation between the animals with an impaired EDR and those without such a change. ET-1 content within aortic wall was increased with concomitant decrease in cyclic GMP production after the intraperitoneal application of authentic monomethylarginine as a NOS inhibitor in the rat. The current results suggest that accumulated endogenous NOS inhibitors and enhanced arginase activity possibly bring about the impaired NO production, thereby attenuating the EDR and contributing to the accumulation of ET-1 within the vessel wall. The accumulated endogenous NOS inhibitors at least partly result from the decreased DDAH activity. These alterations may be relevant to the pulmonary dysfunction after induction of SAH.

Topics: Acetylcholine; Amidohydrolases; Animals; Arginase; Cyclic GMP; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Endothelin-1; Indomethacin; Lung Diseases; Male; Models, Biological; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; omega-N-Methylarginine; Oxadiazoles; Pulmonary Artery; Quinoxalines; Rabbits; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vasoconstriction

Cerebral vasospasm remains a major cause of morbidity and mortality in patients with SAH. Although many pharmacologic agents and chemicals have been used to prevent and treat CV, the pathogenesis of that condition has not been established. We investigated the efficacy of resveratrol, a stilbene polyphenol and tyrosine kinase inhibitor that occurs naturally in grapes and red wine, in a murine basilar artery vasospasm model.. Forty-two Wistar albino rats were used in this study. The rats were divided into 3 groups of 14 animals each: the sham-operated control group (group 1), the vasospasm group (group 2), and the treatment group (group 3). In groups 2 and 3, autologous blood (0.3 mL) was injected into the cisterna magna. After that injection, the rats in group 3 received an intravenous injection of resveratrol (10 mg/kg) for 72 hours. The evaluation of the response to both the injection of autologous blood and treatment was based on biochemical markers in tissue and serum and on light microscopic findings from the basilar artery, which were collected at different intervals after experimental SAH.. Endothelin-1 levels in brain tissue and serum were higher in the vasospasm group than in the control group (P < .05). In group 3 rats, the administration of resveratrol resulted in significantly lower ET-1 values than those in group 2. Brain and serum lipid peroxidation levels were markedly elevated in group 2 rats but decreased significantly after resveratrol treatment in group 3 rats (P < .05). Superoxide dismutase expression in brain tissue and serum was lower in group 2 rats than in sham-operated controls, and a significant increase in the SOD level was associated with resveratrol treatment. On examination via light microscopy 72 hours after SAH, the mean perimeters of the arterial lumen in groups 1, 2, and 3 were 719 +/- 16, 411.6 +/- 9, and 590.1 +/- 5.6 microm, respectively. The mean thickness of the arterial wall was as follows: in group 1, 11.1 +/- 0.8 microm; in group 2, 16.1 +/- 1.2 microm; and (after resveratrol treatment) in group 3, 13.4 +/- 0.6 microm.. The results of our study showed that resveratrol induced the relaxation of smooth muscle in the wall of the basilar artery and may be provided with neuroprotection against cerebral ischemia in a rat model. These effects may be associated with the antioxidant and vasodilatory effects of resveratrol, which could prove to be an agent prophylactic against CV and to be therapeutic for individuals who experience that event.

Topics: Animals; Antioxidants; Basilar Artery; Endothelin-1; Injections, Intravenous; Lipid Peroxidation; Muscle, Smooth, Vascular; Rats; Rats, Wistar; Resveratrol; Stilbenes; Subarachnoid Hemorrhage; Superoxide Dismutase; Vasodilation; Vasospasm, Intracranial; Vertebrobasilar Insufficiency

The pathogenesis of cerebral ischaemia after subarachnoid haemorrhage (SAH) still remains elusive. The purpose of the present study was to examine whether specific protein kinas C (PKC) inhibition in rats could alter the transcriptional SAH induced Endothelin (ET) type B and 5-hydroxytryptamine type 1B (5-HT(1B)) receptor upregulation and prevent the associated cerebral blood flow (CBF) reduction. The PKC inhibitor RO-31-7549 or vehicle was injected intracisternally after the induced SAH in rats (n=3 to 10 in each groups for each method). The involvement of the PKC isoforms was investigated with Western blot; only PKCdelta and PKCalpha subtypes were increased after SAH RO-31-7549 treatment abolished this. At 2 days after the SAH basilar and middle cerebral arteries were harvested and the contractile response to endothelin-1 (ET-1; ET(A) and ET(B) receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT(1) receptor agonist) were investigated with a myograph. The contractile responses to ET-1 and 5-CT were increased (P<0.05) after SAH compared with sham operated rats. In parallel, the ET(B) and 5-HT(1B) receptor mRNA and protein expression were significantly elevated after SAH, as analysed by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Administration of RO-31-7549 prevented the upregulated contraction elicited by application of ET-1 and 5-CT in cerebral arteries and kept the ET(B) and 5-HT(1B) receptor mRNA and protein levels at pre-SAH levels. Regional and global CBF evaluated by an autoradiographic technique were reduced by 60%+/-4% after SAH (P<0.05) and prevented by treatment with RO-31-7549. Our study suggests that PKC plays an important role in the pathogenesis of cerebral ischaemia after SAH.

Topics: Algorithms; Animals; Autoradiography; Basilar Artery; Blotting, Western; Capillaries; Cerebral Arteries; Cerebrovascular Circulation; Endothelin-1; Enzyme Inhibitors; Immunohistochemistry; Indoles; Maleimides; Muscle Contraction; Muscle, Smooth, Vascular; Protein Kinase C; Proteins; Rats; Receptor, Serotonin, 5-HT1B; Reverse Transcriptase Polymerase Chain Reaction; Serotonin; Serotonin Receptor Agonists; Subarachnoid Hemorrhage; Up-Regulation

Cerebral vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). It is a sustained constriction of the cerebral arteries that can be reduced by endothelin (ET) receptor antagonists. Voltage-gated Ca(2+) channel antagonists such as nimodipine are relatively less effective. Endothelin-1 is not increased enough after SAH to directly cause the constriction, so we sought alternate mechanisms by which ET-1 might mediate vasospasm. Vasospasm was created in dogs, and the smooth muscle cells were studied molecularly, electrophysiologically, and by isometric tension. During vasospasm, ET-1, 10 nmol/L, induced a nonselective cation current carried by Ca(2+) in 64% of cells compared with in only 7% of control cells. Nimodipine and 2-aminoethoxydiphenylborate (a specific antagonist of store-operated channels) had no effect, whereas SKF96365 (a nonspecific antagonist of nonselective cation channels) decreased this current in vasospastic smooth muscle cells. Transient receptor potential (TRP) proteins may mediate entry of Ca(2+) through nonselective cationic pathways. We tested their role by incubating smooth muscle cells with anti-TRPC1 or TRPC4, both of which blocked ET-1-induced currents in SAH cells. Anti-TRPC5 had no effect. Anti-TRPC1 also inhibited ET-1 contraction of SAH arteries in vitro. Quantitative polymerase chain reaction and Western blotting of seven TRPC isoforms found increased expression of TRPC4 and a novel splice variant of TRPC1 and increased protein expression of TRPC4 and TRPC1. Taken together, the results support a novel mechanism whereby ET-1 significantly increases Ca(2+) influx mediated by TRPC1 and TRPC4 or their heteromers in smooth muscle cells, which promotes development of vasospasm after SAH.

Topics: Amino Acid Sequence; Angiography; Animals; Base Sequence; Disease Models, Animal; Dogs; Electrophysiology; Endothelin-1; Gene Expression Regulation; Molecular Sequence Data; Muscle, Smooth; Patch-Clamp Techniques; RNA, Messenger; Sensitivity and Specificity; Subarachnoid Hemorrhage; TRPC Cation Channels; Vasospasm, Intracranial

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 microl 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 (AT(1)) receptors] induced increased contractile responses [in the presence of the ANG II type 2 (AT(2)) receptor antagonist PD-123319]. In parallel the ET(B), 5-HT(1B), and AT(1) 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 ET(B), 5-HT(1B), and AT(1) 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.

Topics: Angiotensin II; Animals; Autoradiography; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Immunohistochemistry; Male; Muscle, Smooth, Vascular; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; RNA, Messenger; Serotonin; Subarachnoid Hemorrhage; Time Factors; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents

Upregulation of endothelin B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors via transcription has been found after experimental subarachnoid hemorrhage (SAH), and this is associated with enhanced phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1/2). In the present study, we hypothesized that inhibition of ERK1/2 alters the ET(B) and 5-HT(1B) receptor upregulation and at the same time prevents the sustained cerebral blood flow (CBF) reduction associated with SAH. The ERK1/2 inhibitor SB386023-b was injected intracisternally in conjunction with and after the induced SAH in rats. At 2 days after the SAH, cerebral arteries were harvested for quantitative real-time polymerase chain reaction, immunohistochemistry and analysis of contractile responses to endothelin-1 (ET-1; ET(A) and ET(B) receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT1 receptor agonist) in a sensitive myograph. To investigate if ERK1/2 inhibition had an influence on the local and global CBF after SAH, an autoradiographic technique was used. At 48 h after induced SAH, global and regional CBF were reduced by 50%. This reduction was prevented by treatment with SB386023-b. The ERK1/2 inhibition also decreased the maximum contraction elicited by application of ET-1 and 5-CT in cerebral arteries compared with SAH. In parallel, ERK1/2 inhibition downregulated ET(B) and 5-HT(1B) receptor messenger ribonucleic acid and protein levels compared with the SAH. Cerebral ischemia after SAH involves vasoconstriction and subsequent reduction in the CBF. The results suggest that ERK1/2 inhibition might be a potential treatment for the prevention of cerebral vasospasm and ischemia associated with SAH.

