endothelin-1 and Brain-Ischemia

Ischemic stroke is one of the leading causes of morbidity and mortality globally. Hundreds of clinical trials have proven ineffective in bringing forth a definitive and effective treatment for ischemic stroke, except a myopic class of thrombolytic drugs. That, too, has little to do with treating long-term post-stroke disabilities. These studies proposed diverse options to treat stroke, ranging from neurotropic interpolation to venting antioxidant activity, from blocking specific receptors to obstructing functional capacity of ion channels, and more recently the utilization of neuroprotective substances. However, state of the art knowledge suggests that more pragmatic focus in finding effective therapeutic remedy for stroke might be targeting intricate intracellular signaling pathways of the 'neuroinflammatory triangle': ROS burst, inflammatory cytokines, and BBB disruption. Experimental evidence reviewed here supports the notion that allowing neuroprotective mechanisms to advance, while limiting neuroinflammatory cascades, will help confine post-stroke damage and disabilities.

Topics: Aldehydes; Blood-Brain Barrier; Brain Ischemia; Cytokines; Drug Discovery; Endothelin-1; Gene Expression Regulation; Humans; Malondialdehyde; Microglia; Molecular Targeted Therapy; Neuroinflammatory Diseases; Neuroprotective Agents; Nitric Oxide; Oxidation-Reduction; Reactive Oxygen Species; Receptors, Cytokine

In brain disorders, reactive astrocytes, which are characterized by hypertrophy of the cell body and proliferative properties, are commonly observed. As reactive astrocytes are involved in the pathogenesis of several brain disorders, the control of astrocytic function has been proposed as a therapeutic strategy, and target molecules to effectively control astrocytic functions have been investigated. The production of brain endothelin-1 (ET-1), which increases in brain disorders, is involved in the pathophysiological response of the nervous system. Endothelin B (ET

Topics: Alzheimer Disease; Animals; Astrocytes; Brain Diseases; Brain Injuries; Brain Ischemia; Endothelin-1; Humans; Neuralgia; Receptor, Endothelin B

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

Endothelin (ET)-1 is a potent vasoconstrictor peptide produced by endothelial cells and associated with vascular dysfunction and cardiovascular disease. Lys198Asn is a single-nucleotide polymorphism (SNP) of gene encoding ET-1 (EDN1). It is hypothesized that it might have a role in altering ET-1 and ultimately leading to vascular dysfunction and ischemic stroke. We therefore conducted a meta-analysis to investigate the association between Lys198Asn polymorphism of EDN1 gene and susceptibility of ischemic stroke.. This meta-analysis was conducted according to the guidance of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. We searched PubMed, Google Scholar, Embase, Web of Science, J-STAGE, and China National Knowledge Infrastructure (CNKI) for relevant studies. The association between Lys198Asn polymorphism and ischemic stroke susceptibility was evaluated by calculating the pooled ORs and 95% CIs.. Our analysis included 1,291 cases and 2,513 controls. Meta-analysis established a significant association between Lys198Asn SNP of EDN1 gene and ischemic stroke when assuming either recessive model (OR: 1.30; 95% CI: 1.02-1.65; p = .03; I. The present meta-analysis suggests that Lys198Asn polymorphism of EDN1 gene is associated with an increased risk for ischemic stroke.

Topics: Brain Ischemia; Endothelin-1; Genetic Predisposition to Disease; Humans; Polymorphism, Single Nucleotide; Stroke

The exact pathogenesis of multiple sclerosis (MS) is incompletely understood. Although auto-immune responses have an important role in the development of hallmark focal demyelinating lesions, the underlying mechanism of axonal degeneration, the other key player in MS pathology and main determinant of long-term disability, remains unclear and corresponds poorly with inflammatory disease activity. Perfusion-weighted imaging studies have demonstrated that there is a widespread cerebral hypoperfusion in patients with MS, which is present from the early beginning to more advanced disease stages. This reduced cerebral blood flow (CBF) does not seems to be secondary to loss of axonal integrity with decreased metabolic demands but appears to be mediated by elevated levels of the potent vasospastic peptide endothelin-1 in the cerebral circulation. Evidence is evolving that cerebral hypoperfusion in MS is associated with chronic hypoxia, focal lesion formation, diffuse axonal degeneration, cognitive dysfunction, and fatigue. Restoring CBF may therefore emerge as a new therapeutic target in MS.

Topics: Blood Flow Velocity; Brain Ischemia; Cerebrovascular Circulation; Cognition Disorders; Diffuse Axonal Injury; Endothelin-1; Humans; Inflammation; Multiple Sclerosis

Ischemic stroke causes vascular paralysis and impaired autoregulation in the brain, the degree of which is dependent on the depth and duration of ischemia and reperfusion (I/R). Ischemic stroke also impairs the myogenic response of middle cerebral arteries (MCA) that may be an underlying mechanism by which autoregulation is impaired. Myogenic responses are affected by I/R through several mechanisms, including production of peroxynitrite, depolymerization of F-actin in vascular smooth muscle, and circulating vasoactive factors. The vascular endothelium is also significantly affected during focal ischemia that has a particularly large influence on vascular tone in the cerebral circulation. Endothelial nitric oxide (NO) and endothelin-1 (ET-1) are important endothelium-dependent vasoactive substances that can influence the level of myogenic tone in cerebral arteries and arterioles that are significantly affected during ischemic stroke. Unlike MCA, brain penetrating arterioles have considerable myogenic tone that appears less affected by focal ischemia. The persistent tone of brain parenchymal arterioles during focal ischemia could contribute to perfusion deficit and infarct expansion. These arterioles within the cerebral cortex are also unique from MCA in that they constrict to small- and intermediate- conductance calcium-activated potassium channel (SKCa and IKCa, respectively) inhibition, suggesting basal endothelium-derived hyperpolarizing factor (EDHF) is preserved during focal ischemia. This review will highlight our current understanding of the effects of I/R on myogenic response in both MCA and parenchymal arterioles and discuss underlying mechanisms by which focal ischemia affects myogenic tone in these different vascular segments.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Drug Delivery Systems; Endothelin-1; Homeostasis; Humans; Muscle, Smooth, Vascular; Nitric Oxide; Stroke; Vasodilator Agents

Alzheimer's disease (AD) and vascular dementia are the major causes of cognitive disorders worldwide. They are characterized by cognitive impairments along with neuropsychiatric symptoms, and that their pathogeneses show overlapping multifactorial mechanisms. Although AD has long been considered the most common cause of dementia, individuals afflicted with AD commonly exhibit cerebral vascular abnormalities. The concept of mixed dementia has emerged to more clearly identify patients with neurodegenerative phenomena exhibiting both AD and cerebral vascular pathologies-vascular damage along with β-amyloid (Aβ)-associated neurotoxicity and τ-hyperphosphorylation. Cognitive impairment has long been commonly explained through a 'neuro-centric' perspective, but emerging evidence has shed light over the important roles that neurovascular unit dysfunction could have in neuronal death. Moreover, accumulating data have been demonstrating astrocytes being the essential cell type in maintaining proper central nervous system functioning. In relation to dementia, the roles of astrocytes in Aβ deposition and clearance are unclear. This article emphasizes the multiple events triggered by ischemia and the cytotoxicity exerted by Aβ either alone or in association with endothelin-1 and receptor for advanced glycation end products, thereby leading to neurodegeneration in an 'astroglio-centric' perspective.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Brain; Brain Ischemia; Dementia, Vascular; Endothelin-1; Humans

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

Valid experimental models and behavioral tests are indispensable for the development of therapies for stroke. The translational failure with neuroprotective drugs has forced us to look for alternative approaches. Restorative therapies aiming to facilitate the recovery process by pharmacotherapy or cell-based therapy have emerged as promising options. Here we describe the most common stroke models used in cell-based therapy studies with particular emphasis on their inherent complications, which may affect behavioral outcome. Loss of body weight, stress, hyperthermia, immunodepression, and infections particularly after severe transient middle cerebral artery occlusion (filament model) are recognized as possible confounders to impair performance in certain behavioral tasks and bias the treatment effects. Inherent limitations of stroke models should be carefully considered when planning experiments to ensure translation of behavioral data to the clinic.

Topics: Animals; Brain Ischemia; Cell- and Tissue-Based Therapy; Cerebrovascular Disorders; Disease Models, Animal; Endothelin-1; Psychomotor Disorders; Rats

Lacunar ischemic stroke accounts for 25% of all ischemic strokes, but the exact etiology is unknown. Numerous pathophysiologies have been proposed, including atheroma and endothelial dysfunction. Models of any of these pathological features would aid understanding of the etiology and help develop treatments for lacunar stroke. We therefore aimed to assess the relevance of all available potential animal models of lacunar stroke.. We systematically reviewed the published literature for animal models that could represent lacunar stroke using validated search strategies. We included studies that could represent an aspect of lacunar stroke as well as those aiming to model conditions with potentially similar pathology and extracted data on species, induction method, and resulting brain and vessel lesions.. From 5670 papers, 41 studies (46 papers) met inclusion criteria representing over 10 different classes of stroke induction. Important data like infarct size and animal numbers were often missing. Many models' infarcts were too large or affected the cortex. Emboli mostly caused cortical but not small subcortical lesions. Most models focused on creating ischemic lesions in brain tissue. Only one (spontaneous lesions in spontaneously hypertensive stroke-prone rats) also mimicked small vessel pathology. Here, the precursor to small vessel and brain damage was blood-brain barrier failure.. Some animal models produce small subcortical infarcts, but few mimic the human small vessel pathology. Models of small vessel disease could help improve understanding of human lacunar disease, particularly to clarify factors associated with the small vessel morphological changes preceding brain damage. Much lacunar stroke may arise after blood-brain barrier disruption.

Topics: Animals; Arterial Occlusive Diseases; Brain Ischemia; Cerebral Arteries; Data Interpretation, Statistical; Disease Models, Animal; Endothelin-1; Humans; Infarction, Middle Cerebral Artery; Intracranial Embolism; Stroke

Focal cerebral ischemia is an important cause of death and disability. Various experimental models were designed to study the pathophysiological mechanisms underlying ischemic damage and to evaluate putative neuroprotective strategies. The lack of correlation between the results of experimental and clinical studies resulted in much criticism of animal models of stroke. However, experimental models are undoubtedly the most important source of information on stroke pathophysiology, and will definitely remain an integral part of stroke research in the future. Due to much variability between different models, it is important to have a clear understanding of their pathophysiological characteristics - it is essential for correct interpretation of the experimental results. This aim of this review was to summarize the current knowledge about the mechanisms underlying the models of photothrombosis and endothelin-mediated focal ischemia.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Endothelin-1; Photic Stimulation

Vascular damage caused by cerebral ischemia leads to edema, hemorrhage formation, and worsened outcomes in ischemic stroke patients. Therapeutic interventions need to be developed to provide vascular protection. The purpose of this review is to identify the pathophysiologic processes involved in vascular damage after ischemia, which may lead to strategies to provide vascular protection in ischemic stroke patients.. The pathologic processes caused by vascular injury after an occlusion of a cerebral artery can be separated into acute (hours), subacute (hours to days), and chronic (days to months). Targets for intervention can be identified for all 3 stages. Acutely, superoxide is the predominant mediator, followed by inflammatory mediators and proteases subacutely. In the chronic phase, proapoptotic gene products have been implicated.. Pharmacological agents designed to target specific pathologic and protective processes affecting the vasculature should be used in clinical trials of vascular protection after acute ischemic stroke.

Topics: Acute Disease; Angiopoietins; Angiotensin II; Antioxidants; Apoptosis; Blood-Brain Barrier; Brain Edema; Brain Ischemia; Cerebral Arteries; Cerebral Hemorrhage; Chronic Disease; Endothelin-1; Endothelium, Vascular; Humans; Inflammation Mediators; Matrix Metalloproteinases; Neuroprotective Agents; Neutrophils; Oxidative Stress; Reactive Oxygen Species; Vascular Endothelial Growth Factor A; Vasculitis

Multiple endocrine abnormalities have been reported in stroke patients. In the past few years, it has been claimed that some of these abnormalities may play a role in worsening the neurological deficit and the outcome of stroke. Several mechanisms have been hypothesised, including a direct effect on the development of neuronal cell death, vasospasm, and development of brain edema. In this brief review, we discuss the current knowledge concerning the role of endothelin-1, arginine vasopressin, and cortisol in the pathogenesis of stroke. Finally, we discuss the possibility that leptin, the OB gene product, may be the link of some of these endocrine abnormalities, and that its abnormal secretion during stroke may contribute to the eating disorders and poor nutritional status often seen in these patients.

Topics: Arginine Vasopressin; Brain Ischemia; Endocrine System Diseases; Endothelin-1; Humans; Hydrocortisone; Hypothalamus; Leptin; Pituitary-Adrenal System; Stroke

Endothelial cells play a key role in the local regulation of the vascular smooth muscle tone by producing and releasing relaxing and contracting factors. Endothelin (ET)-1, one of the most potent endogenous vasoconstrictor substances known, is produced by endothelial cells. In the cerebral vasculature ET-1 is thought to be involved in several pathological conditions, including vasospasm following subarachnoid hemorrhage and stroke. This review contains evidence suggesting that endothelial dysfunction may contribute to the development of ischemic stroke and discusses the current knowledge concerning the role of ET-1 in the pathogenesis of stroke in animal models and in humans.

Topics: Animals; Aspartic Acid Endopeptidases; Brain Ischemia; Cerebrovascular Circulation; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelium, Vascular; Genetic Predisposition to Disease; Humans; Metalloendopeptidases; Receptors, Endothelin; Stroke

A minimized perfusion circuit (MPC) has proven to be superior to the conventional circulatory perfusion bypass (CCPB) as it reduces the blood-material interaction and hemodilution. Until now not much is known about impact these different perfusion systems have on the brain. The objective of this study is to determine carnosinase and brain-type fatty binding protein (BFABP) activity as novel specific biomarkers for ischemic brain tissue damage and how their activity differs during and after MPC and CCPB as well as to compare the inflammatory response of both perfusion systems. In a prospective pilot study, 28 patients undergoing coronary artery bypass grafting were randomly divided into an MPC group (n = 14) and a CCPB group (n = 14). Blood samples were taken before, during, and after operation until the fifth postoperative day. The brain biomarker carnosinase was determined by measuring the rate of histidine production from the substrate homocarnosine, whereas BFABP and interleukin-6 were determined by enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) and endothelin-1 were determined by enzyme immunoassay. The mean serum carnosinase activity was significantly higher in MPC (0.57 ± 0.34 nM histidine/mL/min) as compared with the CCPB group (0.36 ± 0.13 nM histidine/mL/min) at the end of operation (P = 0.02). The BFABP did not show any difference between the two groups in the immediate postoperative period until the second postoperative day. From that time point onward, it showed a steep increase in the CCPB group (581.3 ± 157.11 pg/mL) as compared with the concentrations in the MPC group (384.6 ± 39 pg/mL) (P = 0.04). The inflammation markers interleukin-6 and CRP showed a similar pattern in both groups without significant difference. In contrast, the leukocyte count on operation day and endothelin-1 on the first postoperative day were significantly higher in the CCPB group (P = 0.01, P = 0.03, respectively). MPC showed a significant higher and stable serum carnosinase activity during extracorporeal circulation as compared with the CCPB due to less hemodilution and a better preserved oxygen capacity. As a consequence, the antioxidant stress during MPC is limited as compared with CCPB, which means less brain tissue damage reflected by a lower BFABP release. Except endothelin-1 and leukocyte count, the inflammatory response of the MPC and CCPB was equal.

Topics: Aged; Biomarkers; Brain Ischemia; C-Reactive Protein; Cardiopulmonary Bypass; Carrier Proteins; Coronary Artery Bypass; Dipeptidases; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Fatty Acid-Binding Protein 7; Female; Germany; Humans; Immunoenzyme Techniques; Inflammation Mediators; Interleukin-6; Male; Middle Aged; Perfusion; Pilot Projects; Prospective Studies; Time Factors; Treatment Outcome; Tumor Suppressor Proteins

For almost 50 years it has been known that hemolysed blood can increase blood pressure. Although preclinical studies suggest that this pressor response is due to an interaction of hemoglobin with endothelium-derived vasoactive substances, its mechanism in humans is unknown. We investigated the involvement of endothelin-1 in the blood pressure response to the oxygen carrier diaspirin cross-linked hemoglobin (DCLHb) in stroke patients.. In a randomized phase II study, increasing doses of DCLHb (25, 50 and 100 mg/kg, n=8, 8 and 11, respectively) or placebo (n=26) were infused intravenously every 6 h for 72 h to patients with an acute ischemic stroke. Blood pressure and heart rate were measured every 15 min and plasma concentrations of endothelin-1, catecholamines, renin, vasopressin and atrial natriuretic peptide were measured before and 24 and 66 h after the start of the infusions.. In the placebo group, mean arterial pressure (MAP) was 112 (109-115) mmHg (mean and 95% confidence interval) at baseline and decreased spontaneously by 11.4 (5.4-17.5) and 12.5 (5.4-19.5) mmHg after 24 and 66 h, respectively. This decrease in MAP was attenuated in patients treated with DCLHb, reaching statistical significance in the highest dose group. The plasma endothelin-1 concentration decreased slightly in the placebo group, from 4.2 (3.1-5.3) pg/ml (median and range) at baseline to 2.4 (1.9-3.7) pg/ml after 24 h (P=0.0044) and 2.8 (1.9-3.7) pg/ml after 66 h (P=0.0042), but increased dose-dependently in response to DCLHb infusion. With the highest dose of DCLHb, the plasma endothelin-1 concentration rose from 4.8 (0.1-7.8) pg/ml at baseline to 21.2 (13.4-53.2) pg/ml after 24 h (P< 0.001) and to 27.6 (11.9-47.8) pg/ml after 66 h (P< 0.001). The increases in the plasma endothelin-1 concentration and in MAP were correlated (r=0.30, P=0.02). Other vasoactive hormones were not affected by the DCLHb infusion.. Infusion of DCLHb in patients with acute ischemic stroke was associated with a dose-dependent increase in plasma endothelin-1 concentration. This may underlie the attenuation by DCLHb of the natural decrease in blood pressure that we observed in these patients.

Topics: Acute Disease; Aged; Aspirin; Blood Pressure; Blood Substitutes; Brain Ischemia; Dose-Response Relationship, Drug; Endothelin-1; Female; Follow-Up Studies; Hemoglobins; Humans; Infusions, Intravenous; Male; Middle Aged; Safety

The development of ischemic lesions has primarily been studied in horizontal cortical space. However, how ischemic lesions develop through the cortical depth remains largely unknown. We explored this question using direct current coupled recordings at different cortical depths using linear arrays of iridium electrodes in the focal epipial endothelin-1 (ET1) ischemia model in the rat barrel cortex. ET1-induced impairments were characterized by a vertical gradient with (i) rapid suppression of the spontaneous activity in the superficial cortical layers at the onset of ischemia, (ii) compartmentalization of spreading depolarizations (SDs) to the deep layers during progression of ischemia, and (iii) deeper suppression of activity and larger histological lesion size in superficial cortical layers. The level of impairments correlated strongly with the rate of spontaneous activity suppression, the rate of SD onset after ET1 application, and the amplitude of giant negative ultraslow potentials (∼-70 mV), which developed during ET1 application and were similar to the tent-shaped ultraslow potentials observed during focal ischemia in the human cortex. Thus, in the epipial ET1 ischemia model, ischemic lesions develop progressively from the surface to the cortical depth, and early changes in electrical activity at the onset of ET1-induced ischemia reliably predict the severity of ischemic damage.

Topics: Animals; Brain Ischemia; Cortical Spreading Depression; Endothelin-1; Humans; Iridium; Ischemia; Rats

Topics: Animals; Brain; Brain Ischemia; Endothelin-1; Heart Diseases; Infarction, Middle Cerebral Artery; Inflammation; Mice; Molecular Imaging; Positron-Emission Tomography; Stroke; Vasoconstrictor Agents

Stroke is a leading cause of death in the general population, and it occurs three times more frequently in diabetic patients, necessitating extensive research into new therapeutics. The reproducibility, similarity, and technical limitations of current animal models are limited.. We developed a stroke induction model using pink zebra-Danio-rerio. Diabetes was induced in zebrafish by giving them D-glucose (111 mM) for 14 days, and those with blood glucose levels higher than 100 mg/dl were included in the study. In Zebrafish, an experimental stroke was induced by a single oral administration of Endothelin-1 (ET-1, 3µl/gm). Swimming, behavioural patterns, and cognitive performance were all recorded and analysed using UMA Tracker. The brains were removed for histopathological analysis.. In both the normal and diabetic groups, ET-1 administration resulted in a statistically significant change in swimming pattern and movements. Furthermore, changes in swimming pattern and recovery time were statistically significant in the diabetic ET-1 treatment group. In the neurocognitive assessment paradigm, the behavioural study of ET-1 treated groups revealed a disturbed cognitive profile and locomotor coordination, with an increase in the number of errors and a decrease in total distance travelled. Histopathological analysis of ET-1 treated groups revealed cortical lesions, shrunken neuronal cells, and thrombocytes in spheroid form with disturbed normal architecture of brain tissue when compared to normal control groups in tectum opticum and telencephalon. In terms of stability, reproducibility, and genetic similarity to human stroke, the current experimental model outperforms other available rodent stroke models.. The ET-1 induced experimental zebrafish stroke model opens up new avenues for diabetes-related stroke research due to its novelty, reproducibility, and ability to overcome technical errors found in other recent models.

Topics: Animals; Behavior, Animal; Blood Glucose; Blood Platelets; Brain Ischemia; Cognition; Diabetes Mellitus, Experimental; Disease Models, Animal; Endothelin-1; Female; Glucose; Ischemic Stroke; Male; Neurons; Psychomotor Performance; Swimming; Zebrafish

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia-reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

Topics: Animals; Apoptosis; Brain Ischemia; Endothelin-1; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Receptor-Interacting Protein Serine-Threonine Kinases

Neonatal arterial ischemic stroke is one of the more severe birth complications. The injury can result in extensive neurological damage and is robustly associated with later diagnoses of cerebral palsy (CP). An important part of efforts to develop new therapies include the on-going refinement and understanding of animal models that capture relevant clinical features of neonatal brain injury leading to CP. The potent vasoconstrictor peptide, Endothelin-1 (ET-1), has previously been utilised in animal models to reduce local blood flow to levels that mimic ischemic stroke. Our previous work in this area has shown that it is an effective and technically simple approach for modelling ischemic injury at very early neonatal ages, resulting in stable deficits in motor function. Here, we aimed to extend this model to also examine the impact on cognitive function. We show that focal delivery of ET-1 to the cortex of Sprague Dawley rats on postnatal day 0 (P0) resulted in impaired learning in a touchscreen-based test of visual discrimination and correlated with important clinical features of CP including damage to large white matter structures.

Topics: Animals; Animals, Newborn; Association Learning; Atrophy; Brain Ischemia; Cell Count; Cerebral Cortex; Cerebral Palsy; Cognition Disorders; Corpus Striatum; Disease Models, Animal; Endothelin-1; Inflammation; Injections; Microglia; Movement Disorders; Neurons; Perceptual Disorders; Rats; Rats, Sprague-Dawley; Rotarod Performance Test; Vasoconstrictor Agents; White Matter

Post-stroke depression (PSD) and post-stroke anxiety (PSA) are usually undertreated and many cases may remain undiagnosed, indicating a need for a better understanding of the underlying mechanisms. Current animal models of PSD and PSA using the middle cerebral artery occlusion model may be associated with motor deficits that can interfere with behavioral tests of depression- and anxiety-like behavior. Unilateral lesions of the medial prefrontal cortex (mPFC) have been reported to induce a depression- and anxiety-like phenotype in mice. The aim of this study was to examine the effects of unilateral microinjections of the vasoconstrictor endothelin-1 (ET-1) in the mPFC alone or in combination with the nucleus accumbens (NAc) on the behavior of rats after 2 and 6 weeks. Specifically, we measured anxiety- and depressive-like behavior, locomotion, and cognition. ET-1 injections in the mPFC and NAc resulted in replicable and localized lesions. Lesions to the mPFC and NAc resulted in more time spent in the open arms of the Elevated Plus Maze compared to sham-operated animals at 2 weeks post stroke, indicating decreased anxiety. This effect did not persist until 6 weeks post injection. No differences in locomotion, cognition and depressive-like behavior were found at either time point. In summary, unilateral lesions of mPFC and NAc did not produce a reliable and persistent anxiety and depression phenotype in rats.

