Insights into cytoprotection from ground squirrel hibernation, a natural model of tolerance to profound brain oligaemia.

Progression of acute ischaemic brain damage is complex and multifactorial. Also, evidence suggests that participating molecules and signal transduction pathways can function differently in different cellular contexts. Hibernation torpor, a model of natural tolerance to profoundly reduced blood flow and oxygen delivery to brain, along with models of induced ischaemic tolerance can guide efforts to identify cytoprotective mechanisms that are multifactorial and that target multiple mechanisms in multiple cellular contexts. Post-translational modification of proteins by conjugation with the SUMO (small ubiquitin-related modifier) is massively increased in hibernation and may be such a mechanism.

Elevated pro-inflammatory CD4+CD28- lymphocytes and stroke recurrence and death.

OBJECTIVE: To determine if the CD4+CD28- T-cell subset is expanded in patients with recurrent stroke or death after acute ischemic stroke. This subset of the peripheral blood T-cell lymphocyte population has a strong pro-inflammatory and tissue-damaging potential. METHODS: Consecutive patients within the first 48 hours of ischemic stroke were prospectively studied. Peripheral blood CD4+CD28- cells were quantified by flow cytometry. The study endpoint was recurrent stroke or death from any cause during 1 year of follow-up. RESULTS: One hundred six patients (mean age 75.0 +/- 13.5 years; 50 women) were studied. The median CD4+CD28- cell count was 4.5% (range 0.2 to 72.2%). Twenty-seven endpoints (10 recurrent strokes and 17 deaths) occurred during follow-up. Stroke recurrence/death rates were significantly associated with increasing CD4+CD28- counts, rising from 14.2% in patients with CD4+CD28- levels of <1.0 to 48.1% for those with CD4+CD28- counts of >8.0% (p = 0.003, Cochran linear test of trend). Higher CD4+CD28- counts were also present in patients with a history of prior stroke (p = 0.03). After adjustment for age, admission NIH Stroke Scale score, prior stroke, and atrial fibrillation, CD4+CD28- counts of >8.0% were associated with a cumulative hazard ratio of 5.81 (95% CI: 1.58 to 21.32) for stroke recurrence or death. CONCLUSIONS: Rising counts of circulating CD4+CD28- cells are associated with an increasing risk of stroke recurrence and death, in addition to an observed association with prior stroke. Expansion of this T-cell subset presumably represents a biomarker and possibly a contributory pathogenic mechanism of recurrent stroke and death after ischemic stroke.

ICP monitoring following bilateral carotid occlusion in GFAP-null mice.

To investigate the possible role of glial fibrillary acidic protein (GFAP) in cerebral ischemia, we have monitored the intracranial pressure (ICP) and local cerebral blood flow (ICBF) following bilateral carotid artery occlusion (BCAO) in GFAP-null mice and their wild type littermates. GFAP-null mice (B6, 129-GfaptmlMes) were obtained from Jackson Laboratories. The ICP and ICBF was continuously monitored during 15 minutes BCAO and reperfusion. The variation of the circle of Willis was also investigated in both GFAP-null and wild type mice. The breakdown of blood brain barrier (BBB) was assessed by immunohistochemical staining against mouse immunogloblins (IgG). A significantly more profound and immediate decrease in ICBF after BCAO was observed in GFAP-null mice (p < 0.04, ANOVA). GFAP-null mice also showed a significant increase (% change) in ICP after reperfusion (p < 0.05, ANOVA). There were no gross differences in the circle of Willis between GFAP-null and wild type mice. No abnormal IgG immuno-reactivity was observed in the forebrain of both animals. These results indicate a high susceptibility to cerebral ischemia in GFAP-null mice and suggest an important role for astrocytes and GFAP in the progress of ischemic brain damage and increased ICP after cerebral ischemia with reperfusion.

Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

A new method to catheterize a femoral artery in mice using a nylon suture as a 'guide wire'.