Topics: Animals; Autoradiography; Brain; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Immunohistochemistry; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Regional Blood Flow; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serotonin Receptor Agonists; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction

Clazosentan, an endothelin ETA antagonist, is under development by Actelion (formerly Axovan), under license from F Hoffman-La Roche, for the potential prevention of cerebral infarction and ischemia induced by cerebral vasospasm following subarachnoid hemorrhage. Results from the phase IIb portion of a phase IIb/III clinical study are expected in the first half of 2006.

Topics: Clinical Trials, Phase III as Topic; Dioxanes; Endothelin-1; Humans; Molecular Conformation; Pyridines; Pyrimidines; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Vasoconstriction; Vasoconstrictor Agents; Vasospasm, Intracranial

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.

Topics: Animals; Disease Models, Animal; Endothelin-1; Estradiol; Intracranial Aneurysm; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vasospasm, Intracranial

The substantial role of endothelin-1 (ET-1) in the development of cerebral vasospasm (CVS) after subarachnoidal hemorrhage (SAH) has been demonstrated by numerous experimental and, recently, clinical investigations. Whether the expression or function of the ET(B) receptor is altered in CVS is still unclear, however. The aim of the present study was, therefore, to characterize the cerebroarterial ET(B) receptor function during CVS. Experimental CVS was induced by the rat double-hemorrhage model. Reduction of the cerebral blood flow (CBF) was confirmed by magnetic resonance perfusion-weighted imaging. Animals were sacrificed on days 3 (d3) and 5 (d5) after CVS induction. The basilar arteries (BA) were dissected, cut into ring segments, and prepared for measurement of isometric force in an organ bath. Concentration-effect curves (CECs) were constructed by cumulative application of ET-1, acetylcholine (Ach), or sarafotoxin S6c (S6c). Segments with (E+) endothelial function were used. CECs were compared by the maximum effect (E(max)), the pD2, and the shift calculated on the pD2 level. The pD2 is the negative decadic logarithm of the concentration producing the half maximal effect (-log10EC50). After SAH, the relative regional CBF in the d3 and d5 groups was reduced to 63% and 32%, respectively, of the CBF in controls. ET-1 induced a dose-dependent contraction of segments with and segments without CVS. In E+ segments, the E(max) for ET-1 was not significantly changed after SAH (mean values [ +/- SEM] of 104% +/- 4% for the control group, 106% +/- 4% for the d3 group, and 104% +/- 3% for the d5 group). The CECs, however, were significantly shifted to the left versus the control by factors of 2.4 in the d3 group and 3.6 in the d5 group. Relaxation by S6c was significantly reduced after SAH (E(max:) 73% +/- 11% in the control group, 21% +/- 13% in the d3 group, and 13% +/- 8% in the d5 group), whereas relaxation associated with Ach was not significantly changed (E(max): 45% +/- 7% in the control group, 56% +/- 6% in the d3 group, and 43% +/- 6% in the d5 group). Significant contraction by S6c was not observed in E+ and E - segments in any of the study groups. The present data indicate the loss of the ET(B) receptor-mediated relaxation of the cerebral arteries in cases of CVS, which is independent of the endothelial nitric oxide synthase level.

Topics: Acetylcholine; Animals; Basilar Artery; Cholinergic Agents; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; In Vitro Techniques; Isometric Contraction; Male; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; RNA, Messenger; Serotonin; Subarachnoid Hemorrhage; Vasoconstrictor Agents; Vasospasm, Intracranial; Viper Venoms

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.

Topics: Adrenomedullin; Cell Line; Constriction, Pathologic; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Humans; Intracranial Aneurysm; Intracranial Arteriovenous Malformations; Protein Transport; RNA, Messenger; Rupture, Spontaneous; Subarachnoid Hemorrhage; von Willebrand Factor

Endothelin-1 (ET-1) is postulated to play an important role in the development of cerebral vasospasm (CVS) following SAH. This study was conducted to investigate the time course of ET-release in three different sources: CSF, plasma and microdialysate.. In a prospective study ET-1-concentrations were measured in plasma, cisternal CSF and microdialysate in 20 patients with aneurysmal SAH for at least 8 days after hemorrhage.. ET-1 concentration in microdialysate was almost four times higher compared to CSF and plasma. (p<0.001) Only in CSF ET-1-release showed a significant increase over time with highest values on day 5 post ictus (p = 0.03). This was parallel to the increase of transcranial Doppler velocities. ET-1 in plasma and microdialysate did not change over time.. ET-1 may have a different biological function in different biological tissues. Only ET-1 in CSF seemed to be associated with CVS.

Topics: Aged; Brain; Brain Infarction; Cerebral Arteries; Cerebrospinal Fluid; Endothelin-1; Extracellular Fluid; Female; Humans; Male; Microdialysis; Middle Aged; Predictive Value of Tests; Prospective Studies; Subarachnoid Hemorrhage; Subarachnoid Space; Ultrasonography, Doppler, Transcranial; Vasospasm, Intracranial

Subarachnoid haemorrhage (SAH), occurring with a delay of 4-10 days is linked to cerebral vasospasm (CVS), a pathological constriction of the cerebral arteries. Several agents have been suggested as being responsible - amongst these perhaps 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) are the most prominent, given their ability to elicit powerful constriction of arteries. Investigating both 5-HT and ET receptors we observed distinct changes in the receptor phenotype after experimental SAH - namely upregulation of the ETB and 5-HT1B receptors - linked to a higher sensitivity to the endogenous agonists. This multiple receptor upregulation may explain the failure in treating CVS using single receptor antagonists, and may also significantly change our understanding of the effector mechanism behind CVS. So far only the ET and 5-HT receptors have been studied in this regard, but other receptor systems may also undergo changes.

Topics: Animals; Endothelin-1; Phenotype; Rats; Receptors, Endothelin; Receptors, Serotonin; Subarachnoid Hemorrhage; Up-Regulation; Vasospasm, Intracranial

Endothelin-1 (ET-1) is one of the major inducers of vasospasm following subarachnoid hemorrhage (SAH). It is generally accepted that extracellular signal-regulated kinase 1 and 2 (ERK1/2) are involved in ET-1-induced vascular contraction. In addition, ET-1 transactivates epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK), which leads to ERK1/2 stimulation. Therefore, the authors examined whether EGFR-PTK transactivation contributes to ET-1-induced vascular contraction in this study.. Mitogen-activated protein kinase inhibitor, PD98059, inhibited ET-1-induced ERK1/2 stimulation in rabbit basilar artery (BA) vascular smooth-muscle cells (VSMCs). Moreover, PD98059 inhibited ET-1-induced contraction of rabbit BA rings. A specific inhibitor of EGFR PTK, AG1478, inhibited ET-1-induced EGFR-PTK transactivation, ERK1/2 stimulation, and contraction of BA rings in a concentration-dependent manner. The concentration of AG1478 required for 50% inhibition of the ET-1-induced contraction of BA rings was similar to that for ET-1-induced EGFR-PTK transactivation. Furthermore, AG1478 also inhibited ET-1-induced BA vasospasm in vivo.. The results indicate that EGFR-PTK transactivation pathway plays an important role in ET-1-induced vascular contraction.

Topics: Animals; Endothelin-1; ErbB Receptors; Rabbits; Signal Transduction; Subarachnoid Hemorrhage; Transcriptional Activation; Vasospasm, Intracranial

Inspired by organ culture-induced changes in the vascular endothelin (ET) receptor population, we investigated whether such changes occur in cerebral arteries in a rat subarachnoid hemorrhage (SAH) model.. SAH was induced with injection of 250 microl of blood into the prechiasmatic cistern. After 2 days, the middle cerebral artery, basilar artery, and posterior communicating artery were harvested. Pharmacological studies were performed in vitro, and levels of messenger ribonucleic acid (mRNA) were quantified in real-time reverse transcriptase-polymerase chain reaction assays.. In the middle cerebral artery and basilar artery from rats with induced SAH, enhanced biphasic responses to ET-1 were observed. The -log(50% effective concentration) value for the high-affinity phase was approximately 12, compared with approximately 8.5 for sham-operated animals. At a concentration of ET-1 of 10(-9) mmol/L (approximately equal to the physiological concentration of ET-1 in the plasma), submaximal contractions of 50 to 75% of the contraction obtained through stimulation with 60 mmol/L K(+) were now observed. Quantitative mRNA studies with the same arteries demonstrated significant increases in the number of copies of ET(B) receptor mRNA but not ET(A) receptor mRNA. Evidence of functional ET(B) receptors was provided in antagonist studies. The posterior communicating artery did not exhibit significant changes.. The altered receptor profile observed may represent the final stage in the series of events leading from SAH to actual spasm of the artery. The pharmacological data for the ET(B) receptor suggest complex interactions between normally present ET(A) receptors and up-regulated ET(B) receptors.

Topics: Animals; Basilar Artery; Disease Models, Animal; Endothelin-1; Gene Expression; In Vitro Techniques; Male; Middle Cerebral Artery; Posterior Cerebral Artery; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Subarachnoid Hemorrhage

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C.