Topics: Animals; Anxiety; Behavior, Animal; Brain Ischemia; Cognition; Depression; Disease Models, Animal; Endothelin-1; Locomotion; Male; Nucleus Accumbens; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Vasoconstrictor Agents

Pharmacological therapies for interrupting biochemical events of the ischaemic cascade and protecting against stroke in humans are as yet unavailable. Up to now, the neuroprotective activity in cerebral ischaemia of phycocyanobilin (PCB), a tetrapyrrolic natural antioxidant, has not been fully examined. Here, we evaluated if PCB protects PC12 neuronal cells against oxygen and glucose deprivation plus reperfusion, and its protective effects in a rat model of endothelin-1-induced focal brain ischaemia. PCB was purified from the cyanobacteria Spirulina platensis and characterized by spectrophotometric, liquid and gas chromatography and mass spectrometry techniques. In Wistar rats, PCB at 50, 100 and 200 μg/kg or phosphate-buffered saline (vehicle) was administered intraperitoneally at equal subdoses in a therapeutic schedule (30 minutes, 1, 3 and 6 hours after the surgery). Brain expression of myelin basic protein (MBP) and the enzyme CNPase was determined by immunoelectron microscopy. PCB was obtained with high purity (>95%) and the absence of solvent contaminants and was able to ameliorate PC12 cell ischaemic injury. PCB treatment significantly decreased brain infarct volume, limited the exploratory behaviour impairment and preserved viable cortical neurons in ischaemic rats in a dose-dependent manner, compared to the vehicle group. Furthermore, PCB at high doses restored the MBP and CNPase expression levels in ischaemic rats. An improved PCB purification method from its natural source is reported, obtaining PCB that is suitable for pharmacological trials showing neuroprotective effects against experimental ischaemic stroke. Therefore, PCB could be a therapeutic pharmacological alternative for ischaemic stroke patients.

Topics: Animals; Brain Injuries; Brain Ischemia; Endothelin-1; Male; PC12 Cells; Phycobilins; Phycocyanin; Rats; Rats, Wistar

Ischemic stroke accounts for 70-80% of stroke cases worldwide and survivors are frequently left with compromising sensorimotor deficits localized to one or more body regions. Most animal models of stroke involve transient or permanent occlusion of one or more major vessels such as the middle cerebral artery and are characterized by widespread damage to cortical and subcortical structures that result in deficits that can confound studies of neuroprotection and neurorehabilitation. Localized microinjections of the vasoconstricting peptide endothelin-1 (ET-1) into specific brain regions are becoming increasingly popular for such studies, but the pharmacology of endothelin-induced ischemic damage is poorly understood. To test the hypothesis that NMDA receptors, and particularly those containing the NR2B subunit, are involved in ET-1-mediated excitotoxicity and functional impairment, male CD1 rats (N = 32) were pre-treated with either the non-competitive NMDA antagonist MK-801 or the NR2B-selective antagonist Ro25-6981 (or vehicle) prior to unilateral microinjections of endothelin-1 into the somatosensory cortex and striatum. Rats were then tested using 4 established tests of sensory and/or motor function over 14 days. Lesion volumes were quantified post-mortem using standard histology and image analysis. Results confirmed reproducible lesions and significant deficits in all tests in vehicle-treated rats that were significantly reduced in both drug groups but were not different between drugs, providing evidence that endothelin-induced ischemic damage is mediated almost exclusively by NR2B-containing NMDA receptors.

Topics: Animals; Brain Ischemia; Dizocilpine Maleate; Endothelin-1; Excitatory Amino Acid Antagonists; Forelimb; Male; Microinjections; Phenols; Piperidines; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

We have demonstrated previously that activation of either the ET

Topics: Animals; Brain Ischemia; Endothelin-1; Hippocampus; Male; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Seizures

Focal brain ischemia is best studied in neocortex and striatum. Both show highly vulnerable neurons and high susceptibility to spreading depolarization (SD). Therefore, it has been hypothesized that these two variables generally correlate. However, this hypothesis is contradicted by findings in cerebellar cortex, which contains highly vulnerable neurons to ischemia, the Purkinje cells, but is said to be less susceptible to SD. Here, we found in the rat cerebellar cortex that elevated K

Topics: Animals; Brain Ischemia; Cerebellum; Cortical Spreading Depression; Disease Models, Animal; Endothelin-1; Male; Purkinje Cells; Rats; Rats, Wistar

Cerebral microdialysis can be used to detect mitochondrial dysfunction, a potential target of neuroprotective treatment. Cyclosporin A (CsA) is a mitochondrial stabiliser that in a recent clinical stroke trial showed protective potential in patients with successful recanalisation. To investigate specific metabolic effects of CsA during reperfusion, and hypothesising that microdialysis values can be used as a proxy outcome measure, we assessed the temporal patterns of cerebral energy substrates related to oxidative metabolism in a model of transient focal ischaemia. Transient ischaemia was induced by intracerebral microinjection of endothelin-1 (150 pmol/15 µL) through stereotaxically implanted guide cannulas in awake, freely moving rats. This was immediately followed by an intravenous injection of CsA (NeuroSTAT; 15 mg/kg) or placebo solution during continuous microdialysis monitoring. After reperfusion, the lactate/pyruvate ratio (LPR) was significantly lower in the CsA group vs placebo (n = 17, 60.6 ± 24.3%, p = 0.013). Total and striatal infarct volumes (mm

Topics: Animals; Brain Ischemia; Cell Respiration; Cyclosporine; Endothelin-1; Infarction; Ischemic Attack, Transient; Male; Microdialysis; Mitochondria; Oxidative Stress; Rats; Rats, Sprague-Dawley; Stroke

Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.

Topics: Animals; Brain Ischemia; Caspase 3; Cell Death; Cerebral Cortex; Disease Models, Animal; Endothelin-1; Gene Knockout Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons

The caudal primate prefrontal cortex (PFC) is involved in target selection and visually guided saccades through both covert attention and overt orienting eye movements. Unilateral damage to the caudal PFC often leads to decreased awareness of a contralesional target alone, referred to as "neglect," or when it is presented simultaneously with an ipsilesional target, referred to as "extinction." In the current study, we examined whether deficits in contralesional target selection were due to contralesional oculomotor deficits, such as slower reaction times. We experimentally induced a focal ischemic lesion in the right caudal PFC of 4 male macaque monkeys using the vasoconstrictor endothelin-1 and measured saccade choice and reaction times on double-stimulus free-choice tasks and single-stimulus trials before and after the lesion. We found that

Topics: Animals; Attention; Behavior, Animal; Brain Ischemia; Choice Behavior; Endothelin-1; Extinction, Psychological; Macaca mulatta; Male; Ocular Motility Disorders; Prefrontal Cortex; Reaction Time; Recovery of Function; Saccades; Vasoconstrictor Agents

It is well-known that in ischemia-induced hypoxia, hypoxia-inducible factor -1α (HIF-1α) is critical in triggering expression of its downstream target genes to produce several products, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), nitric oxide synthesis (NOS), glucose transportor-1 (GLUT-1), insulin-like growth factor (IGF), which further promote erythropoiesis, angiogenesis, vasodilation and capitalization of glucose to overcome hypoxia. Meanwhile, as the factors with opposite effects on blood vessels, endothelin-1 (ET-1) and brain natriuretic peptide (BNP) also stand out strikingly in ischemic pathophysiology. To this day, several preconditioning manners have been used to induce tolerance to ischemia. During our research, exercise preconditioning was applied and it was demonstrated that HIF-1α triggered expression of ET-1 and BNP, which confirmed their downstream target genes for HIF-1α. And ET-1 may influcence expression of BNP to some degree but not the only factor which regulates BNP expression. Therefore, our findings suggest exercise preconditioning may provide protection to the ischemic brain tissue via HIF-1α which in turn increases expression of BNP to cause vasodilation in cooperation with some other factors, such as VEGF and EPO, to increase the blood flow in the ischemic area and then relieve the injuries induced by ischemia.

Topics: Animals; Brain Ischemia; Corpus Striatum; Endothelin-1; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Natriuretic Peptide, Brain; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley

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

Stroke patients often suffer from delayed disturbances of mood and cognition. In rodents, the prefrontal cortex (PFC) is involved in both higher order cognition and emotion. Our objective was to determine if bilateral focal ischaemic lesions restricted to the medial prefrontal cortex (mPFC) could be used to model post-stroke anxiety and/or cognitive deficits.. Groups of adult male Sprague-Dawley rats (n=9) received bilateral injections of either endothelin-1 (ET-1) (400 pmol) or vehicle (artificial cerebrospinal fluid) into the mPFC and were tested at various times using both a test of temporal order memory and in an elevated plus maze. Lesions were verified histologically.. ET-1 lesioned rats had reduced mobility on post-surgery day 8 that had resolved by day 29 at which time they spent significantly more time in the closed arm of the plus maze CONCLUSION: We conclude that ischaemic lesions localised to the mPFC can be used to model post-stroke anxiety in rats.

Topics: Animals; Anxiety; Behavior, Animal; Brain Ischemia; Cognitive Dysfunction; Disease Models, Animal; Endothelin-1; Male; Maze Learning; Memory; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Stroke

Stroke is a leading cause of death worldwide and inflicts serious long-term damage and disability. The vasoconstrictor Endothelin-1, presenting long-term neurological deficits associated with excitotoxicity and oxidative stress is being increasingly used to induce focal ischemic injury as a model of stroke. A DJ-1 based peptide named ND-13 was shown to protect against glutamate toxicity, neurotoxic insults and oxidative stress in various animal models. Here we focus on the benefits of treatment with ND-13 on the functional outcome of focal ischemic injury. Wild type C57BL/6 mice treated with ND-13, after ischemic induction in this model, showed significant improvement in motor function, including improved body balance and motor coordination, and decreased motor asymmetry. We found that DJ-1 knockout mice are more sensitive to Endothelin-1 ischemic insult than wild type mice, contributing thereby additional evidence to the widely reported relevance of DJ-1 in neuroprotection. Furthermore, treatment of DJ-1 knockout mice with ND-13, following Endothelin-1 induced ischemia, resulted in significant improvement in motor functions, suggesting that ND-13 provides compensation for DJ-1 deficits. These preliminary results demonstrate a possible basis for clinical application of the ND-13 peptide to enhance neuroprotection in stroke patients.

Topics: Animals; Brain Ischemia; Cell-Penetrating Peptides; Disease Models, Animal; Endothelin-1; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Protein Deglycase DJ-1; Recovery of Function; Stroke; Vasoconstrictor Agents

Reach training in concert with environmental enrichment provides functional benefits after experimental stroke in rats. The present study extended these findings by assessing whether intensive task-specific reach training or enrichment initiated alone would provide similar functional benefit. Additionally, we investigated whether the 70% recovery rule, or a combined model of initial poststroke impairment, cortical infarct volume, and rehabilitation intensity, could predict recovery in the single-pellet task, as previously found for the Montoya staircase.. Rats were trained on single-pellet reaching before middle cerebral artery occlusion via intracerebral injection of ET-1 (endothelin-1). There were 4 experimental groups: stroke+enrichment, stroke+reaching, stroke+enrichment+reaching, and sham+enrichment+reaching. Reaching rehabilitation utilized a modified Whishaw box that encouraged impaired forelimb reaching for 6 hours per day, 5 days per week, for 4 weeks. All treatment paradigms began 7 days after ischemia with weekly assessment on the single-pellet task during rehabilitation and again 4 weeks after rehabilitation concluded.. Rats exposed to the combination of enrichment and reaching showed the greatest improvement in pellet retrieval and comparable performance to shams after 3 weeks of treatment, whereas those groups that received a monotherapy remained significantly impaired at all time points. Initial impairment alone did not significantly predict recovery in single-pellet as the 70% rule would suggest; however, a combined model of cortical infarct volume and rehabilitation intensity predicted change in pellet retrieval on the single-pellet task with the same accuracy as previously shown with the staircase, demonstrating the generalizability of this model across reaching tasks.. Task-specific reach training and environmental enrichment have synergistic effects in rats that persist long after rehabilitation ends, and this recovery is predicted by infarct volume and rehabilitation intensity.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Endothelin-1; Infarction, Middle Cerebral Artery; Male; Motor Skills; Rats, Sprague-Dawley; Recovery of Function; Stroke; Stroke Rehabilitation

There are a lot of convincing evidences about the involvement of endothelin pathway proteins in the pathogenesis of atherosclerosis and its fatal complications. In this study, the analysis of a possible association between

Topics: Aged; Brain Ischemia; Carotid Artery Diseases; Endothelin-1; Female; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Receptor, Endothelin A; Stroke; Ukraine

The association of poor outcome and mortality with low levels of hemoglobin (Hb) and hematocrit (HcT) in patients admitted after acute Ischemic Stroke (IS) was recently demonstrated. The mechanisms behind this still remain unclear. Our study aims to find out whether mRNA expressions and plasma concentrations of endothelin-1 (ET-l), endothelin-2 (ET-2) and endothelin-3 (ET-3) remain different in IS sufferers with low HcT and Hb levels in comparison with those whose HcT i Hb levels during a severe IS episode remain within the norm. The study included 60 patients treated consecutively for first-time IS. The assessment of mRNA gene expression and plasma concentration of ET-1, ET-2, ET-3 was conducted in the first, third and seventh day following the onset of stroke using qRT-PCR method and ELISA tests. We demonstrated that patients whose initial HcT and Hb levels were below the norm presented a deeper neurologic deficit on 1, 3 and 7 day following stroke with noticeable improvement no earlier than between day 3 and 7. We also found a negative correlation between the initial HcT and Hb and concentration of plasma ET-1 on the same days. The patients whose HcT and Hb levels were within normal limits showed a significant improvement in their neurologic condition on each consecutive day of the observation. Reduced levels of Hb and HcT combined with an increased plasma concentration of vasoconstrictive endothelin-1 are strongly associated with poor outcome and high mortality in acute ischemic stroke.

Topics: Aged; Aged, 80 and over; Blood Pressure; Brain Ischemia; C-Reactive Protein; Calcitonin; Endothelin-1; Female; Hematocrit; Hemoglobins; Humans; Male; Middle Aged; RNA, Messenger; Stroke; Tomography Scanners, X-Ray Computed; Vasoconstriction

Research into stroke is driven by frustration over the limited available therapeutics. Targeting a single aspect of this multifactorial disease contributes to the therapeutic boundaries. To overcome this, we devised a novel multifactorial-cocktail treatment, using lentiviruses encoding excitatory amino acid transporter 2 (EAAT2(, glutamate dehydrogenase 2 (GDH2), and nuclear factor E2-related factor 2 (Nrf2) genes, that acts synergistically to address the effected excito-oxidative axis. Here, we used the vasoconstrictor endothelin-1 (ET-1) to induce focal ischemic injury in mice by direct injection into the striatum. Mice treated with the mixture of these three genes show significant improvement in body balance, motor coordination, and decreased motor asymmetry compared to each gene separately. These results demonstrate that overexpression of the combined EAAT2, GDH2, and NRF2 genes can provide neuroprotection after ischemic injury.

Topics: Animals; Brain Ischemia; Endothelin-1; Excitatory Amino Acid Transporter 2; Genetic Therapy; Glutamate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Stroke

Stroke is a leading cause of long-term disability, though current rehabilitative strategies fail to yield complete recovery. Focused training of the impaired limb improves functional outcome in rodents, but these strategies require intensive training that is difficult to practice in humans. Because aerobic exercise has been found to induce beneficial changes in the brain, it is a promising rehabilitative strategy after stroke. The current study investigated the effect of voluntary poststroke aerobic exercise on functional outcome in young and aged mice. Mice were trained on a skilled reaching task before receiving focal ischemic stroke and being subdivided into 3 different groups for rehabilitative training: traditional skilled reach rehabilitation, aerobic exercise, and control procedures. Both young and aged mice benefited from aerobic exercise after stroke, though the behavioral profile somewhat differed. Aerobic exercise in young mice yielded poststroke performance levels that were equivalent to preinjury levels. In aged mice, aerobic exercise accelerated improvement in motor performance without an effect on the absolute level of performance compared with controls. Our results suggest that aerobic exercise may be an effective alternative or adjunctive rehabilitative strategy after stroke. Potential mechanisms of this effect need to be further investigated. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Topics: Aging; Animals; Brain Ischemia; Disease Models, Animal; Endothelin-1; Learning; Male; Mice, Inbred C57BL; Motor Activity; Recovery of Function; Stroke Rehabilitation; Volition

Ischemic brain injury is one of the leading causes of death and disability, where lack of disease modifying treatment strategies make us rely on symptomatic relief. Treatment principles from traditional systems of medicine may fill this gap and its validation in modern medicine perspective is important to bring them to mainstream. Here, we evaluated the neuroprotective efficacy of Ayurvedic medicinal herb Pluchea lanceolata in treating ischemic hippocampal injury. Focal hippocampal ischemia was modeled in Wistar rats through stereotaxic intrahippocampal injection of endothelin-1 (ET-1). Post-surgery, hydroalcoholic extract of the rhizome of Pluchea lanceolata (HAPL) was administered orally, once in a day for 14 consecutive days to ischemic rats. There were two treatment groups based on the HAPL dosage; HAPL200 (200 mg/kg body weight) and HAPL400 (400 mg/kg body weight). Comparisons were made with the ET-1 ischemic rats which received only the vehicle, and the normal surgical control. Ischemic hippocampal injury led to severe cognitive deficits as evaluated by Morris water maze and open field test, along with locomotory dysfunction noted in actophotometer test. HAPL treatment significantly attenuated these behavioural deficits in a dose dependent manner. Loss of pyramidal cells and degenerative phenotype of shrunken hyperdensed soma with pyknotic nuclei in CA1 and CA3 hippocampal neurons in ischemia were reversed after HAPL treatment. We provide first evidence for loss of dendritic architecture in ET-1 induced focal ischemic hippocampal injury using Golgi impregnation, where HAPL could salvage the dendritic branching and intersections. Intriguingly, it enhanced the dentritic arborization beyond what is noted in normal rats. Ability of HAPL to reverse oxidative stress, especially through maintaining glutathione peroxidase levels and lipid peroxidation in ischemic condition evidences that it may exert neuroprotection through its antioxidant properties. Thus, Pluchea lanceolata and its constituents provide potential alternative/adjuvant treatment strategy for ischemic hippocampal stroke.

Topics: Animals; Brain Ischemia; Cognition; Dendrites; Endothelin-1; Hippocampus; Medicine, Ayurvedic; Motor Activity; Neurons; Neuroprotective Agents; Oxidative Stress; Plant Extracts; Pyramidal Cells; Rats; Rats, Wistar; Spatial Memory

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body β-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Topics: 3-Hydroxybutyric Acid; Acetoacetates; Animals; Astrocytes; Blood Glucose; Brain; Brain Ischemia; Cell Death; Cerebrovascular Circulation; Disease Models, Animal; Electrophysiological Phenomena; Endothelin-1; Hemodynamics; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Microinjections; Neurons; Rats; Reactive Oxygen Species; Sensorimotor Cortex

Maintenance of white matter integrity in health and disease is critical for a variety of neural functions. Ischemic stroke in the white matter frequently results in degeneration of oligodendrocytes (OLs) and myelin. Previously, we found that toll-like receptor 2 (TLR2) expressed in OLs provides cell-autonomous protective effects on ischemic OL death and demyelination in white matter stroke. Here, we identified high-mobility group box-1 (HMGB1) as an endogenous TLR2 ligand that promotes survival of OLs under ischemic stress. HMGB1 rapidly accumulated in the culture medium of OLs exposed to oxygen-glucose deprivation (OGD). This conditioned medium exhibited a protective activity against ischemic OL death that was completely abolished by immunodepletion of HMGB1. Knockdown of HMGB1 or application of glycyrrhizin, a specific HMGB1 inhibitor, aggravated OGD-induced OL death, and recombinant HMGB1 application reduced the extent of OL death in a TLR2-dependent manner. We confirmed that cytosolic translocation of HMGB1 and activation of TLR2-mediated signaling pathways occurred in a focal white matter stroke model induced by endothelin-1 injection. Animals with glycyrrhizin coinjection showed an expansion of the demyelinating lesion in a TLR2-dependent manner, accompanied by aggravation of sensorimotor behavioral deficits. These results indicate that HMGB1/TLR2 activates an autocrine trophic signaling pathways in OLs and myelin to maintain structural and functional integrity of the white matter under ischemic conditions.

Topics: Animals; Brain Ischemia; Cells, Cultured; Demyelinating Diseases; Endothelin-1; HMGB1 Protein; Male; Mice; Mice, Inbred C57BL; Myelin Sheath; Oligodendroglia; Signal Transduction; Stroke; Toll-Like Receptor 2; White Matter

Alzheimer disease (AD) and stroke coexist and interact; yet how they interact is not sufficiently understood. Both AD and basal ganglia stroke can impair behavioural flexibility, which can be reliably modeled in rats using an established operant based set-shifting test. Transgenic Fischer 344-APP21 rats (TgF344) overexpress pathogenic human amyloid precursor protein (hAPP) but do not spontaneously develop overt pathology, hence TgF344 rats can be used to model the effect of vascular injury in the prodromal stages of Alzheimer disease. We demonstrate that the injection of endothelin-1 (ET1) into the dorsal striatum of TgF344 rats (Tg-ET1) produced an exacerbation of behavioural inflexibility with a behavioural phenotype that was distinct from saline-injected wildtype & TgF344 rats as well as ET1-injected wildtype rats (Wt-ET1). In addition to profiling the types of errors made, interpolative modeling using logistic exposure-response regression provided an informative analysis of the timing and efficiency of behavioural flexibility. During set-shifting, Tg-ET1 committed fewer perseverative errors than Wt-ET1. However, Tg-ET1 committed significantly more regressive errors and had a less efficient strategy change than all other groups. Thus, behavioural flexibility was more vulnerable to striatal ischemic injury in TgF344 rats.

Topics: Amyloid beta-Protein Precursor; Analysis of Variance; Animals; Attention Deficit Disorder with Hyperactivity; Brain Ischemia; Conditioning, Operant; Corpus Striatum; Cues; Discrimination, Psychological; Disease Models, Animal; Endothelin-1; Food Deprivation; Humans; Mental Disorders; Mutation; Rats; Rats, Inbred F344; Rats, Transgenic; Spatial Processing

Pericytes are contractile vascular mural cells overlying capillary endothelium, and they have been implicated in a variety of functions including regulation of cerebral blood flow. Recent work has suggested that both in vivo and ex vivo, ischaemia causes pericytes to constrict and die, which has implications for microvascular reperfusion. Assessing pericyte contractility in tissue slices and in vivo is technically challenging, while in vitro techniques remain unreliable. Here, we used isolated cultures of human brain vascular pericytes to examine their contractile potential in vitro using the iCelligence electrical impedance system. Contraction was induced using the vasoactive peptide endothelin-1, and relaxation was demonstrated using adenosine and sodium nitroprusside. Endothelin-1 treatment also resulted in increased proliferation, which we were able to monitor in the same cell population from which we recorded contractile responses. Finally, the observation of pericyte contraction in stroke was reproduced using chemical ischaemia, which caused a profound and irreversible contraction clearly preceding cell death. These data demonstrate that isolated pericytes retain a contractile phenotype in vitro, and that it is possible to quantify this contraction using real-time electrical impedance recordings, providing a significant new platform for assessing the effects of vasoactive and vasculoprotective compounds on pericyte contractility.

Topics: Brain; Brain Ischemia; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Electric Impedance; Endothelin-1; Endothelium, Vascular; High-Throughput Screening Assays; Humans; Muscle Contraction; Muscle, Smooth, Vascular; Pericytes; Vasoconstriction; Vasodilation

Brain plasticity following focal cerebral ischaemia has been observed in both stroke survivors and in preclinical models of stroke. Endogenous neurovascular adaptation is at present incompletely understood yet its potentiation may improve long-term functional outcome. We employed longitudinal MRI, intracranial array electrophysiology, Montoya Staircase testing, and immunofluorescence to examine function of brain vessels, neurons, and glia in addition to forelimb skilled reaching during the subacute stage of ischemic injury progression. Focal ischemic stroke (~100mm

Topics: Animals; Brain; Brain Ischemia; Brain Waves; Encephalitis; Endothelin-1; Hypercapnia; Magnetic Resonance Imaging; Male; Motor Skills; Neuroglia; Neurons; Physical Stimulation; Rats, Sprague-Dawley; Recovery of Function; Sensorimotor Cortex; Somatosensory Cortex; Stroke; Touch Perception; Vascular Remodeling

Delayed cerebral hypoperfusion is a secondary complication found in the days after transient global cerebral ischaemia that worsens the ischaemic damage inflicted by the initial transient episode of global cerebral ischaemia. A recent study demonstrated increased cerebral vasoconstriction in the large arteries on the brain surface (pial arteries) after global cerebral ischaemia. However, smaller arterioles inside the brain (parenchymal arterioles) are equally important in the regulation of cerebral blood flow and yet their pathophysiology after global cerebral ischaemia is largely unknown. Therefore, we investigated whether increased contractility occurs in the intraparenchymal arterioles.. Global cerebral ischaemia was induced in male Wistar rats by bilateral common carotid occlusion for 15 min combined with hypovolaemia. Regional cerebral blood flow was determined by quantitative autoradiography. Intraparenchymal arterioles were isolated and pressurized, and concentration-response curves to endothelin-1 with and without the endothelin B receptor-selective antagonist BQ788 was generated. Endothelin B receptor expression was investigated by quantitative flow cytometry and immunohistochemistry.. We observed increased endothelin-1-mediated contractility of parenchymal arterioles correlating with reduced cerebral blood flow of the cortex, hippocampus and caudate nucleus 48 h after global cerebral ischaemia. The increased endothelin-1-mediated contractility was abolished by BQ788, and the vascular smooth muscle cell-specific expression of endothelin B receptors was significantly increased after global cerebral ischaemia.. Increased endothelin-1-mediated contractility and expression of endothelin B receptors in the intraparenchymal vasculature contributes to the development of delayed cerebral hypoperfusion after global cerebral ischaemia in combination with vascular changes of the pial vasculature.