Mice are commonly used in laboratory experiments. Their femoral arteries are so tiny that catheterization is quite difficult. We describe a new method to catheterize the femoral artery in mice. The key feature of this new method is the use of a nylon suture as a 'guide wire'. The full catheterization system consists of two sizes of polyethylene tubes (PE-10, PE-50) and a 4-0 nylon suture. We have been able to repeatedly catheterize mouse femoral arteries (n = 57) successfully and easily with this new system. We believe that this new method can facilitate vascular catheterization in small animals such as mice.

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.

Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels.

Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.

Proinflammatory cytokine expression contributes to brain injury provoked by chronic monocyte activation.

BACKGROUND: We have proposed that an increased interaction between monocyte/macrophages and blood vessel endothelium predisposes subjects to strokes. The effect of chronic monocyte activation on the development of cerebral infarcts was thus studied in rats after provocation of a modified local Swartzman reaction, in brain vasculature. MATERIALS AND METHODS: Two weeks after an IV bolus of bacillus Calmette-Guérin (BCG), we studied spontaneous superoxide production, integrin expression, endothelial adhesion of monocytes and the neurological symptoms, brain histology, and cytokine immunoreactivity after a provocative dose of LPS (30-300 microg/rat i.c.v.). RESULTS: Monocyte migration into the brain was stimulated by BCG priming. The incidence of paralysis and death in response to LPS was markedly increased in BCG-primed rats. Histological evaluation of the brains of neurologically impaired and moribund animals revealed intravascular thrombosis and pale and hemorrhagic infarcts. Infiltrates of leukocytes expressing immunoreactive IL-1:, IL-6, and TNF-alpha were found around blood vessels, cerebral ventricles, and meninges, and were accompanied by a profound microglial expression of IL1P, endothelial expression of IL-6, and expression of TNF-alpha and TNF-R 1 in glia and neurons of cortex and hippocampus. Treatment (2 x 100 microg/10 ,I, i.c.v.) with recombinant human (rh-)TNF 55kDa receptor completely prevented, and treatment with rh-IL- I receptor antagonist significantly decreased the incidence of paralysis and death in response to BCG + LPS. The improvement of neurological symptoms was accompanied by reduced histological damage and supppression of IL-1P/ expression in the brain tissue. CONCLUSIONS: The data demonstrate that chronic monocyte activation predisposes subjects to thrombosis and hemorrhage via an exaggerated release of proinflammatory cytokines.

Cell permeable exogenous ceramide reduces infarct size in spontaneously hypertensive rats supporting in vitro studies that have implicated ceramide in induction of tolerance to ischemia.

Previous work in primary cell culture has shown that TNF-alpha and ceramide are involved in the signaling that induces tolerance to brain ischemia (Ginis et al., 1999; Liu et al., 2000). To validate the in vitro studies, the authors administered cell permeable analogs of ceramides intracisternally or intravenously to examine their effect on neuroprotection after focal cerebral ischemia. Permanent middle cerebral artery occlusion (MCAO) was performed in spontaneously hypertensive rats. Infarct volumes were assessed at 24 hours after surgery. D-erythro-N-acetylsphingosine (C2-ceramide) or its vehicle was infused intracisternally for 1 hour before MCAO. In a second set of studies, D-erythro-N-octanoylsphingosine (C8-ceramide) or its vehicle was injected intravenously 48 or 24 hours before MCAO to mimic preconditioning (PC) and was also injected 5 minutes after MCAO. C2-ceramide infusion significantly reduced infarct volumes by approximately 14% (P < 0.05). C8-ceramide injection reduced infarct volumes approximately 17% compared with controls. This effect was constant and significant compared with controls over the time periods examined (P < 0.01). This work supports findings in primary brain cell cultures that implicate ceramide as a downstream signal that is proximate to development of tolerance to brain ischemia. Because the degree of protection represents approximately 50% of the maximal infarct reduction observed in this model, there are probably additional signaling pathways that subserve tolerance.