Topics: Alkaloids; Animals; Basilar Artery; Benzophenanthridines; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Male; Phenanthridines; Protease Inhibitors; Protein Kinase C; Rabbits; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction; Vasospasm, Intracranial

This study aimed to clarify the characteristics of the basilar arteries of rabbits two and seven days after subarachnoid haemorrhage (SAH) and to investigate the influence of hypoxia on vascular responses to acetylcholine and endothelin-1.. Thirty four New Zealand white rabbits were divided into 3 groups: 1) control (n=6), 2) 2 days post SAH (n=15), and 3) 7 days post SAH (n=13). Rabbits in the groups 2 and 3 underwent vertebral angiography before sacrifice. Harvested vascular rings were suspended in a small vessel myograph system for isometric tension recording in response to vasodilatory and vasoconstrictory stimulation in oxygenated (95% O2/5% CO2) and hypoxic (95% N2/5% CO2) conditions. To investigate the response to a vasodilator, acetylcholine (ACh) (10(-7) to 3 x 10(-4) M) was applied to a basilar artery pre-contracted with histamine (10(-6) approximately 10(-5) M). To investigate the response to a vasoconstrictor, the effect of endothelin-1 (ET-1) (10(-11) to 3 x 10(-8) M) was observed in quiescent rings.. Seven of 15 rabbits that underwent angiography two days after operation showed vasospasm to 64.3+/-11.2% of the original diameter; likewise seven of the 13 rabbits studied seven days after operation showed vasospasm to 64.9+/-10.9% of the original diameter. Hypoxic conditions significantly reduced the relaxation response to ACh but did not influence basilar artery contraction to ET-1. Responses to ACh and ET-1 did not differ significantly in vessels from animals with or without vasospasm at 2 days. In contrast, at 7 days vascular responses to ACh and ET-1 were significantly less in vessels from animals with vasospasm than those without (p=0.029, 0.032), and also less than in vessels from animals with vasospasm at day 2 (p=0.002, 0.004). There was not a significant difference in the vascular responses of basilar arteries from animals without vasospasm.. The markedly lower vascular responses of basilar arteries of rabbits to ACh and ET-1 found seven days after SAH may relate to delayed vasospasm in patients with SAH.

Topics: Acetylcholine; Animals; Basilar Artery; Coronary Angiography; Disease Models, Animal; Endothelin-1; Hypoxia, Brain; Rabbits; Subarachnoid Hemorrhage; Time Factors; Vasodilator Agents; Vasospasm, Intracranial

This study investigated the effects of Ginkgo biloba extract (EGb-76l), an anti-oxidant and platelet-activating factor antagonist, on basilar artery vasospasm in an experimental canine subarachnoid hemorrhage model. Morphometric analyses were performed, and serum and cerebrospinal fluid endothelin-l levels were measured by radioimmunoassay. Comparisons were made between treated and untreated groups. Twenty-four mongrel dogs were randomly assigned to three groups. The animals in group 1 (n = 8) were not subjected to subarachnoid hemorrhage and received no treatment. In this group, serum and cerebrospinal fluid endothelin-l levels were measured daily for 8 days. On day 9, the animals were killed and their basilar arteries were excised for histopathological examination. In group 2 (n = 8), subarachnoid hemorrhage was produced using autologous arterial blood, and daily intravenous boluses of saline were administered for the next 8 days. Assessments of endothelin-l levels and the basilar arteries were performed as described for group 1. In group 3 (n = 8), subarachnoid hemorrhage was produced using autologous arterial blood, and daily intravenous boluses of EGb-761 were administered for 8 days. Endothelin-1 levels and the basilar arteries were assessed as described above. The groups' serum endothelin-1, cerebrospinal fluid endothelin-1, and histopathological findings were compared. In group 1, the serum and cerebrospinal fluid endothelin-1 levels did not change significantly over the 8 days, and histopathological examination of the basilar arteries revealed no abnormalities. In group 2, the serum and cerebrospinal fluid endothelin-1 levels increased abruptly and significantly on day 2, and remained high to the end of the study period (day 8). Histopathological examination revealed marked vasospasm. In group 3, the serum and cerebrospinal fluid endothelin-1 levels followed the same pattern observed in group 2; however, the arteries showed significantly less vasospasm than that observed in group 2. The study findings did not provide information about the mechanism of action of the platelet-activating factor-antagonist EGb-761, but they clearly show that this agent decreases morphologic vasospasm in the dog basilar artery.

Topics: Animals; Basilar Artery; Dogs; Endothelin-1; Ginkgo biloba; Male; Plant Extracts; Platelet Activating Factor; Subarachnoid Hemorrhage; Vasospasm, Intracranial

This study was aimed at investigating the effects of extract of Ginkgo biloba (EGb) on cerebral vasospasm and microcirculatory perfusion after subarachnoid hemorrhage (SAH). An endovascular piercing method was used to induce Wistar rat SAH models, and animals were divided into sham-operated, vehicle controls, and EGb-treated groups. EGb was injected intraperitoneally 30 minutes before operation and was repeated every 6 hours, with a single dose of 15 mg/kg bw. Diameters of basilar arteries before and after operation were measured. Microcirculatory blood perfusion of parietal lobe cortex was detected using a laser Doppler flow-meter probe within 24 hours. Endothelin-1 levels in both plasma and brain tissue were detected at different time points. The results showed that SAH caused an immediate drop in microcirculatory blood flow in vehicle controls, which persisted for 24 hours. Endothelin-1 levels in both plasma and brain tissue increased after SAH. EGb partly reversed spasms of the basilar artery and antagonized a drop in microcirculatory blood flow. EGb also prevented an increase in endothelin-1 both in plasma and in brain tissue. In conclusion, EGb, by antagonizing the overproduction of endo- thelin-1, partly reverses cerebral vasospasm and improves microcirculation, and thus relieves secondary ischemic brain injury after experimental SAH.

Topics: Animals; Basilar Artery; Brain Ischemia; Cerebrovascular Circulation; Drug Evaluation, Preclinical; Endothelin-1; Female; Gene Expression Regulation; Ginkgo biloba; Injections, Intraperitoneal; Male; Microcirculation; Models, Animal; Parietal Lobe; Phytotherapy; Plant Extracts; Plants, Medicinal; Rats; Rats, Wistar; Subarachnoid Hemorrhage; Vasoconstriction; Vasodilator Agents; Vasospasm, Intracranial

The purpose of this study is to investigate the effect of L-arginine (L-Arg) on cerebral blood perfusion and vasomotion (perfusion motion) in microvessels following subarachnoid hemorrhage (SAH). Rat noncraniotomy SAH models were used and animals were divided into sham-operated, saline-treated, and L-Arg-treated groups. L-Arg was injected intraperitoneally 30 minutes before the operation and repeated every 6 hours, with a single dose of 0.5 g/kg bw. Dynamic changes in regional cerebral blood flow (CBF) and vasomotion within 24 hours were measured using a laser Doppler flow-meter probe. Serum nitric oxide (nitrite/nitrate) and plasma endothelin-1 levels were also measured at different time points within 24 hours. Morphologic changes in neurons in the hippocampus CA1 region were examined. SAH gave rise to an immediate and persistent decrease in CBF in saline-treated rats. Abnormal vasomotions with decreased frequency and amplitude were observed. Serum nitric oxide decreased, while plasma endothelin-1 increased significantly. Neurons in the hippocampus CA1 region were severely damaged. The above pathological alterations in the L-Arg-treated group were alleviated. It was concluded that L-Arg, which increases cerebral blood perfusion and improves vasomotions of microvessels by enhancing nitric oxide levels and decreasing endothelin-1 levels in blood, exerts a protective effect on secondary cerebral ischemic injury following experimental SAH.

Topics: Animals; Arginine; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Female; Hippocampus; Injections, Intraperitoneal; Laser-Doppler Flowmetry; Male; Microcirculation; Neurons; Nitric Oxide; Pyramidal Cells; Rats; Rats, Wistar; Subarachnoid Hemorrhage; Vasodilation; Vasospasm, Intracranial

Plasma endothelin (ET) is elevated in patients with vasospasm following subarachnoid haemorrhage (SAH). However, systemic levels provide no indication regarding local production in the brain, and late elevation may be a consequence rather than a cause of vasospasm. We measured arteriojugular (AJ) gradients of ET-1 in 17 patients over the first week after SAH, and related these to the subsequent development of vasospasm. Daily, paired arterial and jugular bulb blood samples were obtained up to seven days post SAH, and assayed for ET-1 using radioimmunoassay. Systemic levels and AJ gradients were compared in patients with and without vasospasm. Significant AJ gradients were observed for ET-1 (P<0.01). These differences remained significant in the subgroup of patients who developed vasospasm (0.12+/-0.05 pmol/l; P<0.05), in whom AJ gradients represented 25+/-7% of systemic levels (0.84+/-0.05 pmol/l). AJ gradients did not reach significance in patients who did not develop vasospasm (0.09+/-0.07 pmol/l; P=0.2). Systemic ET-1 levels and AJ gradients were unrelated to SAH grade, surgical or endovascular interventions, or extracranial complications. AJ gradients in the first week following SAH suggest early production of ET-1 in the cerebrovascular bed. However, early systemic ET-1 levels did not discriminate between patients with and without vasospasm. Larger AJ differences may predict vasospasm, but further work is needed to confirm this observation. AJ gradient measurement may provide a useful technique for investigating the role of other peptides in acute brain injury.