Topics: Animals; Arterioles; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Male; Rats; Rats, Wistar; Receptor, Endothelin B; Vasoconstriction

Recovery of hand function following lesions in the primary motor cortex (M1) is associated with a reorganization of premotor areas in the ipsilesional hemisphere, and this reorganization depends on the size of the lesion. It is not clear how lesion size affects motor representations in the contralesional hemisphere and how the effects in the 2 hemispheres compare. Our goal was to study how lesion size affects motor representations in the ipsilesional and contralesional hemispheres. In rats, we induced lesions of different sizes in the caudal forelimb area (CFA), the equivalent of M1. The effective lesion volume in each animal was quantified histologically. Behavioral recovery was evaluated with the Montoya Staircase task for 28 days after the lesion. Then, the organization of the CFA and the rostral forelimb area (RFA)--the putative premotor area in rats--in the 2 cerebral hemispheres was studied with intracortical microstimulation mapping techniques. The distal forelimb representation in the RFA of both the ipsilesional and contralesional hemispheres was positively correlated with the size of the lesion. In contrast, lesion size had no effect on the contralesional CFA, and there was no relationship between movement representations in the 2 hemispheres. Finally, only the contralesional RFA was negatively correlated with chronic motor deficits of the paretic forelimb. Our data show that lesion size has comparable effects on motor representations in premotor areas of both hemispheres and suggest that the contralesional premotor cortex may play a greater role in the recovery of the paretic forelimb following large lesions.

Topics: Animals; Brain Ischemia; Brain Mapping; Chronic Disease; Disease Models, Animal; Endothelin-1; Forelimb; Functional Laterality; Motor Activity; Motor Cortex; Movement Disorders; Neural Pathways; Neuronal Plasticity; Random Allocation; Rats; Rats, Sprague-Dawley; Recovery of Function

Topics: Animals; Brain Ischemia; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Humans; Nitric Oxide Synthase Type III; Peptides, Cyclic; Rats; Vasoconstriction

Cortical injections of the vasoconstrictor endothelin-1 (ET1) have widely been used to induce focal circumscribed ischemic lesions in the motor cortex of rodents in the context of stroke recovery studies. In order to apply this model correctly, it is essential to understand the time course of regional flow changes and of the development of penumbra and infarction.. Multitracer micro-PET of ET1 focal ischemia in rats was performed using [11C]-flumazenil ([11C]FMZ) as a flow- and viability tracer and [18F]-fluoromisonidazole ([18F]FMISO) as hypoxia marker in order to characterize the physiological time-course of this model. Nine adult Sprague-Dawley rats received stereotaxic injections of ET1 into the right primary motor cortex, 3 served as controls. PET imaging was started 2, 3 and 20 h after the last ET1 injection. Histology was obtained at the end of the scans. Standardized uptake value ratios reflecting cerebral blood flow (CBF), [11C]FMZ-binding and [18F]FMISO-retention were calculated for the region of hypoperfusion and the normoperfused cortex.. CBF in the hypoperfused cortex was significantly reduced (p < 0.01) at 5 h (0.58 ± 0.025), 6 h (0.54 ± 0.043) and 23 h (0.66 ± 0.024) compared to controls (1.00 ± 0.011) and moderately reduced (p < 0.05) in the remainder of the affected hemisphere at 5 h (0.93 ± 0.036). [11C]FMZ-binding was within the control range at all time points. Significant [18F]FMISO-retention (1.16 ± 0.091, p < 0.05) was observed only after 6 h in the ischemic core that later turned into infarct.. ET1 injections yield reproducible, slowly developing ischemic lesions with constant levels of hypoperfusion. This multitracer micro-PET study suggests that the ET1 model is appropriate for inducing chronic circumscribed ischemic lesions but seems to be less suited for studying acute stroke pathophysiology.

Topics: Animals; Brain; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Flumazenil; Infarction, Middle Cerebral Artery; Ischemia; Male; Models, Animal; Motor Cortex; Rats, Sprague-Dawley; Stroke

BACKGROUND Ischemic stroke is widely recognized as a major health problem and social burden worldwide, and it usually leads to dementia. In this study, we aimed to better understand the pathogenesis in the development of dementia following ischemic stroke. MATERIAL AND METHODS We exploited miRNA database to search for the target for miR-125a and validated the found target using luciferase assay. Further, we performed real-time PCR and Western blot analysis to examine the expression of miR-125a and its target in the tissue samples. In addition, a polymorphism was genotyped and its association with post-stroke dementia was analyzed. RESULTS We identified enthothelin-1 (ET-1) as a target of miR-125a, and this relationship was validated using luciferase assay. Furthermore, transfection of miR-125a inhibitor substantially upregulated the expression of ET-1, while miR-125a and ET-1 siRNA caused downregulation of ET-1 in endothelial cells. In addition, we found that a polymorphism (rs12976445) interferes with the expression of miR-125a, which in turn caused an increase in the expression of ET-1 in human endothelial cells. Logistic regression analysis showed that rs12976445 is significantly associated with the risk of dementia after ischemic stroke. CONCLUSIONS Our study demonstrated the pathogenesis mechanism during the development of dementia after ischemic stroke by investigating the relationship between miR-125a and its target ET-1, promising a potential pathological solution for post-stroke dementia in the future.

Topics: 3' Untranslated Regions; Base Sequence; Brain Ischemia; Case-Control Studies; Dementia; Demography; Endothelin-1; Gene Expression Regulation; Genetic Predisposition to Disease; Human Umbilical Vein Endothelial Cells; Humans; Luciferases; MicroRNAs; Polymorphism, Single Nucleotide; RNA, Messenger; Stroke; Transfection

Chronic alcohol intoxication (CAI) increases both morbidity and mortality of stroke patients. Despite the high prevalence of CAI and ischemic stroke, studies addressing their comorbidity and/or protective alternatives remain scarce. Thus, the influence of CAI on both stroke outcome and minocycline treatment (recognized for its neuroprotective effect) was investigated. Female Wistar rats (35 days old) were treated with water or ethanol (6.5 g/kg/day, 22.5% w/v) for 55 days. Then, focal ischemia was induced by endothelin-1 in the motor cortex. Two hours later, four doses of 50 mg/kg of minocycline every 12 hours followed by five doses of 25 mg/kg every 24 hours were administered. Behavioral performance (open field and rotarod tests) and immunohistochemical (cellular density, neuronal death, and astrocytic activation) and biochemical (lipid peroxidation and nitrite levels) analyses were performed. CAI increased motor disruption, nitrite and lipid peroxidation levels, and neuronal loss caused by ischemia, whereas it reduced the astrogliosis. Minocycline was effective in preventing the motor and tissue damage caused by stroke. However, these effects were attenuated when CAI preceded stroke. Our data suggest that CAI beginning in adolescence contributes to a worse outcome in ischemic stroke survivors and reduces the benefits of minocycline, possibly requiring adjustments in therapy.

Topics: Alcoholic Intoxication; Animals; Behavior, Animal; Brain Ischemia; Disease Models, Animal; Endothelin-1; Female; Immunohistochemistry; Lipid Peroxidation; Minocycline; Motor Cortex; Neuroprotective Agents; Nitrites; Rats; Rats, Wistar

Neuroblasts from the subventricular zone (SVZ) migrate to striatum following stroke, but most of them die in the ischaemic milieu and this can be related to exacerbated microglial activation. Here, we explored the effects of the non-steroidal anti-inflammatory indomethacin on microglial activation, neuronal preservation and neuroblast migration following experimental striatal stroke in adult rats. Animals were submitted to endothelin-1 (ET-1)-induced focal striatal ischaemia and were treated with indomethacin or sterile saline (i.p.) for 7 days, being perfused after 8 or 14 days. Immunohistochemistry was performed to assess neuronal loss (anti-NeuN), microglial activation (anti-Iba1, ED1) and migrating neuroblasts (anti-DCX) by counting NeuN, ED1 and DCX-positive cells in the ischaemic striatum or SVZ. Indomethacin treatment reduced microglia activation and the number of ED1+ cells in both 8 and 14 days post injury as compared with controls. There was an increase in the number of DCX+ cells in both SVZ and striatum at the same survival times. Moreover, there was a decrease in the number of NeuN+ cells in indomethacin-treated animals as compared with the control group at 8 days but not after 14 days post injury. Our results suggest that indomethacin treatment modulates microglia activation, contributing to increased neuroblast proliferation in the SVZ and migration to the ischaemic striatum following stroke.

Topics: Animals; Brain Ischemia; Cell Proliferation; Corpus Striatum; Doublecortin Protein; Endothelin-1; Humans; Indomethacin; Lateral Ventricles; Microglia; Neural Stem Cells; Neurogenesis; Neurons; Rats; Stroke

Stroke is a leading cause of death and neurological disability worldwide and striatal ischemic stroke is frequent in humans due to obstruction of middle cerebral artery. Several pathological events underlie damage progression and a comprehensive description of the pathological features following experimental stroke in both acute and chronic survival times is a necessary step for further functional studies. Here, we explored the patterns of microglial activation, astrocytosis, oligodendrocyte damage, myelin impairment, and Nogo-A immunoreactivity between 3 and 30 postlesion days (PLDs) after experimental striatal stroke in adult rats induced by microinjections of endothelin-1 (ET-1). The focal ischemia induced tissue loss concomitant with intense microglia activation between 3 and 14 PLDs (maximum at 7 PLDs), decreasing afterward. Astrocytosis was maximum around 7 PLDs. Oligodendrocyte damage and Nogo-A upregulation were higher at 3 PLDs. Myelin impairment was maximum between 7 and 14 PLDs. Nogo-A expression was higher in the first week in comparison to control. The results add important histopathological features of ET-1 induced stroke in subacute and chronic survival times. In addition, the establishment of the temporal evolution of these neuropathological events is an important step for future studies seeking suitable neuroprotective drugs targeting neuroinflammation and white matter damage.

Topics: Animals; Astrocytes; Brain; Brain Ischemia; Disease Models, Animal; Endothelin-1; Immunohistochemistry; Male; Microglia; Microscopy; Myelin Basic Protein; Nogo Proteins; Oligodendroglia; Rats; Rats, Wistar; Stroke; Up-Regulation; White Matter

Levels of cerebral amyloid, presumably β-amyloid (Abeta), toxicity and the incidence of cortical and subcortical ischemia increases with age. However, little is known about the severe pathological condition and dementia that occur as a result of the comorbid occurrence of this vascular risk factor and Abeta toxicity. Clinical studies have indicated that small ischemic lesions in the striatum are particularly important in generating dementia in combination with minor amyloid lesions. These cognitive deficits are highly likely to be caused by changes in the cortex. In this study, we examined the viability and morphological changes in microglial and neuronal cells, gap junction proteins (connexin43) and neuritic/axonal retraction (Fer Kinase) in the striatum and cerebral cortex using a comorbid rat model of striatal injections of endothelin-1 (ET1) and Abeta toxicity. The results demonstrated ventricular enlargement, striatal atrophy, substantial increases in β-amyloid, ramified microglia and increases in neuritic retraction in the combined models of stroke and Abeta toxicity. Changes in connexin43 occurred equally in both groups of Abeta-treated rats, with and without focal ischemia. Although previous behavioral tests demonstrated impairment in memory and learning, the visual discrimination radial maze task did not show significant difference, suggesting the cognitive impairment in these models is not related to damage to the dorsolateral striatum. These results suggest an insight into the relationship between cortical/striatal atrophy, pathology and functional impairment.

Topics: Amyloid beta-Peptides; Amyloidosis; Animals; Brain Ischemia; Cerebral Cortex; Comorbidity; Connexin 43; Corpus Striatum; Disease Models, Animal; Endothelin-1; Male; Maze Learning; Microglia; Nerve Degeneration; Neurites; Neurons; Protein-Tyrosine Kinases; Random Allocation; Rats, Wistar; Stroke; Visual Perception

The incidence of stroke in adulthood increases with advancing age, but there is little understanding of how poststroke treatment should be tailored by age.. The goal of this study was to determine if age and task specificity of rehabilitative training affect behavioral improvement and motor cortical organization after stroke.. Young and aged mice were trained to proficiency on the Pasta Matrix Reaching Task prior to lesion induction in primary motor cortex with endothelin-1. After a short recovery period, mice received 9 weeks of rehabilitative training on either the previously learned task (Pasta Matrix Reaching), a different reaching task (Tray Reaching), or no training. To determine the extent of relearning, mice were tested once weekly on the Pasta Matrix Reaching Task. Mice then underwent intracortical microstimulation mapping to resolve the remaining forelimb movement representations in perilesion motor cortex.. Although aged mice had significantly larger lesions compared with young mice, Pasta Matrix Reaching served as effective rehabilitative training for both age-groups. Young animals also showed improvement after Tray Reaching. Behavioral improvement in young mice was associated with an expansion of the rostral forelimb area ("premotor" cortex), but we failed to see reorganization in the aged brain, despite similar behavioral improvements.. Our results indicate that reorganization of motor cortex may be limited by either aging or greater tissue damage, but the capacity to improve motor function via task-specific rehabilitative training continues to be well maintained in aged animals.

Topics: Aging; Animals; Brain Ischemia; Disease Models, Animal; Electric Stimulation; Endothelin-1; Exercise Therapy; Male; Mice, Inbred C57BL; Motor Cortex; Motor Skills; Neuronal Plasticity; Recovery of Function; Stroke; Stroke Rehabilitation

In preclinical stroke models, improvement in motor performance is associated with reorganization of cortical motor maps. However, the temporal relationship between performance gains and map plasticity is not clear.. This study was designed to assess the effects of rehabilitative training on the temporal dynamics of behavioral and neurophysiological endpoints in a rat model of focal cortical infarct.. Eight days after an ischemic infarct in primary motor cortex, adult rats received either rehabilitative training or were allowed to recover spontaneously. Motor performance and movement quality of the paretic forelimb was assessed on a skilled reach task. Intracortical microstimulation mapping procedures were conducted to assess the topography of spared forelimb representations either at the end of training (post-lesion day 18) or at the end of a 3-week follow-up period (post-lesion day 38).. Rats receiving rehabilitative training demonstrated more rapid improvement in motor performance and movement quality during the training period that persisted through the follow-up period. Motor maps in both groups were unusually small on post-lesion day 18. On post-lesion day 38, forelimb motor maps in the rehabilitative training group were significantly enlarged compared with the no-rehab group, and within the range of normal maps.. Postinfarct rehabilitative training rapidly improves motor performance and movement quality after an ischemic infarct in motor cortex. However, training-induced motor improvements are not reflected in spared motor maps until substantially later, suggesting that early motor training after stroke can help shape the evolving poststroke neural network.

Topics: Animals; Biomechanical Phenomena; Brain Ischemia; Brain Mapping; Cerebral Infarction; Disease Models, Animal; Electric Stimulation; Endothelin-1; Forelimb; Male; Motor Activity; Physical Conditioning, Animal; Psychomotor Performance; Rats; Rats, Long-Evans; Recovery of Function

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

Pathophysiological conditions such as cerebral ischemia trigger the production of new neurons from the neurogenic niche within the subgranular zone (SGZ) of the dentate gyrus. The functional significance of ischemia-induced neurogenesis is believed to be the regeneration of lost cells, thus contributing to post-ischemia recovery. However, the cell signaling mechanisms by which this process is regulated are still under investigation. Here, we investigated the role of mitogen and stress-activated protein kinases (MSK1/2) in the regulation of progenitor cell proliferation and neurogenesis after cerebral ischemia. Using the endothelin-1 model of ischemia, wild-type (WT) and MSK1(-/-)/MSK2(-/-) (MSK dKO) mice were injected with BrdU and sacrificed 2 days, 4 weeks, or 6 weeks later for the analysis of progenitor cell proliferation, neurogenesis, and neuronal morphology, respectively. We report a decrease in SGZ progenitor cell proliferation in MSK dKO mice compared to WT mice. Moreover, MSK dKO mice exhibited reduced neurogenesis and a delayed maturation of ischemia-induced newborn neurons. Further, structural analysis of neuronal arborization revealed reduced branching complexity in MSK dKO compared to WT mice. Taken together, this dataset suggests that MSK1/2 plays a significant role in the regulation of ischemia-induced progenitor cell proliferation and neurogenesis. Ultimately, revealing the cell signaling mechanisms that promote neuronal recovery will lead to novel pharmacological approaches for the treatment of neurodegenerative diseases such as cerebral ischemia.

Topics: Animals; Brain Ischemia; Dentate Gyrus; Disease Models, Animal; Endothelin-1; Female; Green Fluorescent Proteins; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neural Stem Cells; Neurogenesis; Neurons; Ribosomal Protein S6 Kinases, 90-kDa; Stem Cell Niche

Constraint-induced movement therapy (CIMT) after stroke enhances not only functional reorganization but also structural plasticity of the brain in the adult rats. We examined whether forced limb-use which mimicked CIMT could influence ischemia-induced neurogenesis, apoptosis and behavioral recovery in the aged rats. Aged rats were divided into a sham group, an ischemia group, and an ischemia group with forced limb-use. Focal cerebral ischemia was induced by injection of endothelin-1. Forced limb-use began on post-stroke day 7 by fitting a plaster cast around the unimpaired upper limbs of rats for 3 weeks. Behavioral recovery was evaluated by tapered/ledged beam-walking test on postoperative day 32. The expression of doublecortin, neuronal nuclei, glial fibrillary acidic protein and Iba-1 were measured by single or double immunohistochemistry, and apoptosis was measured by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay. The production of neuroblasts in the subventricular zone (SVZ) was significantly increased after stroke. Forced limb-use enhanced the proliferation of newborn neurons in the SVZ, as well as increased the long-term survival of newborn neurons. Furthermore, forced limb-use suppressed apoptosis and improved the motor functions after stroke in the aged rats. Forced limb-use exerted few effects on inflammation. Neither the number nor dendritic complexity of newborn granule cells in the hippocampus was affected by forced limb-use. Forced limb-use is effective in enhancing neurogenesis and behavioral recovery after stroke even in the aged rats.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Proliferation; Disease Models, Animal; Doublecortin Protein; Endothelin-1; Lateral Ventricles; Locomotion; Male; Neurogenesis; Neurons; Physical Therapy Modalities; Rats; Rats, Sprague-Dawley; Restraint, Physical; Stroke; Stroke Rehabilitation

Epidemiological studies imply a significantly positive association between particulate matter (PM) level and ischemic stroke hospitalization. However, considering that PM10 is highly heterogeneous and varies with season within the same location, existing experimental evidence remained low. In the present study, we first treated Wistar rats with PM10 samples collected from different seasons in Taiyuan, a typically coal-burning city of China, and determined ischemia-related markers in the cortex. The results indicated that PM10 exposure caused endothelial dysfunction, inflammatory response, and neuro-functional impairment similar to that of cerebral ischemia with season-dependent properties, and the winter sample presented the most obvious injuries. Then, we detected the chemical composition of PM10 samples followed by analysis of their correlation with the above biomarkers and found that winter PM10, characterized by higher polycyclic aromatic hydrocarbons (PAHs) and carbon load, played the major role in causing brain ischemia-like injuries among different season samples. Furthermore, by setting up an ischemic neuron model in vitro, we confirmed that winter PM10 presented the most serious aggravation on ischemia-produced injury outcome. This study provides experimental evidence for clarifying the association between season-dependent PM10 pollution in the atmospheric environment and an increased risk of ischemia-like injuries.

Topics: Administration, Inhalation; Air Pollutants; Animals; Brain; Brain Ischemia; Carbon; Cyclooxygenase 2; Endothelin-1; Gene Expression; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; Metals; Neurons; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Particulate Matter; Polycyclic Aromatic Hydrocarbons; Rats, Wistar; RNA, Messenger; Seasons

Endothelin-1 (ET-1) induced focal ischemia is increasingly being used as a preclinical model of stroke. Here, we described for the first time, the time course of neuronal death and infarct evolution during the first 7 days following ischemia.. We used hematoxylin and eosin (H&E) staining to evaluate infarct progression and Fluoro-Jade C (FJC) to quantify neuronal degeneration at 24, 48, 72h and 7 days after ET-1 injection to the forelimb motor cortex in Sprague-Dawley rats.. We found that infarct volume and neuronal degeneration are maximal at 24h post-stroke. Neuronal degeneration is also significantly reduced within 7 days of stroke induction.. This study is the first to provide a direct evaluation of both infarct volume evolution and neuronal death time course following ET-1 induced focal ischemia in the forelimb motor cortex.. This study describes the short-term time course of neuronal death and brain injury in the ET-1 stroke model, which provides a significant reference when determining the appropriate time to commence neuroprotective or recovery promoting strategies.

Topics: Animals; Brain Ischemia; Cell Count; Cell Death; Disease Models, Animal; Disease Progression; Endothelin-1; Forelimb; Male; Motor Cortex; Nerve Degeneration; Neurons; Rats, Sprague-Dawley; Stroke; Time Factors

In this study, we aimed to investigate whether changes in cerebrovascular voltage-dependent calcium channels and non-selective cation channels contribute to the enhanced endothelin-1-mediated vasoconstriction in the delayed hypoperfusion phase after experimental transient forebrain ischaemia.. Experimental forebrain ischaemia was induced in Wistar male rats by a two-vessel occlusion model, and the cerebral blood flow was measured by magnetic resonance imaging two days after reperfusion. In vitro vasoreactivity studies, immunofluorescence and quantitative PCR were performed on cerebral arteries from ischaemic or sham-operated rats to evaluate changes in vascular voltage-dependent calcium channels, transient receptor potential canonical channels as well as endothelin-1 receptor function and expression.. The expression of transient receptor potential canonical channels 1 and 6 in the vascular smooth muscle cells was enhanced and correlated with decreased cerebral blood flow two days after forebrain ischaemia. Furthermore, under conditions when voltage-dependent calcium channels were inhibited, endothelin-1-induced cerebrovascular contraction was enhanced and this enhancement was presumably mediated by Ca(2+) influx via upregulated transient receptor potential canonical channels 1 and 6.. Our data demonstrates that endothelin-1-mediated influx of extracellular Ca(2+) activates transient receptor potential canonical channels 1 and 6 in cerebral vascular smooth muscle cells. This seems to have an important role in the enhanced cerebral vasoconstriction in the delayed post-ischaemic hypoperfusion phase after experimental forebrain ischaemia.

Topics: Animals; Blood Flow Velocity; Brain Ischemia; Calcium Signaling; Cerebrovascular Circulation; Endothelin-1; Male; Prosencephalon; Rats; Rats, Wistar; TRPC Cation Channels

To aid in development of chronic stage treatments for sensorimotor deficits induced by ischemic stroke, we investigated the effects of GABA antagonism on brain structure and fine skilled reaching in a rat model of focal ischemia induced via cortical microinjections of endothelin-1 (ET-1). Beginning 7 days after stroke, animals were administered a gamma-aminobutyric acid (GABAA) inverse agonist, L-655,708, at a dose low enough to afford α5-GABAA receptor specificity. A week after stroke, the ischemic lesion comprised a small hypointense necrotic core (6±1 mm(3)) surrounded by a large (62±11 mm(3)) hyperintense perilesional region; the skilled reaching ability on the Montoya staircase test was decreased to 34%±2% of the animals' prestroke performance level. On L-655,708 treatment, animals showed a progressive decrease in total stroke volume (13±4 mm(3) per week), with no change in animals receiving placebo. Concomitantly, treated animals' skilled reaching progressively improved by 9%±1% per week, so that after 2 weeks of treatment, these animals performed at 65%±6% of their baseline ability, which was 25%±11% better than animals given placebo. These data indicate beneficial effects of delayed, sustained low-dose GABAA antagonism on neuroanatomic injury and skilled reaching in the chronic stage of stroke recovery in an ET-1 rat model of focal ischemia.