Brain vessels normally undergo cyclic activation and inactivation: evidence from tumor necrosis factor-alpha, heme oxygenase-1, and manganese superoxide dismutase immunostaining of vessels and perivascular brain cells.

Studies of vascular biology during the past decade have identified an expanding list of agonists and antagonists that regulate local hemostasis, inflammation, and reactivity in blood vessels. Interactions at the blood-endothelial interface are intricate and complex and have been postulated to play a role in the initiation of stroke and the progression of brain injury during early hours of ischemia, particularly in conjunction with reperfusion injury (Hallenbeck, 1996). In the current study of normal and activated vessels in rat brain, immunoreactive tumor necrosis factor-alpha (TNF-alpha), heme oxygenase-1 (HO-1), and manganese superoxide dismutase (MnSOD) exhibit concentric perivascular rings involving vessel wall and surrounding parenchyma that appear to coincide with one another in serial sections. The ring patterns suggest periodic radial expansion of these molecules released through a process of cyclic activation and inactivation of brain vessel segments. In this process, the rings appear randomly scattered instead of affecting all vessels within a high power field (HPF) synchronously. The average number of vessels per HPF (mean +/- SD) with perivascular cuffs of immunoreactive MnSOD increased from 51 +/- 28 in Wistar, 72 +/- 46 in Wistar-Kyoto, and 84 +/- 30 in Sprague Dawley rats (no spontaneous strokes) to 184 +/- 72 in spontaneously hypertensive stroke-prone rats (spontaneous strokes). Perivascular immunoreactive cuffs are also increased in spontaneously hypertensive rats by induction of cytokine expression by lipopolysaccharide (64 +/- 15 vs. 131 +/- 32 /HPF). The patterns of TNF-alpha, HO-1, and MnSOD in naïve animals are interpreted to indicate that focal hemostatic balance normally fluctuates in brain vessels and influences surrounding parenchymal cells. Perivascular immunoreactive cuffs representing this process are more frequent in animals with lipopolysaccharide-induced endothelial activation or genetic stroke proneness.

Lipopolysaccharide-induced ischemic tolerance is associated with increased levels of ceramide in brain and in plasma.

Intravenous administration of lipopolysaccharide (LPS) (0.9 mg/kg) has been shown to induce ischemic tolerance in spontaneously hypertensive rats (SHR). TNF-alpha is believed to play a crucial role in preconditioning as its inhibition with TNF-alpha-binding protein abolished tolerance. Our recent studies (Liu et al., Am. J. Physiol. 278 C144, 2000) have demonstrated that ceramide, a downstream messenger in TNF-alpha signaling, is a mediator of hypoxia-induced tolerance in neuronal cells. To test the hypothesis that ceramide contributes to LPS-induced tolerance in vivo, SHR were injected intravenously with either LPS or saline and the levels of ceramide in brain and in plasma were determined by reversed phase HPLC. LPS injection resulted in a significant increase of ceramide in plasma with a maximum at 24 h (8.32+/-1.14 pmol/microl (LPS) vs. 2.65+/-0.62 pmol/microl (saline)). LPS also induced ceramide upregulation in brain cortex, which started between 6 and 12 h and remained elevated up to 48 h after LPS injection. Fluorescent NBD-C6 ceramide was able to cross blood-brain barrier and was found in brain vessels, perivascular cells and in brain parenchyma 30 min after intravenous injection. These findings demonstrate that LPS preconditioning leads to elevation of ceramide in brain and plasma and, in conjunction with previous work, suggests that ceramide plays a role in LPS-induced protection against brain ischemic injury in vivo.

The protective effect of ceramide in immature rat brain hypoxia-ischemia involves up-regulation of bcl-2 and reduction of TUNEL-positive cells.