Topics: Adult; Aged; Arteries; Blood Specimen Collection; Endothelin-1; Female; Humans; Jugular Veins; Male; Middle Aged; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

The aim of this study was to investigate whether blocking functional endothelin-converting enzyme (ECE) activity may offer a new approach to inhibit the development of cerebral vasopasm after subarachnoid hemorrhage (SAH) by preventing transformation of big Endothelin-1 (big ET-1) to vasoactive Endothelin-1 (ET-1).. In vitro, the effect of potential ECE inhibitors was determined by measurement of isometric contractions, induced by big ET-1, in isolated rat basilar arteries. Endothelium intact (E+) and de-endothelialized (E-) segments were examined after pre-incubated with the putative ECE inhibitors: Phosphoramidon (10(-4) M), Captopril (10(-3) M and 10(-4) M) and [(22)D-Val] big ET-1 (16-38) (10(-5) M and 10(-6) M).. Application of 10(-4) M Phosphoramidon resulted in a statistically significant decrease in big ET-1 induced contraction in endothelium intact (E+) and de-endothelialized (E-) segments; 10(-3) M Captopril in E- segments caused a statistically significant inhibitory effect; 10(-4) M and 10(-3) M Captopril in E+ segments showed no statistically significant effect; 10(-5) M and 10(-6) M [(22)D-Val] big ET-1 (16-38) in E- segments produced no statistically significant effect. The application of 10(-6) M [(22)D-Val] big ET-1 (16-38) in E+ segments caused increased contractions as shown by the shift to the left of the concentration-effect curve (CEC).. The present study indicates the existence of functional ECE activity in rat basilar artery, which is different in the endothelium and the smooth muscle layer. This ECE-activity could be inhibited by Captopril and Phosporamidon, suggesting a potency for prevention and therapy of cerebral vasospasm. However, the structural analogue of big ET-1, [(22)D-Val] big ET-1 (16-38), was ineffective in reducing big ET-1 induced vasoconstriction and rather increased contraction in E+ vessels. Therefore further studies of the biochemical nature of the functional relevant cerebrovascular ECE activity are required for better understanding and development of other efficient ECE inhibitors.

Topics: Animals; Aspartic Acid Endopeptidases; Basilar Artery; Captopril; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Glycopeptides; Male; Metalloendopeptidases; Protein Precursors; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Vascular Resistance; Vasospasm, Intracranial

Pathogenesis of delayed ischemia after aneurysmal subarachnoid hemorrhage (SAH) seems to be complex. An important mediator of chronic vasospasm may be endothelin (ET)-1 with its powerful and long-lasting vasoconstricting activity. In this prospective study the author investigated the correlations between serial plasma concentrations of ET-1 and big ET-1 as well as the ET-1/big ET-1 molar concentration ratio and serum endothelin-converting enzyme (ECE)-1 activity, and ischemic complications after SAH.. To measure plasma ET-1 (51 patients), big ET-1 immunoreactivity (22 patients), and serum ECE-1 activity (13 patients), blood samples were obtained on admission, in the morning after aneurysm surgery, and during the 2nd week after hemorrhage in 51 consecutive patients (28 men and 23 women, with a mean age of 50.8 years) with aneurysmal SAH. Mean plasma concentrations of ET-1 in patients with SAH (mean +/- standard deviation: on admission, 4.2 +/- 2 pg/ml; after surgery, 4.3 +/- 2.2 pg/ml; and during the 2nd week after SAH, 3.7 +/- 1.9 pg/ml) differed from those in healthy volunteers (2.9 +/- 1.2 pg/ml; p < 0.01). Plasma concentrations of ET-1 and big ET-1 as well as the ET-1/big ET-1 ratio did not change significantly with elapsed time following SAH; however, serum ECE-1 activity during the 2nd week after SAH was higher in patients with SAH than that in controls (162 +/- 43 compared with 121 +/- 56 pg/ml, respectively; p = 0.028). Plasma ET-1 concentrations (p < 0.05) and the ET-1/big ET-1 ratios (p = 0.063) were higher but plasma big ET-1 concentrations were lower (p < 0.05) in patients who experienced symptomatic delayed cerebral ischemia, compared with other patients with SAH. In addition, in cases in which follow-up computerized tomography scans or magnetic resonance images demonstrated permanent ischemic lesions attributable to vasospasm, patients had higher ET-1 concentrations than did other patients with SAH.. The plasma ET-1 concentration correlates with delayed cerebral ischemia after SAH, suggesting that an increased ET conversion rate in the endothelium predicts ischemic symptoms. Increased serum ECE-1 activity during the 2nd week may reflect the severity of endothelial injury to cerebral arteries.

Topics: Adult; Aged; Aspartic Acid Endopeptidases; Brain Ischemia; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Female; Humans; Magnetic Resonance Imaging; Male; Metalloendopeptidases; Middle Aged; Predictive Value of Tests; Protein Precursors; Severity of Illness Index; Subarachnoid Hemorrhage; Tomography, X-Ray Computed; Treatment Outcome; Vasospasm, Intracranial

Since isoflurane is known to attenuate endothelium-dependent dilation (EDD) in normal cerebral arterioles, we examined whether the anesthetic has a similar effect and further impairs EDD in vessels exposed to SAH.. Autologous blood was introduced in the subarachnoid space and the parietal lobe harvested. Control animals were sacrificed without introduction of blood. The response of microvessles to the endothelium-dependent dilator adenosine diphosphate (ADP) 10(-9)-10(-4) M, the endothelium-independent dilator nitroprusside 10(-9)-10(-4) M, and ET-1 10(-13)-10(-8) M was measured by videomicroscopy in the presence of 0-2 minimum alveolar concentration (MAC) of isoflurane.. Isoflurane attenuated EDD to ADP in control vessels [66 +/- 5% (control) vs 27 +/- 11% (2 MAC) dilation to ADP 10(-4) M, P < 0.05]. Although SAH was associated with reduced dilation to ADP, exposure to isoflurane did not further impair dilation to ADP after SAH [26 +/- 3% (SAH) vs 21 +/- 5% (SAH/2 MAC) dilation to ADP 10(-4) M, P = NS]. Dilation to nitroprusside was not affected by isoflurane or SAH. Constriction to ET-1 was reduced by 2 MAC of isoflurane [21 +/- 1% (control) vs 13 +/- 5% (2 MAC) constriction to ET-1 10(-8) M, P < 0.05], but not by 1 MAC of isoflurane in control vessels. Constriction to ET-1 was greatly attenuated by 1 or 2 MAC of isoflurane after SAH [32 +/- 5% (SAH) vs 18 +/- 4% (SAH/2 MAC) constriction to ET-1 10(-8) M, P < 0.05].. In rats, isoflurane does not further impair EDD after SAH and modulates the constrictive response to ET-1. Such an effect of isoflurane would not predispose the SAH-exposed vessels to vasospasm.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Anesthetics, Inhalation; Animals; Arterioles; Capillaries; Cerebrovascular Circulation; Endothelin-1; Endothelium, Vascular; Female; Isoflurane; Male; Microscopy, Video; Nitroprusside; Parietal Lobe; Rats; Rats, Wistar; Subarachnoid Hemorrhage; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of Rho kinase. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Y-27632, a Rho kinase inhibitor, relaxed the spasm. However, relaxations to Y-27632 were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that, at least in the rabbit double subarachnoid hemorrhage model, vasospasm does not involve the functional upregulation of Rho kinase.

Topics: Amides; Animals; Basilar Artery; Endothelin-1; Glycopeptides; Intracellular Signaling Peptides and Proteins; Male; Muscle Relaxants, Central; Protease Inhibitors; Protein Serine-Threonine Kinases; Pyridines; Rabbits; rho-Associated Kinases; Subarachnoid Hemorrhage; Vasoconstriction; Vasospasm, Intracranial

Endothelin-1 is the most potent vasoconstrictor known to date. This peptide is believed to play a pathophysiological role in the development of vasospasm, the most important complication of subarachnoid hemorrhage (SAH). In the present study we investigated the release of endothelin-1 in SAH and analyzed the cellular source of this peptide. At a protein and mRNA level we were able to show that endothelin-1 is produced by mononuclear leukocytes. Complementary in vitro studies revealed that aging and subsequent hemolysis of blood is sufficient to induce production of endothelin-1 by mononuclear leukocytes. Thus, cerebrospinal fluid-derived mononuclear leukocytes are a source of endothelin-1 in patients suffering from SAH. This finding may have important therapeutic implications as anti-leukocyte strategies could prevent cerebrovascular complications in SAH patients.

Topics: Adult; Aged; Aneurysm; Blood Flow Velocity; Brain Ischemia; Endothelin-1; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Subarachnoid Hemorrhage

Topics: Acute Disease; Adult; Endothelin-1; Female; Humans; Male; Middle Aged; Osmolar Concentration; Prognosis; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Endothelin-1 (ET-1) was originally identified as a potent vasoconstrictor peptide. Numerous reports have suggested its roles in various disorders. Although there is a great deal of evidence establishing the relationship between ET-1 and cerebral vasospasm in animals, this relationship still remains to be clarified in humans.. The concentration of ET-1 in cerebrospinal fluid (CSF) was measured by radioimmunoassay in 23 subarachnoid hemorrhage patients. CSF samples were collected every 10 days after surgery from the cisternal drainage tube.. Initial concentrations of ET-1 in the CSF collected the first day after operation were all increased compared with the control CSF. In seven of the eight vasospasm patients, the concentrations of ET-1 had increased before the observation of vasospasm and then decreased before the disappearance of the vasospasm. In 13 out of the 15 patients without vasospasm, the concentrations of ET-1 in CSF decreased with time.. We confirmed that the concentration of ET-1 in CSF increased before the onset of cerebral vasospasm caused by subarachnoid hemorrhage. The ET-1 concentration in the CSF could be a useful marker to detect cerebral vasospasm after subarachnoid hemorrhage.