Topics: Animals; Behavior, Animal; Brain Ischemia; Endothelin-1; GABA Antagonists; Heart Rate; Imidazoles; Male; Motor Skills; Necrosis; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Recovery of Function; Respiratory Rate; Stroke; Stroke Volume

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed "paw-dragging". Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia--two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Topics: Animals; Brain Injuries; Brain Ischemia; Disease Models, Animal; Endothelin-1; Forelimb; Male; Mice; Motor Cortex; Rats; Recovery of Function; Stroke

Endothelin-1 (ET-1) is synthesized by endothelial cells and astrocytes in stroke and in brains of Alzheimer's disease patients. Our transgenic mice with ET-1 overexpression in the endothelial cells (TET-1) showed more severe blood-brain barrier (BBB) breakdown, neuronal apoptosis, and glial reactivity after 2-hour transient middle cerebral artery occlusion (tMCAO) with 22-hour reperfusion and more severe cognitive deficits after 30 minutes tMCAO with 5 months reperfusion. However, the role of astrocytic ET-1 in contributing to poststroke cognitive deficits after tMCAO is largely unknown. Therefore, GET-1 mice were challenged with tMCAO to determine its effect on neurologic and cognitive deficit. The GET-1 mice transiently displayed a sensorimotor deficit after reperfusion that recovered shortly, then more severe deficit in spatial learning and memory was observed at 3 months after ischemia compared with that of the controls. Upregulation of TNF-α, cleaved caspase-3, and Thioflavin-S-positive aggregates was observed in the ipsilateral hemispheres of the GET-1 brains as early as 3 days after ischemia. In an in vitro study, ET-1 overexpressing astrocytic cells showed amyloid secretion after hypoxia/ischemia insult, which activated endothelin A (ETA) and endothelin B (ETB) receptors in a PI3K/AKT-dependent manner, suggesting role of astrocytic ET-1 in dementia associated with stroke by astrocyte-derived amyloid production.

Topics: Amyloidogenic Proteins; Animals; Astrocytes; Brain Edema; Brain Ischemia; Cognition; Dementia; Endothelin-1; Hippocampus; Humans; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Maze Learning; Memory Disorders; Mice; Mice, Transgenic; Nervous System Diseases

Ischemic stroke is one of the leading causes of neurological disability worldwide, and it has been estimated that about one quarter of stroke survivors experience some measurable long-term cognitive impairments. Many higher order cognitive deficits occur because of damage to the prefrontal cortex (PFC), which is one of the main areas of the brain responsible for executive functioning in mammals. Currently, there are few animal models that examine the effects of stroke on executive function. In this study we used bilateral micro-injections (1μl) of the vasoconstricting peptide endothelin-1 (ET-1) into the medial PFC in male Sprague-Dawley rats (or vehicle control, N=17-18 per group) in order to model ischemic lesions in the medial PFC. The effects of these lesions on executive function were assessed using tests of set-shifting and temporal object recognition. ET-1 injections in the medial PFC resulted in replicable and specific lesions within the PFC with an average infarct volume of 16.63±2.71mm(3). The ischemic lesions resulted in specific contextual set-shifting deficits within the maze, including an increased number of trials to criterion and a significant difference in learning curves. However, no deficits in temporal order memory processing were noted between sham and stroke animals. We conclude that ischemic lesions localized to the mPFC result in selective but not generalized deficits in executive function in rats.

Topics: Analysis of Variance; Animals; Attention; Brain Infarction; Brain Ischemia; Cognition Disorders; Endothelin-1; Executive Function; Male; Maze Learning; Neuropsychological Tests; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Recognition, Psychology; Set, Psychology; Time Factors

The purposes of this pilot study were to create a model of focal cortical ischemia in Macaca fascicularis and to explore contributions of the reticulospinal system in recovery of reaching. Endothelin-1 was used to create a focal lesion in the shoulder/elbow representation of left primary motor cortex (M1) of two adult female macaques. Repetitive microstimulation was used to map upper limb motor outputs from right and left cortical motor areas and from the pontomedullary reticular formation (PMRF). In subject 1 with a small lesion and spontaneous recovery, reaching was mildly impaired. Changes were evident in the shoulder/elbow representations of both the lesioned and contralesional M1, and there appeared to be fewer than expected upper limb responses from the left (ipsilesional) PMRF. In subject 2 with a substantial lesion, reaching was severely impaired immediately after the lesion. After 12 weeks of intensive rehabilitative training, reach performance recovered to near-baseline levels, but movement times remained about 50% slower. Surprisingly, the shoulder/elbow representation in the lesioned M1 remained completely absent after recovery, and there was a little change in the contralesional M1. There was a definite difference in motor output patterns for left versus right PMRF for this subject, with an increase in right arm responses from right PMRF and a paucity of left arm responses from left PMRF. The results are consistent with increased reliance on PMRF motor outputs for recovery of voluntary upper limb motor control after significant cortical ischemic injury.

Topics: Afferent Pathways; Animals; Brain Ischemia; Disease Models, Animal; Electric Stimulation; Endothelin-1; Female; Functional Laterality; Macaca fascicularis; Motor Cortex; Motor Skills Disorders; Pilot Projects; Recovery of Function; Reticular Formation; Spinal Cord; Upper Extremity

Forced limb-use can enhance neurogenesis and behavioral recovery as well as increasing the level of stromal cell-derived factor-1 (SDF-1) in stroke rats. We examined whether the SDF-1/CXCR4 pathway is involved in the enhanced neurogenesis and promoted behavioral recovery induced by forced limb-use in the chronic phase of stroke.. The CXCR4 antagonist, AMD3100, was used to block the SDF-1/CXCR4 pathway in the ischemic rats. Brain ischemia was induced by endothelin-1. One week after ischemia, the unimpaired forelimb of rats was immobilized for 3 weeks. The proliferation, migration, and survival of DCX-positive cells in the subventricular zone (SVZ), and the dendritic complexity of DCX-positive cells in the dentate gyrus (DG), as well as the inflammatory response in the infarcted striatum were analyzed by immunohistochemistry. Functional recovery was assessed in beam-walking and water maze tests.. Forced limb-use enhanced the proliferation, migration, dendritic complexity and the survival of newborn neurons. Furthermore, forced limb-use suppressed the inflammatory response and improved both motor and cognitive functions after stroke. AMD3100 significantly abrogated the enhanced neurogenesis and behavioral recovery induced by forced limb-use without influencing the inflammatory response.. SDF-1/CXCR4 pathway seems to be involved in the enhancement of neurogenesis and behavioral recovery induced by post-stroke forced limb-use.

Topics: Animals; Benzylamines; Brain; Brain Ischemia; Central Nervous System Agents; Chemokine CXCL12; Cyclams; Disease Models, Animal; Doublecortin Protein; Endothelin-1; Forelimb; Heterocyclic Compounds; Immobilization; Male; Maze Learning; Motor Activity; Musculoskeletal Manipulations; Neurogenesis; Neurons; Random Allocation; Rats, Wistar; Receptors, CXCR4; Recovery of Function; Stroke; Stroke Rehabilitation

Endogenous labeling of stem/ progenitor cells via intracerebroventricular injection of micron-sized particles of iron oxide (MPIOs) has become standard methodology for MRI of adult neurogenesis. While this method is well characterized in the naïve rodent brain, it has not been fully investigated in disease models. Here, we describe methodological challenges that can confound data analysis when this technique is applied to a rat model of stroke, the endothelin-1 model of focal cortical ischemia.. We intended to track endogenous neuroblast migration from the subventricular zone to the stroke area using previously described methods for in vivo labeling of endogenous neuroblasts with MPIOs and following migration with high resolution MRI.. MPIOs accumulation in stroke regions of endothelin-1-treated brains involves two dynamic steps: an initial rapid cell independent mechanism, followed by slower MPIOs accumulation. While the latter may in part be attributable to cell dependent delivery of the particles, the cell independent mechanism complicates the interpretation of the data. Strategies aimed at prelabeling the stem cell niche reduced cell independent MPIOs accumulation, but failed to abolish it.. We conclude that MRI-based neural stem/progenitor cell tracking via direct injection of MPIOs into the lateral and third ventricles, requires significant validation in models of brain disease/trauma.

Topics: Animals; Brain Ischemia; Cell Movement; Cerebral Ventricles; Contrast Media; Endothelin-1; Ferrosoferric Oxide; Magnetic Resonance Imaging; Metabolic Clearance Rate; Neural Stem Cells; Particle Size; Rats; Rats, Sprague-Dawley; Tissue Distribution

Small (lacunar) infarcts frequently arise in frontal and midline thalamic regions in the absence of major stroke. Damage to these areas often leads to impairment of executive function likely as a result of interrupting connections of the prefrontal cortex. Thus, patients experience frontal-like symptoms such as impaired ability to shift ongoing behavior and attention. In contrast, executive dysfunction has not been demonstrated in rodent models of stroke, thereby limiting the development of potential therapies for human executive dysfunction. Male Sprague-Dawley rats (n=40) underwent either sham surgery or bilateral endothelin-1 injections in the mediodorsal nucleus of the thalamus or in the medial prefrontal cortex. Executive function was assessed using a rodent attention set shifting test that requires animals to shift attention to stimuli in different stimulus dimensions. Medial prefrontal cortex ischemia impaired attention shift performance between different stimulus dimensions while sparing stimulus discrimination and attention shifts within a stimulus dimension, indicating a selective attention set-shift deficit. Rats with mediodorsal thalamic lacunar damage did not exhibit a cognitive impairment relative to sham controls. The selective attention set shift impairment observed in this study is consistent with clinical data demonstrating selective executive disorders following stroke within specific sub-regions of frontal cortex. These data contribute to the development and validation of a preclinical animal model of executive dysfunction, that can be employed to identify potential therapies for ameliorating cognitive deficits following stroke.

Topics: Animals; Attention; Behavior, Animal; Brain Ischemia; Discrimination Learning; Endothelin-1; Executive Function; Male; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Reversal Learning; Set, Psychology; Stroke

Ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. In order to better understand and exploit the cell signaling mechanisms that regulate ischemia-induced proliferation, we examined the role of the p42/44 mitogen-activated protein kinase (MAPK) cascade effector ribosomal S6 kinase (RSK) in this process. Here, using the endothelin-1 ischemia model in wild type mice, we show that the activated form of RSK is expressed in the progenitor cells of the subgranular zone (SGZ) after intrahippocampal cerebral ischemia. Further, RSK inhibition significantly reduces ischemia-induced SGZ progenitor cell proliferation. Using the neurosphere assay, we also show that both SGZ- and subventricular zone (SVZ)-derived adult neural stem cells (NSC) exhibit a significant reduction in proliferation in the presence of RSK and MAPK inhibitors. Taken together, these data reveal RSK as a regulator of ischemia-induced progenitor cell proliferation, and as such, suggest potential therapeutic value may be gained by specifically targeting the regulation of RSK in the progenitor cell population of the SGZ.

Topics: Adult Stem Cells; Analysis of Variance; Animals; Brain Ischemia; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin-1; Enzyme Inhibitors; Fluoresceins; Gene Expression Regulation; Hippocampus; Ki-67 Antigen; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Ribosomal Protein S6 Kinases; Time Factors

Evidence indicates that angiotensin II type 2 receptors (AT2R) exert cerebroprotective actions during stroke. A selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exert beneficial effects in models of cardiac and renal disease, as well as hemorrhagic stroke. Here, we hypothesize that C21 may exert beneficial effects against cerebral damage and neurological deficits produced by ischemic stroke. We determined the effects of central and peripheral administration of C21 on the cerebral damage and neurological deficits in rats elicited by endothelin-1 induced middle cerebral artery occlusion (MCAO), a model of cerebral ischemia. Rats infused centrally (intracerebroventricular) with C21 before endothelin-1 induced MCAO exhibited significant reductions in cerebral infarct size and the neurological deficits produced by cerebral ischemia. Similar cerebroprotection was obtained in rats injected systemically (intraperitoneal) with C21 either before or after endothelin-1 induced MCAO. The protective effects of C21 were reversed by central administration of an AT2R inhibitor, PD123319. While C21 did not alter cerebral blood flow at the doses used here, peripheral post-stroke administration of this agent significantly attenuated the MCAO-induced increases in inducible nitric oxide synthase, chemokine (C-C) motif ligand 2 and C-C chemokine receptor type 2 mRNAs in the cerebral cortex, indicating that the cerebroprotective action is associated with an anti-inflammatory effect. These results strengthen the view that AT2R agonists may have potential therapeutic value in ischemic stroke, and provide the first evidence of cerebroprotection induced by systemic post stroke administration of a selective AT2R agonist.

Topics: Angiotensin II Type 2 Receptor Blockers; Animals; Brain Infarction; Brain Ischemia; CD11b Antigen; Cerebrovascular Circulation; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Glial Fibrillary Acidic Protein; Imidazoles; Male; Nitric Oxide Synthase Type II; Peroxidase; Pyridines; Rats; Rats, Sprague-Dawley; Stroke; Sulfonamides; Thiophenes; Time Factors

The objective of this study is to investigate the association between SNP polymorphisms of endothelin-1 (EDN1) and endothelin receptor (EDNRA and EDNRB) gene and ischemic stroke (IS) in the Chinese Han population in northern. A case-control study was introduced. We genotyped eight SNPs (rs1800541, rs2070699, and rs5370 in EDN1 gene; rs1801708, rs5333, and rs5335 in EDNRA gene; and rs3818416 and rs5351 in EDNRB gene) and calculated their polymorphic distribution in control group, IS group, and the IS subgroups. In male population, EDN1 gene rs2070699 G allele increased the incidence risk to 1.78 times (P = 0.009; OR 1.78; 95 % CI 1.15-2.75) and the risk of morbidity of rs5370 T allele carrying increased to 1.49 times (P = 0.048; OR 1.49; 95 % CI 1.00-2.21). EDNRA gene mutation rs5335 homozygous CC morbidity risk was significantly lower (P = 0.016; OR 0.52; 95 % CI 0.31-0.88). In the female population, the mutant homozygous AA cancer risk was significantly higher than G allele carriers (P = 0.019; OR 2.65; 95 % CI 1.18-6.00) on EDNRA gene rs1801708. In EDN1 gene, T allele of rs5370 and G allele of rs2070699 may be IS incidence risk factors in Northern Han male population. A allele of rs1801708 in EDNRA gene can increase the risk of IS in Northern Han women population.

Topics: Brain Ischemia; Case-Control Studies; Endothelin-1; Female; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Receptor, Endothelin A; Receptor, Endothelin B; Stroke

Ethanol is an important risk factor for the occurrence of cerebral ischemia contributing to poor prognosis and inefficacy of drug treatments for stroke-related symptoms. Females have a higher lifetime risk for stroke than males. Moreover, female gender has been associated with increased ethanol consumption during adolescence. In the present study, we investigated whether chronic ethanol exposure during adolescence may potentiate the motor impairments and cortical damage induced by focal ischemia in female rats. We also addressed whether these effects can be mitigated by minocycline, which has been shown to be neuroprotective against different insults in the CNS. Female rats were treated with distilled water or ethanol (6.5 g/kg/day, 22.5% w/v) for 55 days by gavage. Focal ischemia was induced by microinjections of endothelin-1 (ET-1) into the motor cortex. Animals of both groups were treated daily with minocycline (25-50 mg/kg, i.p.) or sterile saline (i.p.) for 5 days, and motor function was assessed using open field, inclined plane and rotarod tests. Chronic ethanol exposure exacerbated locomotor activity and motor coordination impairments induced by focal ischemia in rats. Moreover, histological analysis revealed that microinjections of ET-1 induced pyramidal neuron loss and microglial activation in the motor cortex. Minocycline reversed the observed motor impairments, microglial activation and pyramidal neuron loss in the motor cortex of ischemic rats even in those exposed to ethanol. These results suggest that minocycline induces neuroprotection and functional recovery in ischemic female rats intoxicated with ethanol during adolescence. Furthermore, the mechanism underlying this protective effect may be related to the modulation of neuroinflammation.

Topics: Alcohol-Related Disorders; Animals; Brain Ischemia; Central Nervous System Depressants; Disease Models, Animal; Endothelin-1; Ethanol; Female; Microglia; Minocycline; Motor Activity; Motor Cortex; Movement Disorders; Neurons; Neuroprotective Agents; Pyramidal Cells; Rats, Wistar; Recovery of Function

To investigate the protective effect of catalpol on cerebral ischaemia/reperfusion (CI/R) injury in gerbils and further explore the underlying mechanism.. A gerbil model of CI/R was prepared by bilateral common carotid occlusion for 10 min followed by 6 h reperfusion. Catalpol (5, 10 or 20 mg/kg per day) was injected intraperitoneally for 3 days before the carotid occlusion. Stroke index was measured during the reperfusion. The contents of endogenous neuropeptides, endothelin-1 (ET-1) and calcitonin gene-related peptide in plasma were evaluated by radioimmunoassay. Superoxide dismutase (SOD) and malondialdehyde (MDA) in brain tissue homogenate were also examined.. The results showed that catalpol significantly improved the stroke index compared with CI/R control group (P < 0.05 or P < 0.01). Catalpol significantly increased the activity of SOD at the doses of 10 and 20 mg/kg (P ≤ 0.05), decreased the brain MDA content and the plasma level of ET-1 at the doses of 10 and 20 mg/kg (P ≤ 0.01).. These data suggested that the efficacy of catalpol pretreatment on CI/R injury may be attributed to reduction of free radicals and inhibition of lipid peroxidation and ET-1 production.

Topics: Animals; Brain; Brain Ischemia; Calcitonin Gene-Related Peptide; Cerebral Infarction; Disease Models, Animal; Endothelin-1; Female; Free Radicals; Gerbillinae; Injections, Intraperitoneal; Iridoid Glucosides; Lipid Peroxidation; Male; Malondialdehyde; Neuropeptides; Oxidative Stress; Phytotherapy; Plant Extracts; Rehmannia; Reperfusion Injury; Stroke; Superoxide Dismutase

Stroke is an acute cerebrovascular disease with high incidence, morbidity, and mortality. Preischemic treadmill training has been shown to be effective in improving behavioral and neuropathologic indices after cerebral ischemia. However, the exact neuroprotective mechanism of preischemic treadmill training against ischemic injury has not been elucidated clearly. The present study investigated whether preischemic treadmill training could protect the brain from ischemic injury via regulating cerebral blood flow (CBF) and endothelin 1 (ET-1). We analyzed the CBF by laser speckle imaging and ET-1 expression by an enzyme-linked immunosorbent assay using an ischemic rat model with preischemic treadmill training. Generally speaking, ET-1 expression decreased and CBF increased significantly in the pretreadmill group. It is worth noting that ET-1 expression is increased at 24 hours of reperfusion in the pretreadmill group compared with the level of the time after middle cerebral artery occlusion. These changes were followed by significant changes in neurologic deficits and cerebral infarct volume. This study indicated that preconditioning exercise protected brain from ischemic injury through the improvement of CBF and regulation of ET-1 expression, which may be a novel component of the neuroprotective mechanism of preischemic treadmill training against brain injury.

Topics: Animals; Brain; Brain Ischemia; Cerebrovascular Circulation; Disease Models, Animal; Endothelin-1; Infarction, Middle Cerebral Artery; Ischemic Preconditioning; Male; Physical Conditioning, Animal; Rats; Rats, Sprague-Dawley; Stroke

We tested the influence of erythropoietin (EPO), a basic cytokine in erythropoiesis regulation, on the process of motor function and cognition after focal brain ischemia induced by a local application of endothelin. Endothelin-1 (ET-1) induced short lasting strong vasoconstriction, with described impact on the structure and on the function of neuronal cells. Neurological description of motor function and Morris water maze test (the swimming test is one of most widely used methods for studying cognitive functions in rodents) were used to study the process of learning and memory in three-month-old male albino Wistar rats (n=52). Both tests were performed one week before, and three weeks after ischemia induction (endothelin application on the cortex in the area of a. cerebri media dx.). Experimental group received i.p. injection of EPO (5,000 IU/kg body weight, 10 min before endothelin application). Control group of animals received one i.p. injection of saline at the dose of 1 ml/kg body weight at the same time. Only sham surgery was performed in the third group of animals. Rats with EPO pretreatment before the experimental lesion exhibited significantly better motor and cognitive function then those with saline injection. No significant changes in the motor and cognitive function were found in the third group of rats (sham operated controls).

Topics: Animals; Brain Ischemia; Cognition; Endothelin-1; Erythropoietin; Male; Motor Activity; Rats; Rats, Wistar

Despite the availability of numerous transgenic mouse lines to study the role of individual genes in promoting neural repair following stroke, few studies have availed of this technology, primarily due to the lack of a reproducible ischemic injury model in the mouse. Intracortical injections of Endothelin-1 (ET1) a potent vasoconstrictive agent, reliably produces focal infarcts with concomitant behavioral deficits in rats. In contrast, ET1 infarcts in mice are significantly smaller and do not generate consistent behavioral deficits.. We have modified the ET1 ischemia model to target the anterior forelimb motor cortex (aFMC) and show that this generates a reproducible focal ischemic injury in mice with consistent behavioral deficits. Furthermore, we have developed a novel analysis of the cylinder test by quantifying paw-dragging behavior.. ET1 injections which damage deep layer neurons in the aFMC generate reproducible deficits on the staircase test. Cylinder test analysis showed no forelimb asymmetry post-injection; however, we observed a novel paw-dragging behavior in mice which is a positive sign of damage to the FMC.. Previous ET1 studies have demonstrated inconsistent behavioral deficits; however, targeting ET1 injections to the aFMC reliably results in staircase deficits. We show that analysis of paw-dragging behavior in the cylinder test is a more sensitive measure of damage to the FMC than the classical forelimb asymmetry analysis.. We have developed a focal ischemic injury model in the mouse that results in reproducible behavioral deficits and can be used to test future regenerative therapies.

Topics: Animals; Brain Ischemia; Cell Count; Disease Models, Animal; Endothelin-1; Forelimb; Immunohistochemistry; Male; Mice; Motor Cortex; Movement Disorders; Neurons; Random Allocation; Reproducibility of Results; Severity of Illness Index; Stroke

Aging is a major risk factor for cerebrovascular disease. Growth hormone (GH) and its anabolic mediator, insulin-like growth factor (IGF)-1, decrease with advancing age and this decline has been shown to promote vascular dysfunction. In addition, lower GH/IGF-1 levels are associated with higher stroke mortality in humans. These results suggest that decreased GH/IGF-1 level is an important factor in increased risk of cerebrovascular diseases. This study was designed to assess whether GH/IGF-1-deficiency influences the outcome of cerebral ischemia. We found that endothelin-1-induced middle cerebral artery occlusion resulted in a modest but nonsignificant decrease in cerebral infarct size in GH/IGF-1 deficient dw/dw rats compared with control heterozygous littermates and dw/dw rats with early-life GH treatment. Expression of endothelin receptors and endothelin-1-induced constriction of the middle cerebral arteries were similar in the three experimental groups. Interestingly, dw/dw rats exhibited reduced brain edema and less astrocytic infiltration compared with their heterozygous littermates and this effect was reversed by GH-treatment. Because reactive astrocytes are critical for the regulation of poststroke inflammatory processes, maintenance of the blood-brain barrier and neural repair, further studies are warranted to determine the long-term functional consequences of decreased astrocytic activation in GH/IGF-1 deficient animals after cerebral ischemia.

Topics: Aging; Animals; Astrocytes; Brain Edema; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Dwarfism; Endothelin-1; Female; Growth Hormone; Insulin-Like Growth Factor I; Male; Rats; Rats, Inbred Lew; Rats, Mutant Strains

Endothelins are potent vasoconstrictors and signaling molecules. Their effects are broad, impacting processes ranging from neurovascular and cardiovascular health to cell migration and survival. In stroke, traumatic brain injury or subarachnoid hemorrhage, endothelin-1 (ET-1) is induced resulting in cerebral vasospasm, ischemia, reperfusion and the activation of various pathways. Given the central role that ET-1 plays in these patients and to identify the downstream molecular events specific to transient vasoconstriction, we studied the consequences of ET-1-mediated vasoconstriction of the middle cerebral artery in a rat model. Our observations demonstrate that ET-1 can lead to increases in gene expression, including genes associated with the inflammatory response (Ifnb, Il6, Tnf) and oxidative stress (Hif1a, Myc, Sod2). We also observed inductions (>2 fold) of genes involved in eicosanoid biosynthesis (Pla2g4a, Pla2g4b, Ptgs2, Ptgis, Alox12, Alox15), heme metabolism (Hpx, Hmox1, Prdx1) and iron homeostasis (Hamp, Tf). Our findings demonstrate that mRNA levels for the hormone hepcidin (Hamp) are induced in the brain in response to ET-1, providing a novel target in the treatment of multiple conditions. These changes on the ipsilateral side were also accompanied by corresponding changes in a subset of genes in the contralateral hemisphere. Understanding ET-1-mediated events at the molecular level may lead to better treatments for neurological diseases and provide significant impact on neurological function, morbidity and mortality.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Disease Models, Animal; Eicosanoids; Endothelin-1; Gene Expression; Homeostasis; Iron; Iron-Sulfur Proteins; Male; Middle Cerebral Artery; Neuroimmunomodulation; Oxidative Stress; Random Allocation; Rats, Sprague-Dawley; RNA, Messenger; Vasoconstriction; Vasoconstrictor Agents

Stroke causes disability and mortality worldwide and is divided into ischemic and hemorrhagic subtypes. Although clinical trials suggest distinct recovery profiles for ischemic and hemorrhagic events, this is not conclusive due to stroke heterogeneity. The aim of this study was to produce similar brain damage, using experimental models of ischemic (IS) and hemorrhagic (HS) stroke and evaluate the motor spontaneous recovery profile. We used 31 Wistar rats divided into the following groups: Sham (n=7), ischemic (IS) (n=12) or hemorrhagic (HS) (n=12). Brain ischemia or hemorrhage was induced by endotelin-1 (ET-1) and collagenase type IV-S (collagenase) microinjections, respectively. All groups were evaluated in the open field, cylinder and ladder walk behavioral tests at distinct time points as from baseline to 30 days post-surgery (30 PS). Histological and morphometric analyses were used to assess the volume of lost tissue and lesion length. Present results reveal that both forms of experimental stroke had a comparable long-term pattern of damage, since no differences were found in volume of tissue lost or lesion size 30 days after surgery. However, behavioral data showed that hemorrhagic rats were less impaired at skilled walking than ischemic ones at 15 and 30 days post-surgery. We suggest that experimentally comparable stroke design is useful because it reduces heterogeneity and facilitates the assessment of neurobiological differences related to stroke subtypes; and that spontaneous skilled walking recovery differs between experimental ischemic and hemorrhagic insults.