Preconditioning brain with tumor necrosis factor alpha (TNF-alpha) can induce tolerance to experimental hypoxia and stroke and ceramide is a downstream messenger in the TNF-alpha signaling pathway. A hypoxic-ischemic (HI) insult in the immature rat injures brain primarily through apoptosis. Apoptosis is regulated by Bcl-2 family proteins. The authors explored whether ceramide protects against HI in the immature rat, and whether Bcl-2 family protein expression is involved. Hypoxia-ischemia was produced in seven-day-old rats by ligating the right carotid artery, followed by 2 hours of 8% oxygen exposure. Thirty minutes after HI, C2-ceramide (150 microg/kg) was injected intraventricularly. Infarct volume was measured 5 days later. C2-ceramide reduced HI-induced brain damage by 45% to 65% compared with HI/dimethyl sulfoxide (DMSO) (vehicle control) or HI only groups. In separate experiments, brains of sham-operated control and HI only animals and animals subjected to HI plus C2-ceramide or DMSO infusion were sampled 6 hours, 24 hours, and 5 days after treatments and analyzed for Bcl-2, Bcl-xl, and Bax expression (Western blotting), and apoptosis (TUNEL assay). Augmented Bcl-2 and Bcl-xl levels in the C2-ceramide treated group were associated with a significant decrease in TUNEL-positive cells. The results support a protective role for ceramide in neonatal HI.

High susceptibility to cerebral ischemia in GFAP-null mice.

Astrocytes perform a variety of functions in the adult central nervous system (CNS) that contribute to the survival of neurons. Thus, it is likely that the activities of astrocytes affect the extent of brain damage after ischemic stroke. The authors tested this hypothesis by using a mouse ischemia model to compare the infarct volume produced in wild-type mice with that produced in mice lacking glial fibrillary acidic protein (GFAP), an astrocyte specific intermediate filament component. Astrocytes lacking GFAP have been shown to have defects in process formation, induction of the blood-brain barrier. and volume regulation; therefore, they might be compromised in their ability to protect the CNS after injury. The authors reported here that 48 hours after combined permanent middle cerebral artery occlusion (MCAO) and 15 minutes transient carotid artery occlusion (CAO) GFAP-null mice had a significantly (P < 0.001) larger cortical infarct volume (16.7 +/- 2.2 mm3) than their wild-type littermates (10.1 +/- 3.9 mm3). Laser-Doppler flowmetry revealed that the GFAP-null mice had a more extensive and profound decrease in cortical cerebral blood flow within 2 minutes after MCAO with CAO. These results indicated a high susceptibility to cerebral ischemia in GFAP-null mice and suggested an important role for astrocytes and GFAP in the progress of ischemic brain damage after focal cerebral ischemia with partial reperfusion.

Advances in the vascular pathophysiology of ischemic stroke.

The cerebral vascular supply is constructed to protect the cerebral hemispheres and brainstem from the consequences of blood flow cessation. Reversal of blood flow around local obstructions is a feature of the microvascular beds of the striatum and cerebral cortex. Cerebral capillaries of these beds consist of endothelial cells, basal lamina, and astrocyte end-feet that sit in close apposition. The interaction of astrocytes with neurons indicates the close relationship of microvessels to neurons. These relationships are altered when blood flow ceases in the supplying artery. Increased endothelial cell permeability and endocytoses lead to edema formation, and matrix degradation is associated with hemorrhage. Autoregulation is lost. Ischemia initiates leukocyte adhesion receptor expression, which is promoted by cytokine generation from the neuropil and activated monocytes. "Preactivation" may further augment the inflammatory responses to ischemia. The activation of cerebral microvessels by ischemia is heterogeneous, involving alterations in integrin-matrix interactions, leukocyte-endothelial cell adhesion, permeability changes, and the "no-reflow" phenomenon due to platelet activation, fibrin formation, and leukocyte adhesion. Ischemia produces swelling of the microvascular endothelium, and rapid detachment and swelling of the astrocyte end-feet. Ischemic injury targets the microvasculature, where the inflammatory responses are initiated and contribute to tissue injury.

Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia.

Ischemic stroke is a leading cause of death and disability in developed countries. Yet, in spite of substantial research and development efforts, no specific therapy for stroke is available. Several mechanism for neuroprotection have been explored including ion channels, excitatory amino acids and oxygen radicals yet none has culminated in an effective therapeutic effect. The review article on "inflammation and stroke" summarizes key data in support for the possibility that inflammatory cells and mediators are important contributing and confounding factors in ischemic brain injury. In particular, the role of cytokines, endothelial cells and leukocyte adhesion molecules, nitric oxide and cyclooxygenase (COX-2) products are discussed. Furthermore, the potential role for certain cytokines in modulation of brain vulnerability to ischemia is also reviewed. The data suggest that novel therapeutic strategies may evolve from detailed research on some specific inflammatory factors that act in spatial and temporal relationships with traditionally recognized neurotoxic factors. The dual nature of some mediators in reformatting of brain cells for resistance or sensitivity to injury demonstrate the delicate balance needed in interventions based on anti-inflammatory strategies.

Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide.

Brief "preconditioning" ischemia induces ischemic tolerance (IT) and protects the animal brain from subsequent otherwise lethal ischemia. Identification of the signaling steps most proximal to the development of the IT will allow induction of the resistance to ischemia shortly after the onset of stroke. Animal studies demonstrate a key role of tumor necrosis factor-alpha (TNF-alpha) in induction of IT. The sphingolipid ceramide is known as a second messenger in many of the multiple effects of TNF-alpha. We hypothesized that ceramide could mediate IT. We demonstrate that preconditioning of rat cortical neurons with mild hypoxia protects them from hypoxia and O(2)-glucose deprivation injury 24 h later (50% protection). TNF-alpha pretreatment could be substituted for hypoxic preconditioning (HP). HP was attenuated by TNF-alpha-neutralizing antibody. HP and TNF-alpha pretreatment cause a two- to threefold increase of intracellular ceramide levels, which coincides with the state of tolerance. Fumonisin B(1), an inhibitor of ceramide synthase, attenuated ceramide upregulation and HP. C-2 ceramide added to the cultures right before the hypoxic insult mimicked the effect of HP. Ceramide did not induce apoptosis. These results suggest that HP is mediated via ceramide synthesis triggered by TNF-alpha.

Tumor necrosis factor and reactive oxygen species cooperative cytotoxicity is mediated via inhibition of NF-kappaB.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death. MATERIALS AND METHODS: Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies. RESULTS: TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus. CONCLUSIONS: Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.

Endothelin receptor antagonist preserves microvascular perfusion and reduces ischemic brain damage following permanent focal ischemia.

Synthesis and release of the potent vasoconstrictor peptide endothelin-1 (ET-1) increases following cerebral ischemia and has previously been shown to mediate the delayed hypoperfusion associated with transient global ischemia. In this study we assessed the impact of ET-1 on perfusion and infarct volume in a focal model of cerebral ischemia by use of the selective ET(A) receptor antagonist Ro 61-1790 (affinity for ET(A) receptor 1000 fold greater than ETB receptor). Control rats subjected to permanent middle cerebral artery occlusion (MCAO) showed extensive reductions in microvascular perfusion 4 h post-MCAO that were significantly attenuated by Ro 61-1790 pretreatment (10 mg/kg, i.v.). Ro 61-1790 concomitantly and significantly reduced the ischemic lesion volume in the same animals. This effect was maintained 24 h post-MCAO providing that the animals received additional i.v. injections of 5 mg/kg Ro 61-1790 at 5 h and 8 h after MCAO. These findings demonstrate that ET(A) receptor antagonism partially preserves tissue perfusion following focal ischemia and that this effect is associated with significant neuroprotection. The results also support the hypothesis that vasoactive mediators, and ET-1 in particular, are important contributors to the pathogenesis of cerebral ischemic injury.