Topics: Adult; Aged; Aged, 80 and over; Endothelin-1; Female; Humans; Male; Middle Aged; Radioimmunoassay; Subarachnoid Hemorrhage; Vasospasm, Intracranial

The most potent vasoconstrictor known, endothelin-1, is currently considered to mediate cerebral vasospasm in subarachnoid hemorrhage (SAH), which can cause delayed cerebral ischemia. In our study, we performed clinical and in vitro experiments to investigate the origin and the mechanisms of the secretion of endothelin-1 in SAH.. Endothelin-1 and markers of inflammatory host response (interleukin [IL]-1ss, IL-6, and tumor necrosis factor-alpha) were comparatively quantified in the cerebrospinal fluid (CSF) of SAH patients and control subjects, and concentrations were related to clinical characteristics. Furthermore, mononuclear leukocytes isolated from the CSF of SAH patients and control subjects were analyzed regarding their mRNA expression of endothelin-1 and inflammatory cytokines. Finally, complementary in vitro experiments were performed to investigate whether coincubation of blood and CSF can trigger leukocytic mRNA expression and release of these factors.. Activated mononuclear leukocytes in the CSF of SAH patients synthesize and release endothelin-1 in parallel with known acute-phase reactants (IL-1ss, IL-6, and tumor necrosis factor-alpha). Complementary in vitro experiments not only further confirmed this leukocytic origin of endothelin-1 but also showed that aging and subsequent hemolysis of blood is sufficient to induce such endothelin-1 production.. The demonstration that endothelin-1 is produced by activated CSF mononuclear leukocytes suggests that subarachnoid inflammation may represent a therapeutic target to prevent vasospasm and delayed cerebral ischemia after SAH.

Topics: Acute-Phase Proteins; Adult; Aged; Cerebrospinal Fluid; Cytokines; Endothelin-1; Female; Humans; Leukocytes; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Subarachnoid Hemorrhage; Tumor Necrosis Factor-alpha

Endothelium plays a role in the regulation of vascular tone. Endothelin is a family of potent vasoconstrictive peptides, and endothelin-1 (ET-1) produced in the endothelium induces a tonic contraction via specific receptor ET(A). ET-1 has been postulated as an important factor in the development of vasospasm after subarachnoid haemorrhage (SAH). We have previously shown that protein kinase C (PKC) of the cerebral artery plays a pivotal role in the pathogenesis of vasospasm. The purpose of this study is to clarify the relationship between ET-1 and PKC in the development and maintenance of vasospasm. Using a "two-haemorrhage" canine model, chronological changes of angiographic progression of vasospasm, PKC activation, and ET-1 level of the basilar artery were assessed. In an isometric tension study with a control artery, the effects of ET(A)- and ET(A)/ET(B)-antagonists on the tonic contraction induced by ET-1 were examined. The effects of ET-1, ET-1 and an ET(A)-antagonist, and ET-1 and an ET(A)/ET(B)-antagonist on PKC activation were also evaluated. ET-1 level temporarily increased, then decreased to the control level in a later stage of vasospasm. ET-1 induced a tonic contraction and enhancement of PKC activation, but both were inhibited either by an ET(A)- or an ET(A)/ET(B)-antagonist. These results indicate that ET-1 initiates the development of vasospasm through PKC activation, but does not contribute to prolonged vasospasm.

Topics: Animals; Basilar Artery; Cerebral Angiography; Dogs; Endothelin-1; Enzyme Activation; Female; Male; Protein Kinase C; Subarachnoid Hemorrhage; Time Factors; Vasoconstriction; Vasospasm, Intracranial

The purpose of this study is twofold: 1) to test antisense genetic techniques used in the prevention of cerebral vasospasm in a canine model of subarachnoid hemorrhage (SAH), targeting the endothelin-1 (ET-1) gene; and 2) to determine if fibrinolysis of subarachnoid clot with recombinant tissue plasminogen activator (rtPA) could enhance the effect of antisense treatment.. A total of 39 dogs were studied in this experiment. Placebo (six animals), rtPA (six animals), antisense preproET-1 oligodeoxynucleotide (ASOD; five animals), or rtPA plus ASOD (combined treatment; six animals) was injected into the cisterna magna 30 minutes after a second SAH was induced on the 2nd day of the experiment. The animals were observed until Day 7, when they underwent follow-up angiography and then were killed; their basilar arteries were removed for analysis. Control animals included in this study (two animals in each group) received placebo, rtPA, ASOD, or rtPA plus ASOD without induction of SAH, or rtPA with mismatched (nonsense) preproET-1 oligodeoxynucleotide following SAH. Six additional dogs were analyzed earlier following SAH. Dogs that received placebo developed severe vasospasm (51+/-8% of baseline caliber). Administration of ASOD alone resulted in a mild reduction in vasospasm (64+/-13% of baseline caliber) and rtPA alone resulted in a moderate reduction in vasospasm (81+/-5% of baseline caliber); however, the combined therapy of rtPA plus ASOD almost completely prevented vasospasm (95+/-6%, of baseline caliber), which was significantly different from all other groups (p < 0.05). Morphological analysis of the basilar arteries yielded results similar to angiography with respect to vasospasm severity. The ASOD treatment combined with rtPA resulted in reduced ET-1 expression, as demonstrated by immunohistochemical staining of the arteries, and reduced preproET-1 levels on Day 4, as measured by reverse transcription-polymerase chain reaction. Nonsense DNA sequences had no effect on the vessels.. Antisense preproET-1 oligodeoxynucleotide treatment, when combined with clot lysis caused by rtPA, reduced vasospasm in the canine model of SAH, and this effect appeared to be related to reduced ET-1 synthesis. The results of this experiment support a causative role for ET-1 early in the course of vasospasm development in dogs. The apparent additive therapeutic effects of antisense and fibrinolytic treatments could be due to clot lysis, which allows better delivery of oligodeoxynucleotides to arteries within the subarachnoid space.

Topics: Animals; Cerebral Angiography; DNA; Dogs; Endothelin-1; Endothelins; Female; Fibrinolytic Agents; Genetic Techniques; Glyceraldehyde-3-Phosphate Dehydrogenases; Immunohistochemistry; Ischemic Attack, Transient; Male; Microscopy, Electron; Molecular Biology; Oligonucleotides, Antisense; Protein Precursors; Recombinant Proteins; RNA, Messenger; Subarachnoid Hemorrhage; Tissue Plasminogen Activator

We examined the effect of intracisternal application of endothelin-1 (ET-1) on the permeability of fluorescein into the cerebrospinal fluid (CSF) in beagle dogs in order to evaluate its role in disruption of blood-brain barrier (BBB) permeability seen in the subarachnoid hemorrhage animal model. Intracisternal application of their autologous blood for producing a canine two-hemorrhage model revealed an enhanced fluorescein permeability into the CSF together with the development of cerebral vasospasm. A single dose of ET-1 (40 pmol/animal) significantly increased penetration of fluorescein compared with that in normal dogs. Although its magnitude was much less than that in the two-hemorrhage model after the first administration of ET-1, the second challenge of the same dose of ET-1 with a 48-h interval produced marked disruption of BBB permeability similar to those in the animal model. Moreover, the ET-1-induced enhancement of fluorescein permeability into the CSF was completely prevented by intracisternal pretreatment with an endothelin ET(A)-receptor selective antagonist, S-0139 (0.03 mg/kg), as were the ET-1-induced cerebral vasoconstriction and behavioral changes as previously reported. Thus, we conclude that ET-1 acting on the adventitial site of brain in dogs contributes to the disruption of BBB permeability via endothelin ET(A)-receptor mediation.

Topics: Analysis of Variance; Animals; Blood; Blood-Brain Barrier; Caffeic Acids; Cell Membrane Permeability; Cisterna Magna; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Female; Fluorescein; Injections, Intraventricular; Male; Oleanolic Acid; Receptor, Endothelin A; Subarachnoid Hemorrhage

Hemoglobin released from hemolysed erythrocytes has been postulated to be responsible for delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). However, the evidence is indirect and the mechanisms of action are unclear. Cerebrovascular tone is regulated by a dynamic balance of relaxing and contracting factors. Loss of the endothelium-derived relaxing factor-nitric oxide in the presence of oxyhemoglobin and overproduction of endothelin-1 stimulated by oxyhemoglobin have been postulated as causes of delayed cerebral vasospasm after SAH.. The authors aimed to investigate this hypothesis using in vivo microdialysis to examine time-dependent changes in the perivascular concentrations of oxyhemoglobin, deoxyhemoglobin, and methemoglobin in a primate model of SAH.. Nine cynomolgus monkeys underwent right-sided frontotemporal craniectomy and placement of a semipermeable microdialysis catheter adjacent to the right middle cerebral artery (MCA). Saline (control group, three animals) or an arterial blood clot (SAH group, six animals) was then placed around the MCA and the catheter. Arteriographically confirmed vasospasm had developed in all animals with SAH but in none of the control animals on Day 7. The dialysate was collected daily for 12 days. Levels of oxyhemoglobin, deoxyhemoglobin, and methemoglobin were measured by means of spectrophotometry. Perivascular concentrations of oxyhemoglobin, deoxyhemoglobin, and methemoglobin peaked on Day 2 in the control monkeys and could not be detected on Days 5 to 12. Perivascular concentrations of oxyhemoglobin and deoxyhemoglobin peaked on Day 7 in the SAH group, at which time the concentrations in the dialysate were 100-fold higher than in any sample obtained from the control animals. Methemoglobin levels increased only slightly, peaking between Days 7 and 12, at which time the concentration in the dialysate was 10-fold higher than in samples from the control animals.. This study provides in vivo evidence that the concentrations of oxyhemoglobin and deoxyhemoglobin increase in the cerebral subarachnoid perivascular space during the development of delayed cerebral vasospasm. The results support the hypothesis that oxyhemoglobin is involved in the pathogenesis of delayed cerebral vasospasm after SAH and implicate deoxyhemoglobin as a possible vasospastic agent.