Topics: Animals; Brain; Brain Ischemia; Collagenases; Endothelin-1; Intracranial Hemorrhages; Male; Microinjections; Motor Activity; Motor Skills; Rats; Recovery of Function; Stroke

A comparative analysis of clinical, instrumental and laboratory data in groups of patients with Parkinson's disease and chronic brain ischemia (stages I-II) was carried out. The information obtained in the groups showed significant differences in clinical and instrumental data that indicated the presence of chronic brain ischemia. A significant increase in molecular markers of endothelial damage comparable with the group of chronic cerebral ischemia was identified in patients with Parkinson's disease. The increase in indicators including monocyte chemoattractant protein-1, vascular endothelial growth factor and endothelin-1 is important for the verification of the disease before the appearance of symptoms of chronic cerebral ischemia in the examined groups of patients.

Topics: Aged; Aged, 80 and over; Biomarkers; Brain Ischemia; Chemokine CCL2; Disease Progression; Endothelin-1; Endothelium, Vascular; Female; Humans; Male; Middle Aged; Parkinson Disease; Prognosis; Severity of Illness Index; Vascular Endothelial Growth Factor A; Vasoconstriction

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure ('basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca(2+)-sensitive K(+) channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21 ± 4% versus 3 ± 3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphinato iron (III) chloride) (100 μmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

Topics: Animals; Arterioles; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Endothelium, Vascular; Male; Microcirculation; Muscle Tonus; Muscle, Smooth, Vascular; Peroxynitrous Acid; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Receptor, Endothelin B; Reperfusion Injury; Vasodilation

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Male; Microcirculation; Peroxynitrous Acid; Reperfusion Injury

The aim of this study was to test the insulin-like growth factor-I (IGF-I) as a neuroprotective agent in a rat model for ischemic stroke and to compare its neuroprotective effects in conscious normotensive and spontaneously hypertensive rats. The effects of subcutaneous IGF-I injection were investigated in both rat strains using the endothelin-1 rat model for ischemic stroke. Motor-sensory functions were measured using the Neurological Deficit Score. Infarct size was assessed by Cresyl Violet staining. Subcutaneous administration of IGF-I resulted in significantly reduced infarct volumes and an increase in motor-sensory functions in normotensive rats. In these rats, IGF-I did not modulate blood flow in the striatum and had no effect on the activation of astrocytes as assessed by GFAP staining. In hypertensive rats, the protective effects of IGF-I were smaller and not always significant. Furthermore, IGF-I significantly reduced microglial activation in the cortex of hypertensive rats, but not in normotensive rats. More detailed studies are required to find out whether the reduction by IGF-I of microglial activation contributes to an impairment IGF-I treatment efficacy. Indeed, we have shown before that microglia in hypertensive rats have different properties compared to those in control rats, as they exhibit a reduced responsiveness to ischemic stroke and lipopolysaccharide.

Topics: Animals; Body Weight; Brain Ischemia; Endothelin-1; Glial Fibrillary Acidic Protein; Glucose; Humans; Hypertension; Immunohistochemistry; Injections, Subcutaneous; Insulin-Like Growth Factor I; Laser-Doppler Flowmetry; Macrophage Activation; Male; Microglia; Nervous System Diseases; Neuroprotective Agents; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Recombinant Proteins; Stroke; Telemetry

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.

Topics: Animals; Blotting, Western; Brain Ischemia; Disease Models, Animal; Endothelin-1; Hypothermia, Induced; Male; Nerve Tissue Proteins; Proteomics; Rats; Rats, Wistar; Two-Dimensional Difference Gel Electrophoresis

Improved non-invasive magnetic resonance (MR) characterisation of in vivo models of focal ischaemic insults such as transient ischaemic attack (TIA) and perinatal arterial ischaemic stroke (AIS) may assist diagnosis, outcome prediction and treatment design. The classic middle cerebral artery occlusion (MCAO) model of ischaemic stroke is well documented in MR studies but generates extensive and complex lesions involving an acute inflammatory response and de-occlusion that immediately restores circulation. By contrast, intrastriatal microinjection of the potent vasoconstrictor, endothelin-1 (ET-1), induces a focal, reversible and low-flow ischaemia in the absence of a typical inflammatory response, which gradually restores blood flow over several hours and may be more relevant to TIA and AIS pathology. This study presents the first comprehensive longitudinal MR characterisation of the real-time anatomical [T1-weighted (T1-w)/T2-weighted (T2-w)], pathophysiological [apparent diffusion coefficient (ADC), cerebral blood volume, gadolinium contrast imaging of blood-brain barrier (BBB) integrity] and metabolic [phosphorus magnetic resonance spectroscopy (31P MRS)] evolution of a purely ischaemic ET-1-induced lesion within the juvenile and adult rat brain. ET-1-induced cytotoxic oedema was visualised on T2-w magnetic resonance imaging (MRI), inconsistent with the conventional notion that it cannot be detected using anatomical MRI. There was no immunohistochemical evidence of an acute inflammatory response or loss of BBB integrity, thus excluding a vasogenic oedema contribution to the pathology. Maximal T2-w intensity correlated with the lowest ADC value in both age groups, re-emphasising the purely ischaemic nature of the lesion and the absence of vasogenic oedema. Furthermore, extensive acute T1-w hypointensity was observed in the presence of cytotoxic oedema-induced T2-w changes, whereas other authors have shown that increased T1 values following MCAO reflect vasogenic oedema. Intriguingly, the lesion border exhibited hyperintensity on T2-w and ADC MRI at later time points, and the former may be a consequence of phagocytosis-induced fatty droplet deposition by macrophages detected immunohistochemically. In spite of a chronically reduced ADC, typically associated with ischaemia-induced energy failure, a 31P MRS-detectable reduction in the phosphocreatine (PCr) to gamma adenosine triphosphate (γATP) ratio was not observed at any time point in either age group, su

Topics: Analysis of Variance; Animals; Blood Volume; Blood-Brain Barrier; Brain Ischemia; Cerebrovascular Circulation; Diffusion Magnetic Resonance Imaging; Endothelin-1; Injections, Intraventricular; Magnetic Resonance Spectroscopy; Male; Phosphorus Isotopes; Radiopharmaceuticals; Rats; Rats, Wistar; Vasoconstrictor Agents

Neurodegeneration is a hallmark of most of the central nervous system (CNS) disorders including stroke. Recently inflammation has been implicated in pathogenesis of neurodegeneration and neurodegenerative diseases. The aim of this study was analysis of expression of several inflammatory markers and its correlation with development of neurodegeneration during the early stage of experimental stroke. Ischemic stroke model was induced by stereotaxic intracerebral injection of vasoconstricting agent endothelin-1 (ET-1). It was observed that neurodegeneration appears very early in that model and correlates well with migration of inflammatory lymphocytes and macrophages to the brain. Although the expression of several studied chemotactic cytokines (chemokines) was significantly increased at the early phase of ET-1 induced stroke model, no clear correlation of this expression with neurodegeneration was observed. These data may indicate that chemokines do not induce neurodegeneration directly. Upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke. Inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration.

Topics: Animals; Brain; Brain Ischemia; Chemokine CCL2; Chemokine CCL3; Chemokine CCL5; Chemokine CXCL2; Chemokines; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Profiling; Gene Expression Regulation; Inflammation; Lymphocytes; Macrophages; Mice; Neurodegenerative Diseases; Real-Time Polymerase Chain Reaction; Stroke

The occurrence of stroke exhibits a strong circadian pattern with a peak in the morning hours after waking. The factors that influence this pattern of stroke prevalence may confer varying degrees of neuroprotection and therefore influence stroke severity. This question is difficult to address in clinical cases because of the variability in the location and duration of the ischemic event.. The purpose of this study was to determine if time of day affected the severity of stroke targeting the motor cortex in rats. Strokes were produced using topical application of the vasoconstrictor endothelin-1 to motor cortex of unanesthetized animals at 2 time points: early day and early night. Behavioral deficits were measured using reaching, cylinder, and horizontal ladder tasks, and the volume of the lesion was quantified.. Behavior on reaching and horizontal ladder tasks were both severely impaired by endothelin-1 treatment compared to vehicle-treated animals, but deficits did not differ according to time of treatment. Similarly, while endothelin-1 produced larger lesions of the motor cortex than did vehicle treatment, the size of the lesion did not differ according to time of treatment.. These results suggest that while many factors under circadian control can influence the prevalence of stroke, the magnitude of lesion and behavioral deficit resulting from an ischemic event may not be influenced by time of day.

Topics: Animals; Behavior, Animal; Brain Ischemia; Circadian Rhythm; Disease Models, Animal; Endothelin-1; Motor Activity; Motor Cortex; Rats; Rats, Long-Evans; Stroke; Time Factors

We explored whether the modulation of microglia activation with minocycline is beneficial to the therapeutic actions of bone marrow mononuclear cells (BMMCs) transplanted after experimental stroke. Male Wistar adult rats were divided in four experimental groups: ischemic control saline treated (G1, N = 6), ischemic minocycline treated (G2, N = 5), ischemic BMMC treated (G3, N = 5), and ischemic minocycline/BMMC treated (G4, N = 6). There was a significant reduction in the number of ED1+ cells in G3 animals (51.31 ± 2.41, P < 0.05), but this effect was more prominent following concomitant treatment with minocycline (G4 = 29.78 ± 1.56). There was conspicuous neuronal preservation in the brains of G4 animals (87.97 ± 4.27) compared with control group (G1 = 47.61 ± 2.25, P < 0.05). The behavioral tests showed better functional recovery in animals of G2, G3, and G4, compared with G1 and baseline (P < 0.05). The results suggest that a proper modulation of microglia activity may contribute to a more permissive ischemic environment contributing to increased neuroprotection and functional recovery following striatal ischemia.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Brain Ischemia; Cells, Cultured; Endothelin-1; Macrophage Activation; Macrophages; Male; Microglia; Minocycline; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Stroke

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

Constraint-induced movement therapy (CIMT), which forces use of the impaired arm following unilateral stroke, promotes functional recovery in the clinic but animal models of CIMT have yielded mixed results. The aim of this study is to develop a refined endothelin-1 (ET-1) model of focal ischemic injury in rats that resulted in reproducible, well-defined lesions and reliable upper extremity impairments, and to determine if an appetitively motivated form of rehabilitation (voluntary forced use movement therapy; FUMT) would accelerate post-ischemic motor recovery.. Male Sprague Dawley rats (3 months old) were given multiple intracerebral microinjections of ET-1 into the sensorimotor cortex and dorsolateral striatum. Sham-operated rats received the same surgical procedure up to but not including the drill holes on the skull. Functional deficits were assessed using two tests of forelimb placing, a forelimb postural reflex test, a forelimb asymmetry test, and a horizontal ladder test. In a separate experiment ET-1 stroke rats were subjected to daily rehabilitation with FUMT or with a control therapy beginning on post-surgery d 5. Performance and post-mortem analysis of lesion volume and regional BDNF expression were measured.. Following microinjections of ET-1 animals exhibited significant deficits in contralateral forelimb function on a variety of tests compared with the sham group. These deficits persisted for up to 20 d with no mortality and were associated with consistent lesion volumes. FUMT therapy resulted in a modest but significantly accelerated recovery in the forelimb function as compared with the control therapy, but did not affect lesion size or BDNF expression in the ipsilesional hemisphere.. We conclude that refined ET-1 microinjection protocols and forcing use of the impaired forelimb in an appetitively motivated paradigm may prove useful in developing strategies to study post-ischemic rehabilitation and neuroplasticity.

Topics: Animals; Brain; Brain Ischemia; Brain-Derived Neurotrophic Factor; Endothelin-1; Forelimb; Male; Physical Therapy Modalities; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Recovery of Function

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Topics: Aged; Aged, 80 and over; Biomarkers; Brain Ischemia; Cerebral Hemorrhage; Endothelial Cells; Endothelin-1; Female; Humans; Male; Middle Aged; Stem Cells; Stroke; Up-Regulation

Increased level of endothelin-1 (ET-1), a potent vasoconstrictor, has been found in the cerebral spinal fluid (CSF) of patients with multi-infarction dementia, suggesting a possible role of ET-1 in cognitive deficit associated with stroke. Previously, we have reported that synthesis of ET-1 is induced in endothelial cells in hypoxic/ischemic conditions. Transgenic mice over-expressing endothelin-1 in endothelial cells (TET-1) developed systemic hypertension and showed more severe brain damage after transient ischemia. To further understand the significance of endothelial ET-1 in cognitive deficit, we subjected adult TET-1 mice to 30 min middle cerebral artery occlusion (MCAO) with 7 days reperfusion. At baseline, TET-1 mice showed similar locomotor activity, emotion and cognitive function compared to non-transgenic (NTg) mice. However, after 30 min MCAO and 7 days reperfusion, although the sensorimotor function measured by neurological scores was recovered in both genotypes, TET-1 mice showed increased anxiety-like behavior in the open field test and impaired spatial learning and reference memory in the Morris water maze. Parallel with these behavioral changes, TET-1 mice showed more severe brain damage with blood-brain-barrier breakdown (BBB), reactive astrogliosis, increased caspase-3, and increased peroxiredoxin 6 (Prx6) expressions around blood vessels in the ipsilateral hippocampus, compared to that of NTg mice, suggesting that ET-1 over-expression in the endothelial cells leads to more severe BBB breakdown and increased oxidative stress which may resulted in neuronal apoptosis and glial reactivity, which might contribute to the emotional changes and cognitive deficits after short-term ischemia with long-term reperfusion.

Topics: Animals; Anxiety; Behavior, Animal; Blood-Brain Barrier; Brain Ischemia; Caspase 3; Cognition Disorders; Endothelin-1; Hippocampus; Infarction, Middle Cerebral Artery; Memory Disorders; Mice; Mice, Transgenic; Oxidative Stress; Peroxiredoxin VI; Reperfusion Injury

Bradykinesia in upper extremities is associated with a wide variety of motor disorders; however, there are few tasks that assay forelimb movement speed in rodent models. This study describes the bradykinesia assessment task, a novel method to quantitatively measure forelimb speed in rats. Rats were trained to reach out through a narrow slot in the cage and rapidly press a lever twice within a predefined time window to receive a food reward. The task provides measurement of multiple parameters of forelimb function, including inter-press interval, number of presses per trial, and success rate. The bradykinesia assessment task represents a significant advancement in evaluating bradykinesia in rat models because it directly measures forelimb speed. The task is fully automated, so a single experimenter can test multiple animals simultaneously with typically in excess of 300 trials each per day, resulting in high statistical power. Several parameters of the task can be modified to adjust difficulty, which permits application to a broad spectrum of motor dysfunction models. Here we show that two distinct models of brain damage, ischemic lesions of primary motor cortex and hemorrhagic lesions of the dorsolateral striatum, cause impairment in all facets of performance measured by the task. The bradykinesia assessment task provides insight into bradykinesia and motor dysfunction in multiple disease models and may be useful in assessing therapies that aim to improve forelimb function following brain damage.

Topics: Animals; Brain Ischemia; Cerebral Hemorrhage; Conditioning, Operant; Corpus Striatum; Disease Models, Animal; Endothelin-1; Equipment Design; Female; Forelimb; Hypokinesia; Microbial Collagenase; Motor Cortex; Movement Disorders; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Time Factors

Hypothermia is a promising neuroprotective therapy that has been shown to reduce apoptosis after an ischemic insult. This study evaluated the effect of mild hypothermia on activated caspase-3 up to 1 week after the induction of a stroke. Endothelin-1 (Et-1) was used to elicit transient focal cerebral ischemia in rats. Twenty minutes after the ischemic insult, a state of mild hypothermia (33°C) was imposed for a duration of 2h. The functional outcome, infarct volume and activated caspase-3 immunoreactivity (IR) were assessed at 8, 24 and 72h, and one week after the insult. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry. Hypothermia improved the neurological outcome at all of the time points studied compared to the normothermic group, and was associated with a reduction in infarct volume. In both groups, activated caspase-3 IR peaked 24h after the Et-1 induced insult and hypothermia significantly reduced the number of apoptotic cells at 8h, 24h and 1 week after ischemia. Furthermore, the hypothermic treatment did not affect the CBF in the Et-1 model. These findings indicate that in the Et-1 model, hypothermia exerts a long lasting effect on stroke-induced apoptosis.

Topics: Animals; Brain Ischemia; Caspase 3; Cerebral Cortex; Endothelin-1; Hypothermia, Induced; Male; Neurons; Rats; Rats, Wistar; Recovery of Function

NKN reduces brain edema, neuronal death and neurological deficits in rats after cerebral ischemia. In the present study, we investigated whether NKN was effective on brain injury in cerebral ischemia rats.. The middle cerebral artery occlusion (MCAO) model was used to produce experimental cerebral ischemia in adult male Wistar rats. The activity of SOD and concentration of MDA were determined. We also examined the efficacy of NKN on neurological deficit scores, expression of N-methyl-D-aspartate receptor and endothelin-1 mRNA in brain of focal cerebral ischemia rats.. The results show that NKN significantly increased the activity of SOD, decreased the concentration of MDA, decreased neurological deficit scores, inhibited the expression of N-methyl-D-aspartate receptor and endothelin-1 mRNA in brain of focal cerebral ischemia rats.. The results implied NKN could protect brain against injury caused by cerebral ischemia.

Topics: Abietanes; Acetophenones; Animals; Benzoates; Brain Ischemia; Bridged-Ring Compounds; Drug Combinations; Endothelin-1; Glucosides; Immunohistochemistry; In Situ Hybridization; Infarction, Middle Cerebral Artery; Male; Malondialdehyde; Neurologic Examination; Neurons; Neuroprotective Agents; Pyrazines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Stroke; Superoxide Dismutase

Endothelin (ET) receptor A antagonism decreases neuronal damage in experimental models of stroke. Since large arteries like basilar artery contribute significantly to total cerebrovascular resistance and are major determinants of microvascular pressure, dysregulation of basilar artery function may worsen stroke injury. ET-1 is involved in the regulation of basilar constriction. However, whether stroke influences vasoreactivity of basilar artery and to what extent ET-1 contributes to basilar vascular dysfunction after stroke remained unknown. The goal of this study was to test the hypothesis that ET-1 impairs basilar artery vasorelaxation after ischemia/reperfusion (I/R) injury via activation of ET(A) receptor.. Male Wistar rats were subjected to 3h middle cerebral artery occlusion (MCAO) and 21 h reperfusion. One group received ET(A) receptor antagonist atrasentan (5 mg/kg, i.p.) at reperfusion. At 24h, basilar arteries were isolated from control non-stroked, stroked and stroked+atrasentan-treated animals for vascular reactivity measurements using pressurized arteriograph.. Acetylcholine (Ach)-induced maximum relaxation (R(max)) was decreased in stroked animals as compared to non-stroked group and ET(A) antagonism partially restored it. There was also a trend for decreased EC(50) value for the antagonist treatment group indicating improved Ach sensitivity.. These findings suggest that I/R not only affects vessels distal to the occlusion but also impairs relaxation of proximal large vessels. ET-1-mediated basilar artery dysfunction may contribute to neurovascular damage after stroke and early restoration of vascular function by ET receptor antagonism after I/R injury may offer a therapeutic strategy.

Topics: Acetylcholine; Animals; Atrasentan; Basilar Artery; Brain Ischemia; Disease Models, Animal; Endothelin A Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Male; Pyrrolidines; Rats; Rats, Wistar; Receptor, Endothelin A; Reperfusion Injury; Stroke; Vasodilation

Ischemic white matter injury has been relatively little studied despite its importance to the outcome of stroke. To aid such research a new rat model has been developed in vivo and used to assess whether blockade of the sodium/calcium exchanger is effective in protecting central axons from ischemic injury. Vasoconstrictive agent endothelin-1 was injected into the rat spinal cord to induce ischemia. KB-R7943 or SEA0400 was administered systemically to block the operation of the sodium/calcium exchanger. Endothelin-1 caused profound reduction of local blood perfusion and resulted in a prompt loss of axonal conduction. Whereas recovery of conduction following vehicle administration was only to 10.5 ± 9% of baseline (n = 8) 4.5 h after endothelin-1 injection, recovery following KB-R7943 (30 mg/kg, i.a.) administration was increased to 35 ± 9% of baseline (n = 6; P < 0.001). SEA0400 (30 mg/kg, i.a.) was also protective (33.2 ± 6% of baseline, n = 4; P < 0.001). Neither drug improved conduction by diminishing the severity of the ischemia. The protective effect of KB-R7943 persisted for at least 3 days after ischemia, as it improved axonal conduction (76.3 ± 11% for KB-R7943 vs. 51.0 ± 19% for vehicle; P < 0.01) and reduced lesion area (55.6 ± 15% for KB-R7943 vs. 77.9 ± 9% for vehicle; P < 0.01) at this time. In conclusion, a new model of white matter ischemia has been introduced suitable for both structural and functional studies in vivo. Blocking the sodium/calcium exchanger protects central axons from ischemic injury in vivo.