Topics: Analysis of Variance; Animals; Catheterization; Cerebral Angiography; Cerebral Arteries; Craniotomy; Disease Models, Animal; Endothelin-1; Exudates and Transudates; Hemoglobins; Hemolysis; Ischemic Attack, Transient; Macaca fascicularis; Methemoglobin; Microdialysis; Nitric Oxide; Oxyhemoglobins; Sodium Chloride; Spectrophotometry; Subarachnoid Hemorrhage; Time Factors

While it has been widely reported that the vasospasm following subarachnoid hemorrhage (SAH) is prevented/reversed by endothelin (ET) receptor antagonists selective for the ET(A) receptor and by nonselective ET receptor antagonists, ie, antagonists of both the ET(A) and ET(B) receptors, there are no reports on the possible attenuation of the spasm by selective ET(B) receptor antagonists. The purpose of this study was to investigate whether (1) ET(B) receptor antagonists prevent and reverse SAH-induced spasm and (2) attenuation of the spasm results from blockade of smooth muscle ET(B) (ET(B2)) receptor-mediated constriction and/or endothelial ET(B) (ET(B1)) receptor-mediated ET-1-induced ET-1 release.. SAH-induced spasm of the rabbit basilar artery was induced with the use of a double hemorrhage model. In vivo effects of agents on the spasm were determined by angiography after their intracisternal infusion (10 microL/h) by mini osmotic pump. In situ effects of agents on the spasm were determined by direct measurement of vessel diameter after their suffusion in a cranial window.. SAH constricted the basilar artery by 30%. Intracisternal infusion with 10 micromol/L BQ788, an ET(B1/B2) receptor antagonist, reduced the spasm to 10%. To investigate whether BQ788 prevented the spasm by blockade of ET(B1) receptor-mediated ET-1-induced ET-1 release, as opposed to ET(B2) receptor-mediated constriction, we tested whether ET(B1) receptor blockade also prevented the spasm. Indeed, intracisternal infusion with 10 micromol/L RES-701-1, a selective ET(B1) receptor antagonist, reduced the spasm to 10%. Similarly, in situ superfusion with 1 micromol/L BQ788 reversed the spasm by 40%, and 1 micromol/L RES-701-1 reversed the spasm by 50%. However, both BQ788 and RES-701-1 enhanced by 40% to 50% the 3 nmol/L ET-1-induced constriction elicited in spastic vessels previously relaxed with 0.1 mmol/L phosphoramidon, an ET-converting enzyme inhibitor.. These results demonstrate that ET(B) receptor antagonists prevent and reverse SAH-induced cerebral vasospasm in an animal model. The likely mechanism underlying the attenuation of the spasm is blockade of ET(B1) receptor-mediated ET-1-induced ET-1 release of newly synthesized ET-1. These studies provide rationale for the therapeutic use of ET(B1) receptor antagonists to relieve the vasospasm following SAH, as well as other pathophysiological conditions involving possible ET-1-induced ET-1 release.

Topics: Animals; Basilar Artery; Endothelin Receptor Antagonists; Endothelin-1; Injections, Intraventricular; Ischemic Attack, Transient; Male; Muscle, Smooth, Vascular; Oligopeptides; Peptides, Cyclic; Piperidines; Rabbits; Receptor, Endothelin B; Subarachnoid Hemorrhage; Vasoconstriction; Vasodilation

Endothelin participates in regulating the vascular tone, and it is also involved in the pathogenesis of vasospasm following subarachnoid hemorrhage (SAH). Endothelin-1 (ET-1) induced cerebral vasospasm is inhibited by ETA receptors specific antagonist-BQ-123; this protects the neurons from ischemic damage. The present study evaluates the dynamics of ET-1 concentration changes in the plasma of rats in the acute phase of vasospasm after SAH, which was induced by administering 100 microliters non-heparinized fresh autologous arterial blood into the brain cisterna magna (CM). The study also assesses the effect of blocking ETA receptors on the changes in ET-1 level. BQ-123, the specific ETA receptors antagonist, was administered to cerebrospinal fluid (CSF) through a cannula inserted into CM; the antagonist--40 nmol in 50 microliters CSF--was given 20 minutes prior to SAH. In the control group, sham SAH was induced by administering 100 microliters artificial CSF (aCSF) to CM. ET-1 concentration in the plasma of rats in the acute phase of vasospasm was assessed by radioimmunoassay 30 and 60 minutes after SAH or sham SAH. It has been showed that both SAH and sham SAH cause significant increase in the ET-1 concentration (p < 0.05) in the rat plasma after 30 minutes; the concentration returns to an initial value after following 30 minutes, which may suggest that ET-1 released binds to its receptors in the acute phase of the vasospasm. On the other hand, in the two groups of rats with blocked ETA receptors there was a significant rise in ET-1 concentration 30 minutes after SAH or sham SAH, and a still further rise was observed 60 minutes after the procedure. The rise was significantly higher in animals with SAH (p < 0.05). The dynamics of the ET-1 concentration changes observed in rats with blocked ETA receptor suggests that SAH is an ET-1 production stimulator significantly more potent than other factors assessed in the study, such as a rise in the intracranial pressure resulting from administering aCSF to CM. Blocking ETA receptors makes it impossible for the ET-1 released to bind to the receptors, which may be a factor preventing the occurrence of cerebral vasospasm following SAH.

Topics: Animals; Coronary Vasospasm; Endothelin Receptor Antagonists; Endothelin-1; Intracranial Pressure; Male; Peptides, Cyclic; Rats; Rats, Wistar; Receptor, Endothelin A; Subarachnoid Hemorrhage

To investigate the role of endothelin-1 (ET-1) in development of ischemic brain damage after subarachnoid hemorrhage (SAH), and the protective effect of Ginkgo biloba extract (GBE).. Wistar rat noncraniotomy models of SAH were divided into SAH group and GBE treated group, the diameter of basilar artery (BA) and dynamic changes of regional cerebral blood flow (rCBF) and ET-1 content of intracranial plasma within 24 hours after SAH of both groups were determined. And pathological examination of CA1 region of hippocampus was performed 3 days later.. rCBF decreased and ET-1 content increased obviously and retained in 24 hours after SAH. Spasm of BA occurred half an hour after SAH and neurons of hippocampus CA1 region was damaged severely. GBE could antagonize the above-mentioned pathological changes effectively.. Increase of ET-1 is an important factor leading to ischemic brain damage after SAH. GBE exerts its protective effect by antagonizing pathological increase of ET-1.

Topics: Animals; Brain Ischemia; Endothelin-1; Female; Ginkgo biloba; Hippocampus; Male; Phytotherapy; Plants, Medicinal; Random Allocation; Rats; Rats, Wistar; Subarachnoid Hemorrhage

The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) has gained interest since the discovery of EDRF and of endothelin-1 (ET-1). The effect of SAH and both treatment of deferoxamine (DFO) and sympathectomy on endothelium-dependent vasodilation and ET-1-induced vasoconstriction of isolated rabbit carotid artery was examined using an isometric tension recording method. Thirty-five rabbits were divided into four groups: control animals, 7 days after SAH, treatment with DFO after SAH for 7 days and sympathectomy after SAH. Acetylcholine (10(-8) to 10(-5) M) was used to evoke concentration-dependent vasodilation of isolated arterial rings previously contracted by 10(-6) M phenylephrine. In the animals killed 7 days after SAH, acetylcholine-induced relaxation was suppressed and the degree of relaxation of this group was 50% of the initial contractile tone in response to the 10(-5) M acetylcholine. These relaxant responses did not return to control values in carotid arteries obtained from animals treated with DFO and subjected to sympathectomy. In isolated carotid arteries, ET-1 (10(-10) to 10(-8) M) produced concentration-dependent contractions. These contractile responses were significantly enhanced in animals 7 days after SAH compared with controls and did not return to control values in carotid arteries obtained from animals both treated with DFO and sympathectomized for 7 days after SAH. The present experiments suggest that impairment of endothelium-dependent vasodilation and the hyperreactivity of ET-1 of the carotid artery as well as cerebral arteries may be involved in the pathogenesis of cerebral vasospasm. Both treatment with DFO and sympathectomy during the chronic stage for vasospasm after SAH did not affect these vascular responses of the extradural part of the carotid artery to ET-1 and acetylcholine.