Topics: Aniline Compounds; Animals; Axons; Brain; Brain Ischemia; Electrophysiological Phenomena; Endothelin-1; Immunohistochemistry; Neural Conduction; Neuroprotective Agents; Phenyl Ethers; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Thiourea

For successful translation to clinical stroke studies, the Stroke Therapy Academic Industry Round Table criteria have been proposed. Two important criteria are testing of therapeutic interventions in conscious animals and the presence of a co-morbidity factor. We chose to work with hypertensive rats since hypertension is an important modifiable risk factor for stroke and influences the clinical outcome. We aimed to compare the susceptibility to ischemia in hypertensive rats with those in normotensive controls in a rat model for induction of ischemic stroke in conscious animals.. The vasoconstrictor endothelin-1 was stereotactically applied in the vicinity of the middle cerebral artery of control Wistar Kyoto rats (WKYRs) and spontaneously hypertensive rats (SHRs) to induce a transient decrease in striatal blood flow, which was measured by the laser Doppler technique. Infarct size was assessed histologically by cresyl violet staining. Sensory-motor functions were measured at several time points using the neurological deficit score. Activation of microglia and astrocytes in the striatum and cortex was investigated by immunohistochemistry using antibodies against CD68/Iba-1 and glial fibrillary acidic protein.. The SHRs showed significantly larger infarct volumes and more pronounced sensory-motor deficits, compared to the WKYRs at 24 h after the insult. However, both differences disappeared between 24 and 72 h. In SHRs, microglia were less susceptible to activation by lipopolysaccharide and there was a reduced microglial activation after induction of ischemic stroke. These quantitative and qualitative differences may be relevant for studying the efficacy of new treatments for stroke in accordance to the Stroke Therapy Academic Industry Round Table criteria.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Corpus Striatum; Down-Regulation; Endothelin-1; Genetic Predisposition to Disease; Hypertension; Lipopolysaccharides; Male; Microglia; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Stroke

Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

Topics: Animals; Brain; Brain Ischemia; Endothelial Cells; Endothelin-1; Male; Motor Activity; Nerve Fibers, Myelinated; Rats; Rats, Sprague-Dawley; Recovery of Function

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

Stroke remains one of the most common diseases with a serious impact on quality of life but few effective treatments exist. Mild hypothermia (33°C) is a promising neuroprotective therapy in stroke management. This study investigated whether a delayed short mild hypothermic treatment is still beneficial as neuroprotective strategy in the endothelin-1 (Et-1) rat model for a transient focal cerebral ischemia. Two hours of mild hypothermia (33°C) was induced 20, 60 or 120 minutes after Et-1 infusion. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry in the striatum, which represents the core of the infarct. Functional outcome and infarct volume were assessed 24 hours after the insult. In this sub-acute phase following stroke induction, the effects of the hypothermic treatment on apoptosis, phagocytosis and astrogliosis were assessed as well. Apoptosis was determined using caspase-3 immunohistochemistry, phagocytic cells were visualized by CD-68 expression and astrogliosis was studied by glial fibrillary acidic protein (GFAP) staining.. Cooling could be postponed up to 1 hour after the onset of the insult without losing its positive effects on neurological deficit and infarct volume. These results correlated with the caspase-3 staining. In contrast, the increased CD-68 expression post-stroke was reduced in the core of the insult with all treatment protocols. Hypothermia also reduced the increased levels of GFAP staining, even when it was delayed up to 2 hours after the insult. The study confirmed that the induction of the hypothermia treatment in the Et-1 model does not affect the CBF.. These data indicate that in the Et-1 rat model, a short mild hypothermic treatment delayed for 1 hour is still neuroprotective and correlates with apoptosis. At the same time, hypothermia also establishes a lasting inhibitory effect on the activation of astrogliosis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Brain Infarction; Brain Ischemia; Caspase 3; Cell Count; Cerebrovascular Circulation; Disease Models, Animal; Endothelin-1; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Hypothermia, Induced; Laser-Doppler Flowmetry; Male; Movement Disorders; Neurologic Examination; Random Allocation; Rats; Rats, Wistar; Statistics as Topic; Time Factors

Endothelin-1 (ET-1) is involved on the development of cerebral edema in acute ischemic stroke. As edema is a therapeutic target in cerebral ischemia, our aim was to study the effect of antagonists for ET-1 receptors (Clazosentan® and BQ-788, specific antagonists for receptors A and B, respectively) on the development of edema, infarct volume and sensorial-motor deficits in rats subjected to ischemia by occlusion of the middle cerebral artery (MCAO). We used Wistar rats (280-320 g) submitted to ischemia by intraluminal transient (90 min) MCAO. After ischemia, rats were randomized into 4 groups (n = 6) treated with; 1) control group (saline), 2) Clazosentan® group (10 mg/kg iv), 3) BQ-788 group (3 mg/kg iv), and 4) combined treatment (Clazosentan® 10 mg/kg plus BQ-788 3 mg/kg iv). We observed that rats treated with Clazosentan® showed a reduction of edema, measured by MRI, at 72 h (hours) and at day 7 (both p < 0.0001), and a decrease in the serum levels of ET-1 at 72 h (p < 0.0001) and at day 7 (p = 0.009). The combined treatment also induced a reduction of edema at 24 h (p = 0.004), 72 h (p < 0.0001) and at day 7 (p < 0.0001), a reduction on infarct volume, measured by MRI, at 24 and 72 h, and at day 7 (all p < 0.01), and a better sensorimotor recovery at 24 and 72 h, and at day 7 (all p < 0.01). Moreover, Clazosentan® induced a decrease in AQP4 expression, while BQ-788 induced an increase in AQP9 expression. These results suggest that antagonists for ET-1 receptors may be a good therapeutic target for cerebral ischemia.

Topics: Animals; Aquaporins; Blotting, Western; Brain Edema; Brain Ischemia; Dioxanes; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Evoked Potentials, Somatosensory; Image Processing, Computer-Assisted; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Male; Nervous System Diseases; Neuroprotective Agents; Oligopeptides; Piperidines; Pyridines; Pyrimidines; Rats; Rats, Wistar; Stroke; Sulfonamides; Tetrazoles

The markers of regulation vascular tone, such as rennin, endothelin-1, and C-type natriuretic peptide, are of great value for prognosis of hemorrhagic transformation and fatal outcome of ischemic stroke. A change in the vascular tone in case of hemorrhagic transformation at the affected site precedes activation of the coagulation component of hemostasis as a mechanism preventing blood loss and increasing fibrinogen level. This work was aimed to study the balance of the above markers and fibrinogen in the prognosis of hemorrhagic transformation and fatal outcome in the acute period of ischemic stroke. It included 62 patients receiving no thrombolytic therapy. It was shown that symptomatic hemorrhagic transformation was associated with elevated rennin levels without a marked fall in the level of C-type natriuretic peptide and asymptomatic hemorrhagic transformation with elevated endothelin-1 levels and decreased concentration of natriuretic peptide. Fibrinogen level on day 4 of the observation proved to be a reliable predictor of negative prognosis. Asymptomatic hemorrhagic transformation without fatal outcome was associated with systemic and local vasoconstriction and inhibition of local vasodilation. Symptomatic hemorrhagic transformation with the fatal outcome was accompanied by dysregulation of vascular tone in the form of activation of systemic and local vasoconstriction, insufficient inhibition of local vasodilation and compensatory reaction in the form of activation of hemostatic mechanisms manifest as elevated fibrinogen levels on day 4. The lethal outcome without hemorrhagic transformation was associated with systemic vasoconstriction, activation of local vasodilation and vasoconstriction leading to local "biochemical paralysis" of vascular tone regulation.

Topics: Aged; Aged, 80 and over; Biomarkers; Blood Coagulation; Brain Ischemia; Cerebral Hemorrhage; Chymosin; Endothelin-1; Female; Fibrinogen; Humans; Male; Middle Aged; Natriuretic Peptide, C-Type; Predictive Value of Tests; Prognosis; Stroke; Survival Analysis; Vasomotor System

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

Topics: Brain Ischemia; C-Reactive Protein; Endothelin-1; Humans; Stroke

Visual and neurological disturbances have always been reported following liquid sclerotherapy (LS) for venous insufficiency. In 1993 Cabrera introduced foam sclerotherapy (FS) using a detergent sclerosant as Lauromacrogol 400 or sodium tetradecyl sulphate. Several authors have reported with FS an increased incidence of such transient visual disturbances and neurological complications. This has been associated with gas or air used to generate the sclerosing foam. The frequent association of the presence of a patent foramen ovale, a common condition in normal population, and such complications has led several authors to consider neurological and visual disturbances as paradoxical gas embolism.. We are introducing a new pathogenetic hypothesis for sclerotherapy complications. Medical literature shows evidence of a clear relationship among cerebral and retinal vasospasm, migraine and intimal irritation. We think that the irritating sclerosant agent may stimulate a significant release of vasoactive substances from the venous wall, specifically endothelin 1 (ET-1), the most powerful vasoconstricting agent.. We have studied systemic ET-1 levels after LS and FS with Lauromacrogol 400 in a group of 13 rats at one and five minutes after injection.. While ET-1 levels did not change significantly in control and in the LS group, a significant increase was detected after FS at one and five minutes.. We conclude that should the same results be found in patients treated using sclerosing foam (SF), ET-1 levels may closely correlate to the onset of visual or cerebral complications. Due to the bronchoconstrictor activity of ET-1, a relationship with post-treatment cough can be also postulated.

Topics: Animals; Brain Ischemia; Cell Movement; Disease Models, Animal; Endothelin-1; Endothelins; Gases; Humans; Migraine with Aura; Rats; Retina; Sclerotherapy; Vision Disorders

Most work on ischemia-induced neuronal death has revolved around the relative contributions of necrosis and apoptosis, but this work has not accounted for the role of ischemia-induced stress responses. An expanded view recognizes a competition between ischemia-induced damage mechanisms and stress responses in the genesis of ischemia-induced neuronal death. An important marker of post-ischemic stress responses is inhibition of neuronal protein synthesis, a morphological correlate of which is the compartmentalization of mRNA away from ribosomes in the form of cytoplasmic mRNA granules.. Here we assessed the generality of this mRNA granule response following either 10 or 15 minutes global brain ischemia and 1 hour reperfusion, 4 hours focal cerebral ischemia alone, and endothelin 1 intraventricular injection.. Both global and focal ischemia led to prominent neuronal cytoplasmic mRNA granule formation in layer II cortical neurons. In addition, we report here new post-ischemic cellular phenotypes characterized by the loss of nuclear polyadenylated mRNA staining in cortical neurons following endothelin 1 treatment and 15 minutes global ischemia. Both mRNA granulation and loss of nuclear mRNAs occurred in non-shrunken post-ischemic neurons.. Where cytoplasmic mRNA granules generally appear to mark a protective response in surviving cells, loss of nuclear mRNAs may mark cellular damage leading to cell atrophy/death. Hence, staining for total mRNA may reveal facets of the competition between stress responses and damage mechanisms at early stages in post-ischemic neurons.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Endothelin-1; Male; Nerve Degeneration; Phenotype; Rats; Rats, Long-Evans; Reperfusion Injury; RNA, Messenger; Stress, Physiological

Autopsy evidence suggests that the presence of both Alzheimer(')s disease (AD) and cerebral infarction pathology is associated with more severe cognitive impairment than that produced by AD pathology alone. This study aims to investigate the effect of cerebral ischemia on cognitive function in rats with AD constructed by hippocampal injection and to determine its underlying mechanism, which is proposed to be of significance to the treatment of AD.. AD was modeled by injection of aggregated Aβ(1-40), either alone or followed by hippocampal endothelin-1 injection to mimic cerebral ischemia in hippocampus, into the right dentate gyrus (DG) of rats. The Morris water maze was used to evaluate cognitive function. Aβ deposition, neuronal loss and phosphorylated tau expression in hippocampus were examined by Congo red staining, Nissl's staining and immunohistochemistry, respectively. Reactive astrocytes, IL-1β and TNF-α expressions were measured by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction.. Compared with rats treated with either Aβ or endothelin alone, rats treated with both Aβ and endothelin showed more aggravated cognitive impairment and more Aβ deposits, neuron loss, phosphorylated tau expression, reactive astrocytes, IL-1β and TNF-α expressions in hippocampus.. Hippocampal ischemia aggravates cognitive impairment of AD rats by increasing Aβ deposits, neuron loss and tau phosphorylation in hippocampus. The enhanced inflammatory response may be responsible for cerebral ischemia-induced aggravation of cognitive impairment in AD rats. Based on these findings, prevention and treatment of cerebral ischemia may improve clinical symptoms of AD and suppress the progression of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Brain Ischemia; Cognition Disorders; Disease Models, Animal; Drug Therapy, Combination; Endothelin-1; Hippocampus; Interleukin-1beta; Male; Maze Learning; Memory; Neurons; NF-kappa B; Peptide Fragments; Phosphorylation; Rats; Rats, Sprague-Dawley; tau Proteins; Tumor Necrosis Factor-alpha

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

Topics: Animals; Apoptosis; Base Sequence; Brain Ischemia; Caspase 3; Caspase Inhibitors; Cell Line; Cells, Cultured; Endothelin-1; Female; Genetic Therapy; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Nanomedicine; Nanotubes, Carbon; Neurons; Psychomotor Performance; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering

In this study, we validated the use of (99m)Tc-hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) micro-SPECT combined with micro-CT for semiquantification of the infarct size after an experimental stroke in rats and compared our observations with those obtained from histology. This imaging strategy was applied to measure the longitudinal effect of mild hypothermia on the progression of brain damage after stroke in rats.. The endothelin-1 model was used to elicit a transient focal cerebral ischemia in rats. This resulted in a reproducible insult in which the core is represented by the striatum and the penumbra by the cortex. Micro-SPECT and micro-CT images were taken at 1, 3, and 7 d after infusion of endothelin-1 and compared with those taken before the insult. After the last acquisition, rats were sacrificed and the infarct volume was determined via Nissl staining. The results obtained with micro-SPECT and micro-CT were compared with histology at the same time points. Mild hypothermia (33°C) was induced for 2 h, starting 20 min after the insult.. Brain damage was estimated using micro-SPECT and micro-CT and was reproducible with minimal interobserver variability. Normothermic stroke rats had reduced (99m)Tc-HMPAO uptake at 1 and 3 d after the insult, whereas hypothermia improved damage after stroke. These findings corroborate with histology at the same time points. At 1 wk after the insult, no reduction of radioactive uptake was observed in any treatment group.. Micro-SPECT and micro-CT allow quick and reproducible semiquantification of brain damage as an interesting alternative to histology to measure the extent of infarcted tissue in small animals after stroke.

Topics: Animals; Brain Ischemia; Endothelin-1; Hypothermia, Induced; Male; Rats; Rats, Wistar; Reproducibility of Results; Technetium Tc 99m Exametazime; Time Factors; Tomography, Emission-Computed, Single-Photon; X-Ray Microtomography

Approximately 15-20% of patients with giant-cell arteritis (GCA) develop ischaemic complications often preceded by transient ischaemia. The expression of the endothelin (ET) system in GCA lesions was investigated to assess its relationship with the development of ischaemic complications.. Plasma ET-1 was quantified by immunoassay in 61 patients with biopsy-confirmed GCA and 16 healthy donors. ET-1, endothelin-converting enzyme (ECE-1) and endothelin receptor (ET(A)R and ET(B)R) messenger RNA were measured by real-time quantitative reverse transcriptase-PCR in temporal arteries from 35 of these patients and 19 control arteries. Proteins were measured by immunoassay and Western blot.. ET-1 concentration was increased at the protein level in temporal artery samples from GCA patients compared with controls (0.98 (SEM 0.32) vs 0.28 (SEM 0.098) fmol/mg, p = 0.028). ECE-1, ET(A)R and ET(B)R/actin ratios (Western blot) were also significantly higher in GCA patients. Intriguingly, mRNA expression of ET-1, ECE-1 and both receptors was significantly reduced in GCA lesions compared with control arteries. When investigating mechanisms underlying these results, platelet-derived growth factor and IL-1beta, present in GCA lesions, were found to downregulate ET-1 mRNA in cultured human temporal artery-derived smooth muscle cells. Glucocorticoid treatment for 8 days did not result in significantly decreased endothelin tissue concentration (0.87 (SEM 0.2) vs 0.52 (SEM 0.08); p = 0.6). Plasma endothelin concentrations were higher in patients with ischaemic complications (1.049 (SEM 0.48) vs 1.205 (SEM 0.63) pg/ml, p = 0.032).. The endothelin system is increased at the protein level in GCA lesions creating a microenvironment prone to the development of ischaemic complications. Recovery induced by glucocorticoids is delayed, indicating persistent exposure to endothelin during initial treatment.

Topics: Aged; Aged, 80 and over; Aspartic Acid Endopeptidases; Brain Ischemia; Cells, Cultured; Down-Regulation; Endothelin-1; Endothelin-Converting Enzymes; Female; Gene Expression Regulation; Giant Cell Arteritis; Glucocorticoids; Humans; Interleukin-1beta; Male; Metalloendopeptidases; Middle Aged; Muscle, Smooth, Vascular; Optic Neuropathy, Ischemic; Platelet-Derived Growth Factor; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Temporal Arteries

It has been proposed that the immune system is activated during ischemic cerebral events and that brain damage caused by ischemia is increased by this immune activity. Neutrophils (PMNs) are one of the first factors in the chain of reactions of the immune system during focal cerebral ischemia. Experimental and clinical studies have emphasized the important role of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor (TNFalpha), in addition to vasoactive peptide and endothelin-1 (ET-1), in the formation of cerebral ischemia.. The experiments were carried out using Wistar rats that were divided into four groups: three experimental groups (acute and prolonged focal cerebral ischemia and following reperfusion) and one control group (sham). Focal cerebral ischemia was induced by the intraluminal surgical suture method. The oxidative activity of PMNs was measured after stimulation with phorbol myristate acetate, a protein kinase C activator (luminol enhanced chemiluminescence). The concentration of IL-1beta and TNFalpha in rat lymphocyte culture after stimulation with CSF was determined using commercial ELISA kits. The plasma concentration of ET-1 was determined using commercial kits with the RIA method.. We confirmed a statistically significant increase in the oxidative activity of PMNs in rats with acute focal cerebral ischemia (p < 0.00001), prolonged ischemia (p < 0.001) and reperfusion (p < 0.05). An increase in IL-1beta and TNFalpha in lymphocytes following CSF stimulation was observed in the group with prolonged ischemia and in the group with reperfusion after transient ischemia (p < 0.05 for both). An increase in plasma ET-1 concentration was observed with acute and prolonged focal cerebral ischemia (p < 0.05 and p < 0.01, respectively).. Our results show that acute and prolonged focal cerebral ischemia and reperfusion induce statistically significant increases in the oxidative activity of PMNs. The concentration of proinflammatory mediators (IL-1beta, TNFalpha) as well as ET-1 is also increased, indicating the important role of immune reactions in the development of damage to the brain following ischemia.

Topics: Animals; Brain Ischemia; Cells, Cultured; Disease Models, Animal; Endothelin-1; Enzyme Activators; Interleukin-1beta; Male; Neutrophils; Radioimmunoassay; Rats; Rats, Wistar; Reperfusion; Statistics, Nonparametric; Tetradecanoylphorbol Acetate; Time Factors; Tumor Necrosis Factor-alpha; Vasoactive Intestinal Peptide

Despite recent advances, stroke remains a leading cause of neurological disability with the vast majority of victims being the elderly, who exhibit more severe neurological deficits and a reduced capacity to recover from these disabilities in comparison to young stroke survivors. The objective of the present study was to develop a model of focal ischemic stroke in aged rats using endothelin-1 (ET-1) to produce low mortality rates as well as reliable, robust sensorimotor deficits that resemble functional impairments associated with stroke in humans. Here, we studied the functional and histological outcome following unilateral ET-1 infusions into the sensorimotor cortex of aged rats (20-23 months old). This procedure resulted in low mortality rates (13.3%) and no loss in body weight one week following surgery. Functional assessment was performed using a number of reliable behavioural tests: staircase test (fine motor function), horizontal ladder (skilled locomotion), bilateral tactile stimulation test (somatosensory function) and cylinder test (postural weight support). Following ET-1 induced stroke, all tests demonstrated large and sustained sensorimotor deficits in both forelimb and hindlimb function that failed to improve over the 28-day testing period. In addition, histological assessment revealed a substantial loss of retrogradely labelled corticospinal neurons in the ipsilesional hemisphere following stroke. Our results establish a model for the use of aged rats in future preclinical studies, which will enhance assessment of the long-term benefit of potential neural repair and regenerative strategies.

Topics: Aging; Analysis of Variance; Animals; Behavior, Animal; Brain Ischemia; Disease Models, Animal; Endothelin-1; Functional Laterality; Gait Disorders, Neurologic; Male; Neurons; Physical Stimulation; Psychomotor Performance; Pyramidal Tracts; Rats; Rats, Wistar; Statistics, Nonparametric

We reported previously that amoeboid microglial cells (AMC) in the developing brain exhibited endothelins (ETs) expression which diminished with advancing age and was undetected in microglia in the more mature brain. This study sought to explore if microglia in the adult would be induced to express ETs in altered conditions. By immunofluorescence microscopy, ETs and endothelin (ET)-B receptor were undetected in microglial cells in sham-operated and normal control rats. However, in adult rats subjected to middle cerebral artery occlusion (MCAO), lectin labeled activated microglia which occurred in large numbers in the marginal zones in the ischemic cortex at 3 days and 1 week intensely expressed ETs specifically endothelin (ET)-1 and ET-B receptor; ET-3 and ET-A receptor were absent in these cells. By RT-PCR and ELISA, ET-1 and -3 mRNA and protein expression level was progressively increased in the ischemic cerebral cortex after MCAO compared with the controls. ET-A and ET-B receptor mRNA and protein levels were concomitantly up-regulated. It is suggested that increased release of ET-1 following MCAO by massive activated microglia can exert an immediate constriction of local blood vessels bearing ET-A receptor. ET-1 may also interact with activated microglia endowed with ET-B receptor via an autocrine manner that may be linked to chemokines/cytokines production. ET-1, ET-3 and ET-B receptor were also localized in reactive astrocytes along with some oligodendrocytes. We conclude that activated microglia together with other glial cells in the marginal zone after MCAO are the main cellular source of ETs that may be involved in regulation of vascular constriction and glial chemokines/cytokines production. However, dissecting the role of individual component of the endothelin system in the various glial cells, notably activated microglia, would be vital in designing of an effective therapeutic strategy for clinical treatment of stroke in which microglial cells have been implicated.

Topics: Animals; Brain Ischemia; Cell Proliferation; Cytokines; Disease Models, Animal; Encephalitis; Endothelin-1; Endothelin-3; Endothelins; Gliosis; Immunohistochemistry; Infarction, Middle Cerebral Artery; Lectins; Male; Microglia; Rats; Rats, Sprague-Dawley; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Staining and Labeling; Up-Regulation; Vasoconstriction

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

In young adult rats, unilateral lesions of the sensorimotor cortex lead to neuronal structural plasticity and synaptogenesis in the contralateral motor cortex, which is connected to the lesion site by transcallosal fibers. The contralesional neural plasticity varies with lesion size and results from the convergence of denervation-induced reactive plasticity and behavioral asymmetries. It was unknown whether similar effects occur in older animals. Furthermore, the coordination of synaptic responses with that of perisynaptic astrocytes had not been investigated. In this study, middle-aged rats (14-16 months old) were given sham-operations or unilateral ischemic lesions of the sensorimotor cortex. Fifty days later, numerical densities of neurons and synapses and morphological characteristics of astrocytic processes in layer V of the contralesional motor cortex were measured using stereological light and electron microscopy methods. Lesions resulted in behavioral asymmetries, but no significant synapse addition in the contralesional motor cortex. Synapse number per neuron was negatively correlated with lesion size and reduced opposite larger lesions compared with smaller ones. Astrocytic changes were also lesion size-dependent. Astrocytic hypertrophy was observed only after smaller lesions and was associated with greater coverage and greater numbers of synapses. These findings are consistent with those in younger rats indicating an inverse relationship between lesion size and adaptive neuronal restructuring in denervated cortex. However, they indicate that the synaptogenic reaction to this lesion is relatively limited in older animals. Finally, the results indicate that structural plasticity of perisynaptic astrocytes parallels, and could play a role in shaping, synaptic responses to postischemic denervation.

Topics: Animals; Astrocytes; Axons; Behavior, Animal; Brain Ischemia; Cerebral Cortex; Cerebral Infarction; Dendrites; Endothelin-1; Forelimb; Functional Laterality; Male; Microscopy, Electron, Transmission; Motor Cortex; Nerve Degeneration; Neurons; Neuropil; Posture; Rats; Rats, Long-Evans; Somatosensory Cortex; Synapses; Vasoconstrictor Agents

Evidence indicates that accumulation of excitotoxic mediators, such as glutamate, contributes to neuronal damage after an ischaemic insult. It is not clear, however, whether this accumulation is due to excess synaptic release or to impaired uptake. To test a role for synaptic release, here we investigated the neuroprotective potential of the synaptic blocker botulinum neurotoxin E (BoNT/E), that prevents vesicle fusion via the cleavage of the SNARE (soluble NSF-attachment receptor) protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Focal ischaemia was induced in vivo by infusing the potent vasoconstricting peptide endothelin-1 (ET-1) into the CA1 area of the hippocampus in adult rats; BoNT/E or vehicle were administered into the same site 20 min later. Injection of ET-1 was found to produce a transient and massive increase in glutamate release that was potently antagonized by BoNT/E. To assess whether blocking transmitter release translates into neuroprotection, the extent of the ischaemic damage was determined 24 h and 6 weeks after the insult. We found that BoNT/E administration consistently reduced the loss of CA1 pyramidal neurons at 24 h. The neuroprotective effect of BoNT/E, however, was no longer significant at 6 weeks. These data provide evidence that blockade of synaptic transmitter release delays neuronal cell death following focal brain ischaemia, and underline the importance of assessing long-term neuroprotection in experimental stroke studies.

Topics: Animals; Botulinum Toxins; Brain Ischemia; CA1 Region, Hippocampal; Chromatography, High Pressure Liquid; Drug Evaluation, Preclinical; Endothelin-1; Female; Glutamic Acid; Microdialysis; Neuroprotective Agents; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Synaptosomal-Associated Protein 25; Time Factors

Clinical data has shown that stroke exacerbates dementia in Alzheimer's disease (AD) patients. Previous work, combining rat models of AD and stroke have shown that neuroinflammation may be the common mediator between AD and stroke toxicity. This study examined the effects of triflusal (2-acetoxy-4-trifluoromethylbenzoic acid) in APP(23) transgenic mice receiving strokes. Six month-old APP(23) mice over-expressing mutant human amyloid precursor protein (APP) were used to model AD in this study. Unilateral injections of a potent vasoconstrictor, endothelin-1, into the striatum were used to mimic small lacunar infarcts. Immunohistochemical analysis was performed to examine AD-like pathology and inflammatory correlates of stroke and AD. APP(23) mice showed increases in AD-like pathology and inflammatory markers of AD in the cortex and hippocampus. Endothelin-induced ischemia triggered an inflammatory response along with increases in AD pathological markers in the region of the infarct. Triflusal reduced inflammation surrounding the endothelin-induced infarct only. At the dose used, anti-inflammatory treatment may be beneficial in reducing the AD and inflammatory correlates of stroke in a combined AD-stroke mouse model.

Topics: Administration, Oral; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Antigens, CD; Brain Ischemia; Disease Models, Animal; Endothelin-1; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B; Platelet Aggregation Inhibitors; Salicylates; Stroke; tau Proteins; Tumor Necrosis Factor-alpha

Endothelins (ETs) are potent vasoconstrictor and may play a role in the pathophysiology of several cardiovascular diseases. Endothelin-mediated vasoconstriction may enhance ischemic neuronal damage. The study aimed to find out whether the plasma ET-1 levels may serve as marker of early ischemic stroke. Plasma ET-1 levels were tested in 20 patients with acute ischemic stroke, mean age 63.7 +/- 5.03 years, 12 men and 8 women, within 24 hours of stroke onset as compared to 10 sex- and age-matched control subjects; only the patients with normal CT-scan at admission were included in the study. Plasma ET-1 was measured by ELISA. The results were statistically analyzed by Student test and a p < 0.05 (95% CI) was considered statistically significant. ET-1 levels in patients with hemiplegia and normal CT-scan at admission were significantly higher as compared to control group (0.0910 +/- 0.0256 pg/mL vs. 0.0490 +/- 0.0185 pg/mL, p < 0.0001) (95% CI). Ischemic stroke is associated with acute and marked increased levels of ET-1 in plasma. This may reflect enhanced production by damaged endothelial cells within the infarcted lesion. ET-1 may be used as additional marker of cerebral ischemia in selected cases to distinguish between the onset of an ischemic stroke and other non-vascular diseases presenting similar symptoms.

Topics: Aged; Biomarkers; Brain Ischemia; Case-Control Studies; Endothelin-1; Female; Humans; Male; Middle Aged; Stroke

Inhibition of the canonical Wnt pathway has been implicated in the pathophysiology of neuronal death. Here, we report that the secreted Wnt antagonist, Dickkopf-1 (Dkk-1) is rapidly induced in neurons after induction of focal brain ischemia. In rats undergoing transient focal ischemia in response to brain infusion of endothelin-1, Dkk-1 was induced in neurons of the ischemic core and the penumbra region. Induction of Dkk-1 was associated with a reduced expression of beta-catenin (a downstream signaling molecule of the canonical Wnt pathway), and was not observed in neurons expressing the protective protein, heat shock protein-70. Treatment with lithium ions, which, inter alia, rescue the canonical Wnt pathway, was highly protective against ischemic damage. Dkk-1 was also induced in cortical neurons of mice undergoing permanent middle cerebral artery (MCA) occlusion. This model allowed us to compare wild-type mice with doubleridge mice, which are characterized by a reduced expression of Dkk-1. Doubleridge mice showed an attenuated reduction of beta-catenin and a reduced infarct volume in response to MCA occlusion, providing a direct demonstration that Dkk-1 contributes to the pathophysiology of ischemic neuronal damage. These data rise the interesting possibility that Dkk-1 antagonists or drugs that rescue the Wnt pathway might be neuroprotective in stroke.

Topics: Animals; beta Catenin; Brain Ischemia; Cell Death; Cerebral Arteries; Disease Models, Animal; Endothelin-1; Infusions, Intra-Arterial; Intercellular Signaling Peptides and Proteins; Lithium Chloride; Male; Mice; Mice, Inbred C3H; Mice, Transgenic; Rats; Wnt Proteins

A vagus nerve-mediated, efferent cholinergic protective pathway activated by melanocortins is operative in circulatory shock and myocardial ischemia. Moreover, melanocortins have neuroprotective effects against brain damage after ischemic stroke. Here we investigated cerebral and systemic pathophysiologic reactions to focal cerebral ischemia in rats induced by intrastriatal microinjection of endothelin-1, and the possible protective role of the melanocortin-activated vagal cholinergic pathway. In the striatum and liver of saline-treated control rats, the activation of extracellular signal-regulated kinases, c-jun N-terminal kinases, and caspase-3, the increase in tumor necrosis factor-alpha (TNF-alpha) concentration and DNA fragmentation, as well as the increase in TNF-alpha plasma levels, occurred 10 and 20 h after the ischemic insult suggesting an activation of inflammatory and apoptotic responses. Treatment with [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH; 3 or 9 h after stroke) suppressed the inflammatory and apoptotic cascades at central and peripheral level. Bilateral vagotomy and pharmacologic blockade of peripheral nicotinic acetylcholine receptors blunted the protective effect of NDP-alpha-MSH. The present results show that focal brain ischemia in rats causes significant effects not only in the brain, but also in the liver. Moreover, our data support the hypothesis that a protective, melanocortin-activated, vagal cholinergic pathway is likely operative in conditions of ischemic stroke.

Topics: alpha-MSH; Animals; Apoptosis; Blotting, Western; Brain Ischemia; Corpus Striatum; DNA Fragmentation; Endothelin-1; Liver; Male; Rats; Rats, Wistar; Receptors, Nicotinic; Stroke; Tumor Necrosis Factor-alpha; Vagotomy; Vagus Nerve

Stroke is the second most common cause of death and major cause of disability worldwide. Actual treatment involves surgery and/or thrombolytic drugs, but there is an urgent need for new approaches. Periodic acceleration, a rocking headward to footward movement of the whole body, is a non-invasive method to induce pulsatile shear stress on the vascular endothelium eliciting an enhanced production and secretion of endothelium-derived products such as nitric oxide, prostacyclin, prostaglandin E2, tissue plasminogen activator (tPA), and adrenomedullin. All these products have been shown to protect the brain from ischemic injuries. A rat model of focal brain ischemia was treated with application of periodic acceleration for 3 h immediately after the onset of ischemia. Controls remained static for the same period of time. Brain damage was assessed by magnetic resonance imaging (MRI) and biochemical markers. A significant reduction in brain damage was observed, 7 days post-ischemia, in rocked rats when compared with the static controls, through MRI. Furthermore, rocked animals had significantly lower levels of Beclin 1 and fractin than their static counterparts, and some isoforms of nitric oxide synthase were regulated by periodic acceleration. Our results show that periodic acceleration may provide a novel, affordable, non-invasive therapeutic option for the treatment of stroke.

Topics: Acceleration; Animals; Apoptosis Regulatory Proteins; Beclin-1; Brain Injuries; Brain Ischemia; Caspase 3; Disease Models, Animal; Endothelin-1; Exercise Therapy; Gene Expression Regulation; Magnetic Resonance Imaging; Male; Nitric Oxide Synthase; Periodicity; Rats; Rats, Wistar; Time Factors

Endogenous levels of angiotensin II (Ang II) are increased in the cortex and hypothalamus following stroke, and Ang II type 1 receptor blockers (ARBs) have been shown to attenuate the deleterious effects in animal stroke models using middle cerebral artery (MCA) intraluminal occlusion procedures. However, the endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO) model of cerebral ischaemia is thought to more closely mimic the temporal events of an embolic stroke. This method provides rapid occlusion of the MCA and a gradual reperfusion that lasts for 16-22 h. The aim of the present study was to evaluate whether systemic administration of an ARB prior to ET-1-induced MCAO would provide cerebroprotection during this model of ischaemic stroke. Injection of 3 microl of 80 microM ET-1 adjacent to the MCA resulted in complete occlusion of the vessel that resolved over a period of 30-40 min. Following ET-1-inducedMCAO, rats had significant neurological impairment, as well as an infarct that consisted of 30% of the ipsilateral grey matter. Systemic pretreatment with 0.2 mg kg(-1) day(-1) candesartan for 7 days attenuated both the infarct size and the neurological deficits caused by ET-1-induced MCAO without altering blood pressure. This study confirms the cerebroprotective properties of ARBs during ischaemic stroke and validates the ET-1-induced MCAO model for examination of the role of the brain renin-angiotensin system in ischaemic stroke.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Endothelin-1; Infarction, Middle Cerebral Artery; Male; Rats; Rats, Sprague-Dawley; Stroke; Tetrazoles

Intracerebral injection of the vasoconstrictor peptide, endothelin-1 (ET-1), has been used as a method to induce focal ischemia in rats. The relative technical simplicity of this model makes it attractive for use in mice. However, the effect of ET-1 on mouse brains has not been firmly established. In this study, we determined the ability of ET-1 to induce focal cerebral ischemia in four different mouse strains (CD1, C57/BL6, NOD/SCID, and FVB). In contrast to rats, intracerebral injection of ET-1 did not produce a lesion in any mouse strain tested. A combination of ET-1 injection with either CCA occlusion or N(G)-nitro-l-arginine methyl ester (l-NAME) injection produced only a small infarct and its size was strain-dependent. A triple combination of CCA occlusion with co-injection of ET-1 and l-NAME produced a lesion in all mouse strains tested, and this resulted in a significant motor deficit. However, lesion size was still relatively small and strain-dependent. This study shows that ET-1 has a much less potent effect for producing an infarct in mice than rats.

Topics: Animals; Brain Infarction; Brain Ischemia; Carotid Artery Diseases; Cerebral Arteries; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, SCID; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Species Specificity; Vasoconstriction; Vasoconstrictor Agents

Increases in the T(1) of brain tissue, which give rise to dark or hypointense areas on T(1)-weighted images using magnetic resonance imaging (MRI), are common to a number of neuropathologies including multiple sclerosis (MS) and ischaemia. However, the biologic significance of T(1) increases remains unclear. Using a multiparametric MRI approach and well-defined experimental models, we have experimentally induced increases in tissue T(1) to determine the underlying cellular basis of such changes. We have shown that a rapid acute increase in T(1) relaxation in the brain occurs in experimental models of both low-flow ischaemia induced by intrastriatal injection of endothelin-1 (ET-1), and excitotoxicity induced by intrastriatal injection of N-methyl-D-aspartate (NMDA). However, there appears to be no consistent correlation between increases in T(1) relaxation and changes in other MRI parameters (apparent diffusion coefficient, T(2) relaxation, or magnetisation transfer ratio of tissue water). Immunohistochemically, one common morphologic feature shared by the ET-1 and NMDA models is acute astrocyte activation, which was detectable within 2 h of intracerebral ET-1 injection. Pretreatment with an inhibitor of astrocyte activation, arundic acid, significantly reduced the spatial extent of the T(1) signal change induced by intrastriatal ET-1 injection. These findings suggest that an increase in T(1) relaxation may identify the acute development of reactive astrocytes within a central nervous system lesion. Early changes in T(1) may, therefore, provide insight into acute and reversible injury processes in neurologic patients, such as those observed before contrast enhancement in MS.

Topics: Animals; Astrocytes; Brain Ischemia; Endothelin-1; Magnetic Resonance Imaging; N-Methylaspartate; Nervous System Diseases

The study aim was to assess the effects of magnesium sulfate (MgSO(4)) administration on white matter damage in vivo in spontaneously hypertensive rats.. The left internal capsule was lesioned by a local injection of endothelin-1 (ET-1; 200 pmol) in adult spontaneously hypertensive rats. MgSO(4) was administered (300 mg/kg SC) 30 minutes before injection of ET-1, plus 200 mg/kg every hour thereafter for 4 hours. Infarct size was measured by T2-weighted magnetic resonance imaging (day 2) and histology (day 11), and functional recovery was assessed on days 3 and 10 by the cylinder and walking-ladder tests.. ET-1 application induced a small, localized lesion within the internal capsule. Despite reducing blood pressure, MgSO(4) did not significantly influence infarct volume (by magnetic resonance imaging: median, 2.1 mm(3); interquartile range, 1.3 to 3.8, vs 1.6 mm(3) and 1.2 to 2.1, for the vehicle-treated group; by histology: 0.3 mm(3) and 0.2 to 0.9 vs 0.3 mm(3) and 0.2 to 0.5, respectively). Significant forelimb and hindlimb motor deficits were evident in the vehicle-treated group as late as day 10. These impairments were significantly ameliorated by MgSO(4) in both cylinder (left forelimb use, P<0.01 and both-forelimb use, P<0.03 vs vehicle) and walking-ladder (right hindlimb score, P<0.02 vs vehicle) tests.. ET-1-induced internal capsule ischemia in spontaneously hypertensive rats represents a good model of lacunar infarct with small lesion size, minimal adverse effects, and a measurable motor deficit. Despite inducing mild hypotension, MgSO(4) did not significantly influence infarct size but reduced motor deficits, supporting its potential utility for the treatment of lacunar infarct.

Topics: Animals; Anticonvulsants; Blood Pressure; Brain Infarction; Brain Ischemia; Disease Models, Animal; Endothelin-1; Hypertension; Internal Capsule; Magnesium; Magnesium Sulfate; Male; Motor Activity; Nerve Fibers, Myelinated; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley

Following acute and chronic neurodegenerative disorders, a cascade of pathological events including inflammatory response, excitotoxicity and oxidative stress induces secondary tissue loss in both gray and white matter. Axonal damage and demyelination are important components of the white matter demise during these diseases. In spite of this, a few studies have addressed the patterns of inflammatory response, axonal damage and demyelination following focal ischemic damage to the central nervous system (CNS). In the present study, we describe the patterns of inflammatory response, axonal damage and myelin impairment following microinjections of 10 pmol of endothelin-1 into the rat striatum. Animals were perfused at 1 day, 3 days and 7 days after injection. 20 mum sections were stained by hematoxylin and immunolabeled for neutrophils (anti-MBS-1), activated macrophages/microglia (anti-ED1), damaged axons (anti-betaAPP) and myelin (anti-MBP). The evolution of acute inflammation was quantitatively assessed by cell counts in different survival times. There was recruitment of both neutrophils and macrophages to the damaged striatal parenchyma with maximum recruitment at 1 day and 7 days, respectively. Progressive myelin impairment in the striatal white matter tracts has been observed mainly at later survival times. beta-APP+ endbulbs were not present in all evaluated time points. These results suggest that progress myelin impairment in the absence of damage to axonal cylinder is a feature of white matter pathology following endothelin-1-induced focal striatal ischemia.

Topics: Amyloid beta-Peptides; Animals; Axons; Biomarkers; Brain Ischemia; Cerebral Arteries; Chemotaxis, Leukocyte; Corpus Striatum; Demyelinating Diseases; Disease Progression; Encephalitis; Endothelin-1; Male; Microcirculation; Microglia; Microinjections; Myelin Basic Protein; Nerve Fibers, Myelinated; Neutrophils; Rats; Rats, Wistar

Severe cerebral edema is associated with poor outcome in patients with acute stroke. Experimental studies suggest that astrocytic endothelin-1 (ET-1) has deleterious effects on water homeostasis, cerebral edema, and blood brain barrier (BBB) integrity, which contribute to more severe ischemic brain injury. In this study we analyze the association between high serum levels of ET-1 and the development of severe cerebral edema in patients treated with t-PA.. One hundred thirty-four patients treated with t-PA according SITS-MOST (Safe Implementation of Thrombolysis in Stroke Monitoring Study) criteria were prospectively studied. Serum levels of ET-1, matrix metalloproteinase-9 (MMP-9), and cellular fibronectin (c-Fn) were determined by ELISA in serum samples obtained on admission, before t-PA bolus. Severe brain edema was diagnosed if extensive swelling caused any shifting of the structures of the midline was detected on the cranial CT performed at 24 to 36 hours. Stroke severity was evaluated before t-PA administration and at 24 hours by NIHSS. Functional outcome at 3 months was evaluated by the modified Rankin Scale (mRS).. Nineteen patients (14.2%) developed severe brain edema. Median ET-1 (8.4 [6.7, 9.6] versus 1.9 [1.6, 3.2] fmol/mL, P<0.0001) and c-Fn (6.0 [4.1, 6.7] versus 3.2 [2.1, 4.6] mg/L, P<0.0001) serum levels were significantly higher in patients with severe cerebral edema. The best cut-off values for ET-1 and c-Fn serum levels for the prediction of severe brain edema were 5.5 fmol/mL (sensitivity 95% and specificity 94%) and 4.5 mg/L (sensitivity 73% and specificity 77%) respectively. ET-1 serum levels >5.5 fmol/mL before t-PA treatment were independently associated with development of severe brain edema (OR, 139.7; CI95%, 19.3 to 1012.2; P<0.0001), after adjustment for baseline stroke severity, early CT signs of infarction, serum levels of cFn >4.5 mg/L, and cardioembolic stroke subtype.. ET-1 serum levels >5.5 fmol/mL are associated with severe brain edema in acute stroke patients treated with t-PA. These results suggest that ET-1 may be a new diagnostic marker for development of severe brain edema in patients with acute ischemic stroke treated with t-PA.

Topics: Aged; Brain Edema; Brain Ischemia; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Fibronectins; Humans; Male; Matrix Metalloproteinase 9; Middle Aged; Odds Ratio; Stroke; Thrombolytic Therapy; Tissue Plasminogen Activator

To assess plasma endothelin-1 (ET-1), C-reactive protein (CRP) and fibrinogen (FIB) levels in acute ischemic stroke (AIS) and explore the potential association among them.. In 23 consecutive patients with AIS diagnosed clinically and confirmed by CT brain scan, we measured plasma levels of ET-1 by radioimmunoassay, and CRP and FIB on the first and fifth days after the onset of AIS and we compared them with the levels of ten healthy volunteers in the control group.. The mean plasma levels of ET-1 in AIS patients on the first and fifth days were respectively 19.93+/-6.72 and 16.47+/-26.3 pmol/l (p<0.001) compared with 3.68+/-1.2 pmol/l in the control group (p<0.001 versus mean values on the first and fifth days). The mean plasma levels of CRP in the patients on the first and fifth days were respectively 2.7+/-4.7 and 3.0+/-4.4 mg/dl (p>0.05) compared with 0.2+/-0.1 mg/dl in the control group (p<0.05 versus mean values on the first and fifth days). The mean plasma levels of FIB in the patients on the first and fifth days were respectively 361+/-98.89 and 392.7+/-144.89 mg/dl, while in the control group, it was 330.5+/-90.28 mg/dl (p>0.05 versus mean values on the first and fifth days). A positive association was found between the plasma levels of ET-1 and CRP on the fifth day (p<0.05).. ET-1 was found to be significantly elevated in the plasma in the AIS. There is association between ET-1 and CRP on the fifth day after AIS. Plasma levels of ET-1 and its association with CRP levels may be used as additional biomarkers for AIS.

Topics: Acute Disease; Aged; Aged, 80 and over; Biomarkers; Brain; Brain Ischemia; C-Reactive Protein; Endothelin-1; Female; Fibrinogen; Humans; Male; Predictive Value of Tests; Radioimmunoassay; Tomography, X-Ray Computed; Up-Regulation

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P<0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P<0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r(2)=0.86 and 0.80, respectively, P<0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have mis-calculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.

Topics: Animals; Brain Ischemia; Endothelin-1; Fluorescent Antibody Technique; Immunohistochemistry; Infarction, Middle Cerebral Artery; Inflammation; Macrophages; Male; Microscopy, Confocal; Middle Cerebral Artery; Neutrophil Infiltration; Peroxidase; Rats; Rats, Long-Evans; Stereotaxic Techniques

To determine levels of vascular cellular adhesion molecule (VCAM)-1 and endothelin (ET)-1 in patients with stroke.. Thirty-four patients were prospectively studied. Plasma levels of VCAM-1 and ET-1 were measured by ELISA within 72 h of the event, at 7 and 90 days.. Levels of VCAM-1 increased overtime, whereas ET-1 values were initially and persistently elevated.. Increased circulating levels of VCAM-1 and ET-1 are present during stroke.

Topics: Aged; Biomarkers; Brain Ischemia; Endothelin-1; Female; Humans; Longitudinal Studies; Male; Middle Aged; Prospective Studies; Stroke; Vascular Cell Adhesion Molecule-1

The influence of anaesthesia in experimental stroke research is controversial. We addressed this problem using the model of endothelin-1-induced occlusion of the middle cerebral artery (eMCAO). This model provided the opportunity to compare the infarct volumes of rats which were under halothane anaesthesia during eMCAO induction with the lesions of rats which were without anaesthesia during eMCAO. All animals were implanted with guide cannulae which allowed the induction of ischaemia in freely moving animals. For comparison, one group of animals was exposed to halothane during the induction of ischaemia. Seven days after eMCAO, the average infarct volume of halothane-anaesthetised rats was significantly larger than the lesion in freely moving animals. This difference was mainly due to increased cortical damage, whereas the striatum was much less influenced. The cortical infarct volume 21 days after induction of eMCAO under anaesthesia was significantly reduced compared to the infarct volume 7 days after eMCAO under anaesthesia. Our results indicate that halothane anaesthesia during eMCAO can cause a transient cortical increase in ischaemic infarct volume. The influence of volatile anaesthetics on ischaemic pathophysiology should be taken into consideration when preclinically testing potential neuroprotective drugs for clinical applications.

Topics: Anesthetics, Inhalation; Animals; Brain Ischemia; Cerebral Infarction; Endothelin-1; Endothelium, Vascular; Halothane; Male; Middle Cerebral Artery; Rats; Rats, Sprague-Dawley

It has been established that hyperbaric oxygen (HBO) treatment reduces brain edema, decreases infarct volume, contributes to neurological functional recovery and suppresses apoptosis in suture-induced focal cerebral ischemic animal models. In the present study, we evaluated the therapeutic effect of HBO in an endothelin-1-induced focal cerebral ischemia in rats and explored the associated mechanisms of HBO-induced brain protection. One hundred twenty male Sprague-Dawley rats (280 to 320 g) were randomly assigned to sham, focal cerebral ischemia and focal cerebral ischemia treated with HBO groups. Brain water content, neurological function, morphology and molecular biological markers were assessed. HBO (100% O2, 2.5 atmosphere absolute for 2 h) was initiated at 1 h after focal cerebral ischemia. Rats were killed at 24 h to harvest tissues for Western blot or for histology. In HBO-treated animals, an enhanced ratio of Bcl-2 and Bax and a reduced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the hippocampus after focal cerebral ischemia were observed. These results indicate that HBO provides brain protection that is probably associated with the inhibition of HIF-1alpha and the elevation of Bcl-2.

Topics: Analysis of Variance; Animals; bcl-2-Associated X Protein; Brain Edema; Brain Infarction; Brain Ischemia; Disease Models, Animal; Endothelin-1; Gene Expression Regulation; Hyperbaric Oxygenation; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley

The long-term effects of the 5-HT(2A) receptor antagonist ketanserin on deficits in sensorimotor integration (limb placing tests) following transient focal cerebral ischemia in rats were compared to the effects of the NMDA antagonist MK-801. Middle cerebral artery occlusion was induced in conscious rats by microinjection of endothelin-1 in the vicinity of the artery (EMCAO model). The EMCAO/vehicle rats exhibited impaired tactile and proprioceptive limb placing. In contrast to ketanserin, MK-801 exerted severe early behavioral disturbances, but both drugs significantly improved the neurological scores much earlier than the spontaneous recovery of function occurred. The present results suggest that pharmacotherapy by means of ketanserin lacking the severe side effects of the NMDA antagonists can be used to enhance functional recovery after stroke.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain Ischemia; Disease Models, Animal; Dizocilpine Maleate; Endothelin-1; Extremities; Ketanserin; Male; Neurologic Examination; Neuroprotective Agents; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Severity of Illness Index

We investigated the effect of small cortical ischemic lesions, produced by intracerebral injection of the vasoconstrictor endothelin-1, on neurogenesis in the adult mouse subventricular zone. Endothelin-1 (0.5-1 microg) produced infarcts restricted to the cortex, and associated neurobehavioral deficits that largely resolved by 3 days. Bromodeoxyuridine labeling of proliferating cells in the subventricular zone was elevated by about 50% in endothelin-1-treated mice, and cells reactive for doublecortin, a marker for immature neurons, were similarly increased in number. These findings indicate that small ischemic lesions restricted to adult cerebral cortex can stimulate neuroproliferation at a distance.

Topics: Animals; Biomarkers; Brain Ischemia; Cell Differentiation; Cell Proliferation; Cerebral Infarction; Dose-Response Relationship, Drug; Doublecortin Domain Proteins; Endothelin-1; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Nerve Regeneration; Neuronal Plasticity; Neuropeptides; Recovery of Function; Stem Cells; Vasoconstrictor Agents

Stroke affects all age groups from the newborn to the elderly. Previous work from our laboratory has shown that despite a greater susceptibility to brain damage, the immature brain recovers more rapidly and to a greater extent than does the more mature nervous system. In the current study, we examined the influence of environmental enrichment on the effects of age on the brain damaging effects of stroke. Rats aged 10, 63, and 180 days received ischemic insults following stereotactic intra-cerebral injection of endothelin-1, and resulting in injury to the right middle cerebral artery territory. Rats were then housed in either environmentally enriched cages, or standard cages for 60 days, after which they were sacrificed, and brain volumes determined for the extent of neurologic injury. Rats receiving the insult at 10 days of age showed a reduction of pathologic injury when housed in the enriched cages compared to standard. Conversely, rats receiving the insult at 180 days and housed environmentally enriched cages actually showed an increased volume of brain damage compared to controls. Our findings clearly indicate the dramatic influence of age on the extent of stroke and the influence of rehabilitative therapies. Behavioral correlation to morphologic alterations is required. Attempts at therapeutic interventions clearly need to be age-specific.