Topics: Acetylcholine; Animals; Carotid Artery Diseases; Carotid Artery, Common; Deferoxamine; Endothelin-1; Endothelium, Vascular; Female; Ischemic Attack, Transient; Male; Muscle Contraction; Muscle Relaxation; Rabbits; Siderophores; Subarachnoid Hemorrhage; Sympathectomy

Nitric oxide (NO) and endothelin-1 (ET-1) are two endothelium-derived factors probably involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). Our aim was twofold, i.e., to ascertain whether endothelial and nonendothelial NO modulates the contractile response of cerebral arteries to ET-1 and to analyze whether this relationship might be impaired after experimental SAH.. Rings of middle cerebral artery from goats in the control group and from goats with SAH were set up for isometric tension recordings. SAH was induced 3 days before the experiments by infusion of 10 ml of autologous arterial blood through a catheter previously inserted into the subarachnoid space (basal cistern). In goats in the control group, the response to ET-1 was obtained as follows: 1) in control arteries (unrubbed and nonincubated arteries); 2) in rubbed arteries (arteries in which the endothelium was mechanically removed); 3) during incubation with NG-nitro-L-arginine (L-NOArg) alone or plus L- or D-arginine; and 4) in rubbed arteries plus incubation with L-NOArg. In goats with SAH, that response was obtained in control arteries, rubbed arteries, and during incubation with L-NOArg. Specimens of middle cerebral artery were processed for transmission electron microscopy study.. In goats in the control group, ET-1 elicited concentration-dependent contraction of the middle cerebral artery that was significantly potentiated after endothelium denudation or during incubation with L-NOArg. The latter effect was reversed by L-arginine but not by D-arginine. Combined endothelium denudation and incubation with L-NOArg produced a contractile response to ET-1 significantly higher than that induced by each treatment separately. Hyperreactivity to ET-1 was observed in goats with SAH. Endothelium denudation did not alter the enhanced response to ET-1, but it was further significantly increased after incubation with L-NOArg.. These results demonstrate that an ET-1-NO interaction exists in control cerebral arteries in such a way that endothelial and nonendothelial NO partially counteract the contractile response to ET-1 and that although SAH did not modify the effect of nonendothelial NO, the absence of endothelial NO after SAH may contribute to the hyperreactivity of cerebral arteries to ET-1 and, thereby, to the development of cerebral vasospasm.

Topics: Animals; Cerebral Arteries; Endothelin-1; Endothelium, Vascular; Goats; Ischemic Attack, Transient; Microscopy, Electron; Nitric Oxide; Subarachnoid Hemorrhage; Vasoconstriction

Despite years of research, delayed cerebral vasospasm remains a serious complication of subarachnoid hemorrhage (SAH). Recently, it has been proposed that endothelin-1 (ET-1) mediates vasospasm. The authors examined this hypothesis in a series of experiments. In a primate model of SAH, serial ET-1 levels were measured in samples from the perivascular space by using a microdialysis technique and in cerebrospinal fluid (CSF) and plasma during the development and resolution of delayed vasospasm. To determine whether elevated ET-1 production was a direct cause of vasospasm or acted secondary to ischemia, the authors also measured ET-1 levels in plasma and CSF after transient cerebral ischemia. To elucidate the source of ET-1, they measured its production in cultures of endothelial cells and astrocytes exposed to oxyhemoglobin (10 microM), methemoglobin (10 microM), or hypoxia (11% oxygen). There was no correlation between the perivascular levels of ET-1 and the development of vasospasm or its resolution. Cerebrospinal fluid and plasma levels of ET-1 were not affected by vasospasm (CSF ET-1 levels were 9.3 +/- 2.2 pg/ml and ET-1 plasma levels were 1.2 +/- 0.6 pg/ml) before SAH and remained unchanged when vasospasm developed (7.1 +/- 1.7 pg/ml in CSF and 2.7 +/- 1.5 pg/ml in plasma). Transient cerebral ischemia evoked an increase of ET-1 levels in CSF (1 +/- 0.4 pg/ml at the occlusion vs. 3.1 +/- 0.6 pg/ml 4 hours after reperfusion; p < 0.05), which returned to normal (0.7 +/- 0.3 pg/ml) after 24 hours. Endothelial cells and astrocytes in culture showed inhibition of ET-1 production 6 hours after exposure to hemoglobins. Hypoxia inhibited ET-1 release by endothelial cells at 24 hours (6.4 +/- 0.8 pg/ml vs. 0.1 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05) and at 48 hours (6.4 +/- 0.6 pg/ml vs. 0 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05), but in astrocytes hypoxia induced an increase of ET-1 at 6 hours (1.5 +/- 0.6 vs. 6.4 +/- 1.1 pg/ml, control vs. hypoxic astrocytes; p < 0.05). Endothelin-1 is released from astrocytes, but not endothelial cells, during hypoxia and is released from the brain after transient ischemia. There is no relationship between ET-1 and vasospasm in vivo or between ET-1 and oxyhemoglobin, a putative agent of vasospasm, in vitro. The increase in ET-1 levels in CSF after SAH from a ruptured intracranial aneurysm appears to be the result of cerebral ischemia rather than reflecting the cause of cerebral

Topics: Animals; Cells, Cultured; Cerebrospinal Fluid; Disease Models, Animal; Endothelin-1; Macaca; Microdialysis; Subarachnoid Hemorrhage

Although phorbol esters, synthetic activators of protein kinase C (PKC), can stimulate large increases in the binding of cytosolic PKC to form membrane-bound PKC (PKCm, an indicator of PKC activation), the authors report that even small increases in PKCm induced by phorbol esters (8-12% of total PKC content) can be associated with significant PKC-mediated contractions in vitro (50-85% of maximum) in normal canine cerebral arteries. Increases in PKCm of similarly small magnitude were found in vitro when control artery segments were exposed to hemolysate, but only if the arterial smooth-muscle cells were first slightly depolarized by increased extracellular potassium to values of membrane potential similar to those observed in canine cerebral arteries during chronic cerebral vasospasm. These increases in PKCm (6-8% of total PKC content) coincided with a greatly augmented contractile response to hemolysate. These results show that the previous observation of only a small increase in PKCm (approximately 7% of total PKC content) after experimental subarachnoid hemorrhage in the canine model does not preclude a potentially important role for PKC-mediated contraction in the pathogenesis of cerebral vasospasm.

Topics: Animals; Cell Membrane; Cerebral Arteries; Chronic Disease; Cytosol; Dogs; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Hemolysis; Ischemic Attack, Transient; Membrane Potentials; Muscle, Smooth, Vascular; Naphthalenes; Phorbol 12,13-Dibutyrate; Phorbol Esters; Potassium; Protein Binding; Protein Kinase C; Stress, Mechanical; Subarachnoid Hemorrhage; Vasoconstriction; Vasoconstrictor Agents

The role of extracellular Ca2+ in the maintenance of chronic vasospasm after subarachnoid hemorrhage (SAH) is largely unknown. Indeed, studies thus far have been limited to demonstrations that L-type Ca(2+)-channel antagonists were unable to reverse the spasm. This study tested whether SAH-induced vasospasm is maintained, at least in part. through the influx of extracellular Ca2+ and whether the influx of extracellular Ca2+ occurs through L-type Ca2+ channels and possibly, in addition, through store operated channels (SOCs). Furthermore, as there is considerable evidence in the literature to suggest that the spasm is mediated through endothelin-1 (ET-1) release, we tested whether the Ca2+ dependency of the spasm was consistent with the mediation of the spasm by ET-1.. Chronic spasm of the basilar artery was induced in a double SAH rabbit model. Relaxation of SAH-, ET-1-, serotonin-, and KC1-constricted basilar artery in response to Ca(2+)-free solution, verapamil, and Ni2+ was measured in situ with the use of a cranial window.. SAH induced 23% constriction of the basilar artery. Ca(2+)-free solution and 1 mumol/L verapamil reversed the constriction of SAH vessels by 60% and 17%, respectively. In contrast, control vessels challenged with 40 to 50 mmol/L KCl, which induced 34% constriction, relaxed in response to Ca(2+)-free solution and verapamil by 98% and 89%, respectively. In SAH vessels, verapamil followed by 0.1 mmol/L Ni2+, which is known to block SOCs, induced a combined relaxation of 67%. Control vessels challenged with 3 nmol/L ET-1, which induced a magnitude of constriction similar to that of SAH (29%), relaxed in response to Ca(2+)-free solution, verapamil, and verapamil plus Ni2+ by 69%, 20%, and 50%, respectively (P > .05) versus respective values in SAH vessels). In contrast, control vessels challenged with 2 to 8 mumol/L serotonin, which induced a magnitude of constriction similar to those of SAH and ET-1 (22%), completely relaxed in response to Ca(2+)-free solution and verapamil.. These results demonstrate that the maintenance of chronic spasm in the two-hemorrhage rabbit model after SAH is due to smooth muscle cell contractile mechanisms partly dependent on the influx of extracellular Ca2+. The influx of extracellular Ca2+ results from the opening of L-type Ca2+ channels and an additional channel or channels. We speculate that the L-type Ca2+ channel-independent influx of extracellular Ca2+ results from the opening of SOCs. The Ca(2+)-dependent characteristics of the spasm likely reflect the mediation of the spasm by ET-1.

Topics: Animals; Basilar Artery; Calcium; Endothelin-1; Extracellular Space; Male; Nickel; Potassium Chloride; Rabbits; Serotonin; Solutions; Subarachnoid Hemorrhage; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents; Verapamil

The potent vasoconstrictor peptide, endothelin-1 (ET-1), has been implicated in the pathophysiology of cerebral vasospasm that occurs after subarachnoid hemorrhage (SAH). This peptide is synthesized as a large prepropeptide that requires a series of modifying steps for its activation. The last of these steps involves the proteolytic conversion of a relatively inactive propeptide, Big ET-1, to its active, 21-amino acid peptide form. The enzyme responsible for converting Big ET-1 to ET-1 is a metalloprotease called endothelin-converting enzyme (ECE). In the present study the authors examined the effects of a newly developed inhibitor of ECE on responses to ET peptides in the normal basilar artery and on pathophysiological constriction in the spastic basilar artery after SAH. In the first series of experiments the authors examined normal basilar arteries in the rabbit, which were exposed transclivally and measured on-line using videomicroscopy. Intravenous administration or topical application of an active inhibitor of ECE, CGS 26303, blocked vasoconstrictor responses to topically applied Big ET-1 but not to ET-1. In contrast, topical application of a structurally related compound that does not inhibit ECE, CGS 24592, was ineffective in blocking vasoconstriction that was elicited by a topical application of Big ET-1. These findings indicate that CGS 26303 when administered systemically is capable of blocking the conversion of Big ET-1 to ET-1 in the basilar artery without affecting the ability of the vessel to respond to ET-1. In the second series of experiments the authors examined the effects of the ECE inhibitor on cerebral vasospasm after experimental SAH. Intraperitoneal administration of CGS 26303 via osmotic minipumps significantly attenuated the delayed spastic response of the basilar artery to an intracisternal injection of autologous blood. This study provides the first evidence that systemic administration of an inhibitor of ECE is capable of preventing cerebral vasospasm after SAH. The results reinforce a growing body of evidence that ETs play a critical role in the development of spastic constriction after SAH. Moreover, the findings indicate that blocking the conversion of Big ET-1 to its active ET-1 form using CGS 26303 may represent a feasible strategy for ameliorating cerebral vasospasm.

Topics: Animals; Disease Models, Animal; Endothelin-1; Endothelins; Ischemic Attack, Transient; Organophosphonates; Protease Inhibitors; Protein Precursors; Rabbits; Subarachnoid Hemorrhage; Tetrazoles; Time Factors

We investigated the changes in endothelial function of pulmonary as well as basilar artery after experimental subarachnoid hemorrhage (SAH) induced by cisternal blood injection in rabbits. The animals were killed on day 2 and day 7, and the mechanical responses were determined on transverse strips of the main pulmonary artery and rings harvested from the basilar artery. To examine the role of the vagal nerve, we cut the left vagal nerve immediately before injecting cisternal blood. Relaxation response of pulmonary and basilar arteries to acetylcholine (ACh) and A23187 were abolished after endothelial removal and reality inhibited by either NG-nitro-L-arginine (NOARG) or methylene blue (MB). Indomethacin failed to modify these relaxation responses. Relaxation response of the main pulmonary artery strips to ACh was significantly (p < 0.05 and p < 0.01) attenuated on day 2 in 6 of 12 rabbits. In the remaining 6 rabbits, relaxation was not affected. No change in relaxation responses to A23187 and sodium nitroprusside (SNP) was observed in any of the 12 cases. In the vagotomized rabbits no decreased response to ACh was observed. On day 7, relaxation response to ACh returned to normal. ACh also produced decreased relaxation in basilar artery rings on day 2 with no change in A23187- and SNP-induced relaxation. Contractile responses of pulmonary and basilar arteries to norepinephrine (NE), endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and U46619 were not affected after SAH. These results suggest that less functional muscarinic receptors that produce/release less endothelium-derived relaxation factor [EDRF/nitric oxide (NO)] are involved in causing the reduced relaxation of pulmonary and basilar arteries after SAH and the vagal nerve may play a role in regulating the receptor-mediated, endothelium-dependent relaxation in the main pulmonary artery after experimental SAH.

Topics: Acetylcholine; Animals; Basilar Artery; Calcimycin; Endothelin-1; Endothelium, Vascular; Male; Nitroprusside; Pulmonary Artery; Rabbits; Subarachnoid Hemorrhage; Vagotomy; Vasodilation

Topics: Acoustic Stimulation; Adenosine Triphosphate; Animals; Basilar Artery; Brain; Cerebrovascular Circulation; Endothelin-1; Epilepsy; Muscle Contraction; Muscle Relaxation; Rats; Rats, Inbred Strains; Rats, Wistar; Seizures; Species Specificity; Subarachnoid Hemorrhage

Plasma big endothelin (BE) levels were studied in 17 patients during the first 2 weeks following spontaneous intracerebral hemorrhage (ICH). Three patients had experienced an infratentorial hemorrhage of the pons and cerebellum. The group of 14 patients with supratentorial hemorrhage presented with 8 hematomas of the putamen, 4 of the capsula interna and 2 in the thalamus. Significant elevations of BE during the first 2 weeks after the initial ICH, significant differences between the different localisations and between patients with different prognoses were not apparent (mean BE level: 2.95 +/- 1.39, control group: 2.68 +/- 1.29 fmol/ml). These results support the theory that endothelins do not play a significant pathophysiological role in acute severe ICH.

Topics: Adult; Aged; Cerebral Hemorrhage; Cerebral Ventricles; Endothelin-1; Endothelins; Female; Glasgow Coma Scale; Humans; Hypertension; Male; Middle Aged; Protein Precursors; Radioimmunoassay; Subarachnoid Hemorrhage

Endothelin peptides are potent vasoconstrictors and thus are seen as potential cause of cerebral vasospasm after subarachnoid hemorrhage (SAH). Earlier reports showed elevated or normal endothelin levels in plasma and cerebrospinal fluid in patients suffering from SAH. The present study was designed to determine whether endothelin is a causal factor in SAH.. We studied 11 patients with acute SAH. Seven of these 11 patients had a proven aneurysm and six had experienced vasospasm. Big endothelin levels were determined by a radioimmunoassay recognizing the C-terminal peptide (normal range, 1-11 fmol/ml).. There were no elevations of big endothelin in the 59 plasma samples and the 17 simultaneously estimated cerebrospinal fluid samples. Differences between plasma and cerebrospinal fluid did not reach significant levels. Big endothelin values between patients with and without vasospasm showed no significant differences.. Our findings suggest that plasma elevation of the endothelins is not reproducible in SAH and that big endothelin is unlikely to be a causal plasma factor in the complex multifactorial development of vasospasm after SAH.

Topics: Adult; Aged; Aneurysm; Cerebral Arteries; Constriction, Pathologic; Endothelin-1; Endothelins; Female; Humans; Male; Middle Aged; Protein Precursors; Subarachnoid Hemorrhage

Since the discovery of endothelin-1 (ET-1), its involvement in cerebral vasospasm after subarachnoid hemorrhage (SAH) has been suspected. We performed various experiments, first to demonstrate the presence of ET in both patients and dogs with SAH, and second to examine the effects of ET synthesis inhibition in experimental vasospasm. Here we report that ET was present in both plasma and cerebrospinal fluid (CSF) in SAH, but did not correlate with vasospasm. However, ET was locally expressed in the vascular endothelium in vasospasm. Several therapeutic approaches causing the inhibition of ET synthesis were effective in preventing the development of vasospasm. Such approaches utilized drugs that inhibited RNA and DNA synthesis. Among them, actinomycin D treatment was most effective. We also utilized phosphoramidon, a recently found conversion inhibitor of big ET to ET. However, this product failed to ameliorate the development of vasospasm. Therefore, although we cannot yet conclude that ET is the main cause of cerebral vasospasm, it may, at least, act as one of the modifying factors in cerebral vasospasm.

Topics: Animals; Antineoplastic Agents; Basilar Artery; DNA; Dogs; Endothelin-1; Endothelins; Glycopeptides; Immunohistochemistry; In Vitro Techniques; Ischemic Attack, Transient; Protein Precursors; RNA, Messenger; Subarachnoid Hemorrhage; Transcription, Genetic

Intra-arterial injection of endothelin-1 (ET-1) (1 nmol/kg; n = 8) or human big endothelin-1 (b-ET-1; 3 nmol/kg; n = 8) into anesthetized rabbits produced a significant rise in left ventricular systolic pressure (LVSP) and caused subarachnoid hemorrhage (SAH) in 75 +/- 17% and 88 +/- 12% of the experiments, respectively. In all animals, the SAH occurred in the subarachnoid space around the distal part of the basilar artery complex. The cyclooxygenase inhibitor indomethacin (5 mg/kg i.v.) significantly potentiated the pressor effect of both peptides, and all animals pretreated with indomethacin prior to ET-1 (n = 3) or b-ET (n = 3) developed SAH. In contrast, rabbits treated with vehicle (saline; n = 7), indomethacin alone (n = 3), or the carboxy-terminal fragment of b-ET (CT 22-38; 3 nmol/kg i.a.; n = 3) developed neither a rise in LVSP nor SAH. A rise in blood pressure alone is unlikely to account for the SAH brought about by the peptides for angiotensin II (1 nmol/kg/min for 30 min; n = 7) produced a significantly greater increment in LVSP than ET-1 or b-ET, but did not cause SAH. In addition, there was no significant correlation between the rise in LVSP produced by ET-1 or b-ET and the severity of the SAH.

Topics: Anesthesia; Animals; Blood Pressure; Endothelin-1; Endothelins; Injections, Intra-Arterial; Male; Protein Precursors; Rabbits; Subarachnoid Hemorrhage