Topics: Aging; Animals; Animals, Newborn; Brain; Brain Infarction; Brain Ischemia; Endothelin-1; Environment, Controlled; Female; Infarction, Middle Cerebral Artery; Male; Neuronal Plasticity; Rats; Recovery of Function

Inflammatory response, axonal damage and demyelination are important components of the pathophysiology of acute neurodegenerative diseases. We have investigated the outcome of these pathological events following an excitotoxic or an ischemic damage to the spinal nucleus of adult rats at 1 and 7 days postinjury. Microinjections of 80 nmol of NMDA or 40 pmol of endothelin-1 into the rat spinal nucleus induced differential histopathological events. NMDA injection induced intense tissue loss in the gray matter (GM) without significant tissue loss in the white matter (WM). There was a mild inflammatory response, with recruitment of a few neutrophils and macrophages. Axonal damage was present in the GM following NMDA injection, with negligible axonal damage in the WM. Myelin impairment was apparent at 7 days. Microinjections of endothelin-1 into the same region induced lesser tissue loss than NMDA injections, concomitant with an intense inflammatory response characterized by recruitment of macrophages, but not of neutrophils. There were more axonal damage and early myelin impairment after endothelin-1 injection. These results were confirmed by quantitative analysis. Microcysts were present in the WM of the trigeminothalamic tract at 7 days following injection of endothelin-1. These results show that an ischemic damage to the spinal nucleus affects both GM and WM with more bystander inflammation, axonal damage and myelin impairment, while excitotoxic damage induces effects more restricted to the GM. These pathological events may occur following acute damage to the human brain stem and can be an important contributing factor to the underlying functional deficits.

Topics: Amyloid beta-Protein Precursor; Analysis of Variance; Animals; Axons; Brain Ischemia; Cell Count; Demyelinating Diseases; Ectodysplasins; Endothelin-1; Inflammation; Male; Myelin Basic Protein; N-Methylaspartate; Neurotoxins; Rats; Rats, Wistar; Time Factors; Trigeminal Nucleus, Spinal

The goal of this study was to develop a new model of ischemia-induced seizures in immature rats using injection of vasoconstrictor Endothelin-1 (ET-1) into the brain. ET-1 (10, 20, or 40 pmol) was infused into the left dorsal hippocampus of freely moving Wistar rats 12 (P12) and 25 (P25) days old. Animals were then video/EEG-monitored for 100 min and monitoring was repeated 22 h later. Parameters of electrographic seizures (frequency and mean duration) as well as pattern of their behavioral correlates were evaluated. The pattern of behavioral seizures was used to develop model-specific scoring system. Cresyl violet and Fluoro Jade-B-staining were used to evaluate brain damage. Extension of the lesion was correlated with seizure severity. After ET-1-injection, seizures occurred in 83-100% animals of all age-and-dose groups and persisted for 24 h except P12 rats with 10 pmol. There were no differences in average seizure duration (18-40 s) or seizure frequency (3-7 seizures/100 min) among individual dose-groups. Between the 1st and 2nd observation period, total seizure duration decreased in 71% of P12 and 47% of P25 rats. Electrographic seizure activity was most frequently accompanied by clonus, incidence of more severe convulsions (barrel rolling or generalized clonic seizures) increased with dose of ET-1. Morphologic examination did not reveal any dose-related difference in damage severity, hippocampal damage was however more extensive in P12 compared to P25 animals. Seizure severity correlated positively with severity of the damage in both age groups. Our study presents focal injection of ET-1 into the brain as a new and practical model of ischemia-induced seizures in immature rats.

Topics: Animals; Animals, Newborn; Brain Ischemia; Disease Models, Animal; Electroencephalography; Endothelin-1; Epilepsy; Hippocampus; Injections; Male; Rats; Rats, Wistar; Videotape Recording

The absence of effective treatments for stroke presents a critical need for novel strategies that can reduce ischemic injury. Neuroinflammation following focal ischemia induces secondary injury in the region surrounding the insult, thus anti-inflammatory agents are potential neuroprotectants. Minocycline is one such agent possessing neuroprotective properties, however many studies examining minocycline after ischemia have used minimal delays between ischemia and treatment, short survival periods, and lack measures of functional outcome. Such studies do not distinguish whether minocycline provides sustained protection or merely delays cell death. This study was designed to address some of these concerns. Male Sprague-Dawley rats were treated with multiple doses of minocycline (45 mg/kg i.p.) or vehicle beginning 2.5 h after endothelin-1-induced focal ischemia. Measures of forelimb asymmetry and skilled reaching (staircase test) were used to determine functional outcome 7, 15 and 28 days after ischemia. Long-term functional assessment indicates that minocycline provides limited benefit in the staircase test, but confers long-term benefit in the forelimb asymmetry test. Subcortical and whole hemisphere infarct volumes were reduced by 41 and 39% respectively in minocycline-treated animals. Further analysis revealed that minocycline attenuated long-term white matter damage adjacent to the striatal injury core, which correlated with sustained functional benefits. This study indicates that delayed minocycline treatment improves long-term functional outcome which is linked to protection of both white and gray matter.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain Infarction; Brain Ischemia; Disease Models, Animal; Drug Administration Schedule; Endothelin-1; Functional Laterality; Male; Minocycline; Neuroprotective Agents; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Recovery of Function; Severity of Illness Index; Time Factors; Treatment Outcome

Endothelin-1 (ET-1), a potent vasoconstrictor, reduces local blood flow to levels that produce ischemic injury when injected directly into brain tissue. The purpose of this study was to compare 4 different methods of inducing focal ischemia with ET-1: (1) topical application to the forelimb motor region of the cortex, (2) intracerebral injection into the forelimb motor region of the cortex, (3) a combination of intracortical and intrastriatal injections and 4. injection of ET-1 adjacent to the middle cerebral artery (MCA). We examined the effect of delivery method and dose of ET-1 on lesion size, inter-animal variability and behavioral outcome on 3 separate tests of motor function and limb preference. We calculated success rate as the percentage of animals that survived surgery and developed a significant impairment (>20% decrease in performance post-surgery) in the staircase-reaching test. All 4 methods produced similar deficits in the staircase, balance beam, and cylinder tests, but the application of ET-1 adjacent to the MCA, though widely used, provided the lowest success rate. The combined cortical and striatal ET-1 produced a high success rate and consequently we examined cerebral blood flow (CBF), the apparent diffusion coefficient (ADC) and T2-weighted magnetic resonance imaging (MRI) changes for this model. We found that infarct volume measured using T2-weighted MRI correlated with histological measurements and that ADC and CBF together predicted which areas will suffer permanent injury. The combined cortical and striatal injection model offers a number of advantages for studies of recovery of function.

Topics: Animals; Behavior, Animal; Brain Infarction; Brain Ischemia; Cerebral Cortex; Corpus Striatum; Drug Administration Routes; Endothelin-1; Injections; Injections, Intraventricular; Magnetic Resonance Imaging; Male; Motor Cortex; Psychomotor Performance; Rats; Rats, Long-Evans

Stroke is a health hazard that affects all age groups, however the impact of age on brain injury following ischemia remains largely unexamined. We examined the extent to which age, from the newborn to mature adult, affects behavioral recovery following similar degrees of ischemic brain damage. We utilized a model that produces comparable volumes of brain damage between the different ages. Endothelin-1, a potent vasoconstrictor, was injected into the brain of 10, 63 and 180-day-old rats, at the level of the right middle cerebral artery. On days 3, 7, 14, 28 and 56 post-insult, behavioral tests including rota-rod, foot-fault, open-field, inclined screen, tape-removal test and postural reflex were performed. Control animals underwent sham surgery, but ischemia was not induced. Neuropathology was assessed on day 63 post-insult. Volume of damage was determined for each brain as a percentage of the contralateral hemisphere (which remains undamaged). Our results indicated that the volume of damage for each age group was 22.97, 19.97, and 18.85% for 10, 63 and 180-day-old rats, respectively, and were not significantly different from each other. Overall, ischemic animals did significantly more poorly on behavioral testing than did controls. When broken down by age, the difference between ischemics and controls was only evident in the 63 and 180-day-old animals. The tape-removal test revealed main effects of age, group, and day (p<0.001). In addition, significant interactions were noted for day of testing by age (p<0.001), day of testing by group with ischemics performing more slowly than controls, and an age by group interaction which indicated that the 63 and 180-day-old ischemic rats did not recover completely during the testing period and remained significantly slower than their controls (p<0.001). In the foot-fault task, the 63 and 180-day-old ischemic animals performed significantly more poorly on days 3, 7, and 14 of recovery, returning to control values by day 28. The 180-day olds performed more poorly on day 3 of recovery, but then returned to control values. For open-field testing, the results indicate an overall difference between ischemics and controls, with the 63 and 180-day-old animals improving with time though they did not achieve control values. In conclusion our data suggest functional performance is poorly and inconsistently correlated with the extent of morphologic injury across all age groups. The immature rat clearly recovers more completely

Topics: Age Factors; Aging; Analysis of Variance; Animals; Animals, Newborn; Behavior, Animal; Brain Injuries; Brain Ischemia; Disease Models, Animal; Endothelin-1; Exploratory Behavior; Motor Activity; Psychomotor Performance; Rats; Rats, Wistar; Recovery of Function; Rotarod Performance Test; Time Factors

Following transient focal stroke, rapid accumulation and activation of neutrophils in the ischaemic region is deleterious due to release of reactive oxygen species and myeloperoxidase (MPO). The purpose of this study was to examine whether AM-36, both a Na+ channel blocker and an antioxidant, afforded neuroprotection by modulating neutrophil accumulation into brain, following endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAo) in conscious rats.. AM-36 was administered at 3 and 24 h after ET-1-induced MCAo. Functional recovery was determined using grid-walking and cylinder tests. Image analysis of brain sections was used to determine infarct volume. The effect of AM-36 on neutrophil infiltration and their interaction with macrophages was examined in rats at 48 h following MCAo by both an MPO assay and double-label immunofluorescence. Blood brain barrier (BBB) breakdown was measured by the area stained by intravenous Evans Blue.. AM-36 reduced functional deficits in both tests such that no difference existed from pre-ischaemic values at 48 h. Neutrophil infiltration, assessed by MPO activity, and infarct volume were significantly reduced following AM-36 administration by 54 and 60% respectively. Similarly, immunofluorescence revealed that AM-36 reduced neutrophil infiltration by approximately 50% in selected brain regions, when compared to controls, and also modulated macrophage phagocytosis of neutrophils. Breakdown of the BBB was significantly reduced by 60% following AM-36 treatment.. These findings suggest that AM-36 can directly modulate the neutrophil inflammatory response and reduce BBB breakdown following MCAo.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Endothelin-1; Fluorescent Antibody Technique; Inflammation; Male; Microscopy, Confocal; Neutrophils; Peroxidase; Piperazines; Rats; Rats, Long-Evans

Stroke represents one of the leading causes of death and disability in Western countries, but despite intense research, only few options exist for the treatment of stroke-related infarction of brain tissue. In experimental stroke, cell therapy can partly reverse some behavioural deficits. However, the underlying mechanisms have remained unknown as most studies revealed only little, if any, evidence for neuronal replacement and the observed behavioural improvements appeared to be related rather to a graft-derived induction of a positive response in the remaining host tissue than to cell replacement by the graft itself. The present study was performed to test a murine embryonic stem cell (ESC)-based approach in rats subjected to endothelin-induced middle cerebral artery occlusion. Efficacy of cell therapy regarding graft survival, neuronal yield and diversity, and electrophysiological features of the grafted cells were tested after transplanting ESC-derived neural precursors into the infarct core and periphery of adult rats. Here, we show that grafted cells can survive, albeit not entirely, most probably as a consequence of an ongoing immune response, within the infarct core for up to 12 weeks after transplantation and that they differentiate with high yield into immunohistochemically mature glial cells and neurons of diverse neurotransmitter-subtypes. Most importantly, transplanted cells demonstrate characteristics of electrophysiologically functional neurons with voltage-gated sodium currents that enable these cells to fire action potentials. Additionally, during the first 7 weeks after transplantation we observed spontaneous excitatory post-synaptic currents in graft-derived cells indicating synaptic input. Thus, our observations show that ESC-based regenerative approaches may be successful in an acutely necrotic cellular environment.

Topics: Animals; Brain Ischemia; Cell Differentiation; Cell Division; Cell Movement; Cell Survival; Embryonic Stem Cells; Endothelin-1; Immunohistochemistry; Infarction, Middle Cerebral Artery; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Inbred F344; Stroke

Vascular endothelial cells are important not only for maintaining homeostasis, but also in pathogenesis of vascular disorders. Cerebral capillary and microvascular endothelial cells play an active role in maintaining cerebral blood flow, microvascular tone and blood brain barrier functions. Factors produced and released by endothelial cells, other brain cells and circulating blood cells participate in these regulatory functions. In particular, endothelin-1 (ET-1) and nitric oxide (NO) are known to contribute to the functional vascular changes under pathological conditions (e.g., hypertension, arteriosclerosis, and stroke). This report describes the involvement of endothelial cell mediators in the post-ischemic hypoperfusion induced by brain ischemia and in vitro endothelial responses (Ca(2+) mobilization and cytoskeletal rearrangements) to ET-1 and its interactions with NO or 2-AG. The capacity of NO and endocannabinoids to counteract ET-1-induced cerebral capillary and microvascular endothelial responses indicates that they may actively participate in EC function and implicates them in physiological and pathophysiological conditions.

Topics: Actins; Analysis of Variance; Animals; Arachidonic Acids; Blood-Brain Barrier; Brain; Brain Ischemia; Calcium; Cells, Cultured; Cytoskeleton; Endocannabinoids; Endothelin-1; Endothelium, Vascular; Gerbillinae; Glycerides; Humans; Immunohistochemistry; Microcirculation; Nitric Oxide; Reperfusion Injury

Unilateral sensorimotor cortical (SMC) lesions in rats impair reaching and grasping movements of the contralateral forelimb. These impairments can be improved using motor rehabilitative training on a skilled reaching task, but the training may be far from sufficient to return animals to pre-lesion levels of performance. Because D-amphetamine (AMPH) has been found to promote neuroplastic responses to injury and to be very beneficial when combined with some (but not all) types of rehabilitative training, we asked in this experiment whether it could improve the efficacy of rehabilitative training in skilled reaching. Ten to 14 days after unilateral ischemic (endothelin-1 induced) lesions of the SMC, adult rats were given a 3-week regimen of AMPH (1mg/kg) coupled with daily rehabilitative training on a skilled reaching task, the single pellet retrieval task. AMPH treatment not only dramatically improved reaching performance compared with saline-injected controls, the AMPH treated rats surpassed pre-lesion levels of performance by the end of the rehabilitative training period. The greater performance in AMPH compared to saline-treated rats was still evident at 1 month, but not at 2 and 3 months, after the end of rehabilitative training. Thus, AMPH treatment can greatly enhance the efficacy of rehabilitative training on a skilled reaching task after unilateral SMC lesions, but alternate injection and training regimes may be needed to produce permanent improvements.

Topics: Animals; Brain Ischemia; Central Nervous System Stimulants; Cerebral Infarction; Denervation; Dextroamphetamine; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Male; Motor Cortex; Motor Skills; Movement Disorders; Neuronal Plasticity; Paresis; Physical Conditioning, Animal; Rats; Rats, Long-Evans; Recovery of Function; Treatment Outcome

The expression of corticotrophin-releasing factor (CRF) receptors in cerebral arteries and arterioles suggests that CRF may modulate cerebral blood flow (CBF). In the present study, the effects of CRF, CRF-like peptides and the CRF broad spectrum antagonist DPhe-CRF on CBF have been investigated under normal physiologic conditions and in the margins of focal ischaemic insult. The experiments were carried out in anaesthetised and ventilated rats. Changes in CBF after subarachnoid microapplication of CRF and related peptides were assessed with a laser-Doppler flowmetry (LDF) probe. In the ischaemic animals, agents were injected approximately 60 minutes after permanent middle cerebral artery occlusion (MCAo). Microapplication of CRF and related peptides in normal rats into the subarachnoid space produced sustained concentration-dependent increases in CBF. This effect was attenuated by co-application with DPhe-CRF, which did not alter CBF itself. A second microapplication of CRF 30 min after the first failed to produce increases in CBF in normal animals. Microapplication of CRF in the subarachnoid space overlying the ischaemic cortex effected minor increases in CBF whereas D-Phe-CRF had no significant effect on CBF. Activation of the CRF peptidergic system increases CBF in the rat. Repeated activation of CRF receptors results in tachyphylaxis of the vasodilator response. CRF vasodilator response is still present after MCAo in the ischaemic penumbra, suggesting that the CRF peptidergic system may modulate CBF in ischaemic stroke.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Corticotropin-Releasing Hormone; Endothelin-1; Immunohistochemistry; Injections; Male; Middle Cerebral Artery; Neuropeptides; Rats; Rats, Inbred F344; Subarachnoid Space

Endothelial dysfunction has been implicated in the pathogenesis of cerebral small-vessel disease (SVD). Endothelin (ET), released by the endothelium, plays a crucial role in vasoconstriction in the cerebral circulation and could contribute to the pathogenesis of cerebral SVD. Circulating ET levels may not reflect vascular production of endothelin-1 (ET-1), most of which is abluminal. Studying genetic associations, particularly of functional polymorphisms that alter activity of the ET system, is an attractive method of determining whether ET plays a role in SVD pathogenesis. We determined whether genetic variants in components of the ET system are a risk factor for cerebral SVD.. Three hundred SVD patients and 600 community controls were genotyped. Polymorphisms in the ET-1 gene (K198N), the ET receptor type A (ETA), (-231G>A and +1222C>T), and the ET type B (ETB) receptor (G57S and L277L) were genotyped. Polymorphisms were studied both individually and as haplotypes. With brain imaging, cases were subtyped into those with lacunar infarct without leukoaraiosis and those with leukoaraiosis.. No significant differences were observed between SVD cases and controls for any individual single-nucleotide polymorphism or the ETA haplotype. There were no differences between cases with isolated lacunar infarct or with lacunar infarct and leukoaraiosis.. This study, in a well-phenotyped population, does not support a role for genetic variation in the ET system as a risk factor for cerebral SVD.

Topics: Blood Vessels; Brain; Brain Infarction; Brain Ischemia; Case-Control Studies; Cerebrovascular Circulation; Endothelin-1; Endothelins; Female; Genetic Variation; Genotype; Haplotypes; Humans; Leukoaraiosis; Male; Odds Ratio; Phenotype; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Receptor, Endothelin A; Receptor, Endothelin B; Risk Factors; RNA, Messenger; Sequence Analysis, DNA

This study investigated the effect of amphetamine (AMP) on skilled forelimb use following focal cortical ischaemic lesions in the rat. Unilateral lesions were produced by a novel method of intracortical microinjection of endothelin-1 (ET-1), intended to principally target the forelimb representation zone in primary motor-primary somatosensory cortex. Lesions were placed in the hemisphere contralateral to the preferred limb and produced deficits in skilled forelimb use on two tasks: the paw reach (PR) test and the foot fault (FF) test. Beginning on post-lesion day (D) 2, animals received injections of 2 mg/kg AMP and were injected every third day until D26. Animals were tested both during, and 24 h after, AMP administration. AMP facilitated recovery of skilled forelimb use on the PR test when assessed during drug-free test sessions. No such effect was seen on the FF test. These results demonstrate that sub-acute administration of AMP following a unilateral focal ischaemic lesion of FL can facilitate task-dependent recovery of skilled forelimb use in the rat. They also demonstrate that different behavioural tasks measuring superficially similar behavioural outputs may show different sensitivities to such drug effects.

Topics: Amphetamine; Analysis of Variance; Animals; Brain Injuries; Brain Ischemia; Central Nervous System Stimulants; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Endothelin-1; Forelimb; Functional Laterality; Male; Motor Cortex; Motor Skills; Rats; Rats, Inbred Strains; Recovery of Function; Stroke; Stroke Rehabilitation

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.

Topics: Amino Acids; Animals; Blood-Brain Barrier; Brain Ischemia; Dipeptides; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelin-1; Hypothermia; Male; Microdialysis; Neuroprotective Agents; Rats; Rats, Wistar; Time Factors; Xanthenes

Previously, we showed that treatment with resuscitative, post-ischaemic mild hypothermia (34 degrees C for 2 h) reduced apoptosis in the penumbra (cortex), but not in the core (striatum) of an endothelin-1 (Et-1)-induced focal cerebral infarct in the anaesthetized rat. Therefore, the purpose of this study was to investigate by which pathways resuscitative mild hypothermia exerts its neuroprotective effect in this model. The amino acids glutamate, serine, glutamine, alanine, taurine, arginine and the NO-related compound citrulline were sampled from the striatum and cortex of the ischaemic hemisphere using in vivo microdialysis. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (2,3 DHBA) detection. Caspase-3, neuronal nitric oxide synthase (nNOS) immunoreactivity and the volume of ischaemic damage were determined 24 h after the insult. In both the striatum and the cortex, Et-1-induced increases in glutamate, taurine and alanine were refractory to mild hypothermia. However, mild hypothermia significantly attenuated the ischaemia-induced 2,3 DHBA levels and the nNOS immunoreactivity in the cortex, but not in the striatum. These observations were associated with a decreased caspase-3 immunoreactivity. These results suggest that mild hypothermia exerts its neuroprotective effect in the penumbra partially by reducing nNOS activity and thereby preventing oxidative stress. Furthermore, we confirm our previous findings that the neuroprotective effect of resuscitative hypothermia is not mediated by changes in ischaemia-induced amino acid release as they could not be associated with the ischaemia-induced damage in the Et-1 rat model.

Topics: Amino Acids; Animals; Apoptosis; Body Temperature; Brain Ischemia; Caspase 3; Caspases; Cerebral Cortex; Cerebral Infarction; Endothelin-1; Enzyme Inhibitors; Extracellular Space; Hydroxyl Radical; Hypothermia; Immunohistochemistry; Male; Microdialysis; Neostriatum; NG-Nitroarginine Methyl Ester; Oxidants; Rats; Rats, Wistar

In our study, we examined middle cerebral artery (MCA) contractile responses in two animal models. After hemorrhagic disturbances in rats of Krushinsky-Molodkina strain (KMRs) a decrease in contractile responses to serotonin (5-HT) was observed. During incomplete global cerebral ischemia, MCAs had increased responsiveness to endothelin-1 (ET-1), but reduced responsiveness to serotonin. These findings suggest that cerebral circulation disorders alter cerebrovascular function possibly leading to secondary disturbances in brain circulation.

Topics: Animals; Brain Ischemia; Cerebral Hemorrhage; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Female; Male; Middle Cerebral Artery; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Wistar; Serotonin; Vasoconstriction

To determine the protective effect of ONO-1078, a leukotriene receptor antagonist, on focal cerebral ischemia induced by endothelin-1 in rats.. Slow microinjection of endothelin-1 (120 pmol in 6 microL, for > 6 min) into the region near the middle cerebral artery was used to induce focal cerebral ischemia. ONO-1078 (0.1 mg.kg-1) was i.p. injected 1 h before endothelin-1 injection. Neurological symptoms, brain edema, brain infarction size, and the survival neurons in cortex and striatum were observed 24 h after ischemia.. Intracerebral microinjection of endothelin-1 induced remarkable neurological symptoms, brain infarction, brain edema, and decrease of survival neurons in the cortex and striatum. In rats pretreated with ONO-1078, endothelin-1-induced brain edema and brain infarction size were decreased. The numbers of survival neurons in striatum and cortex were increased significantly. The neurological symptoms were improved but not significantly.. ONO-1078 possesses neuroprotective effect against cerebral ischemic injury induced by endothelin-1, therefore, leukotrienes may play a role in the injury of cerebral ischemia.

Topics: Animals; Behavior, Animal; Brain Edema; Brain Ischemia; Cerebral Cortex; Cerebral Infarction; Chromones; Corpus Striatum; Endothelin-1; Leukotriene Antagonists; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

A comprehensive evaluation of the effects of neuroprotection, neurogenesis, and compensatory mechanisms on the outcome of ischemic insults requires assessment of morphological and functional parameters. Behavioural tests are essential when recording performance throughout the time course of an experiment and the results bear predictive value in preclinical animal models. The goal of this study was to establish a behavioural test procedure for a model of transient focal ischemia induced by injection of endothelin-1 (eMCAO) that results in relatively mild behavioural deficits. The test protocol used in the present study allows evaluation of quantitative and qualitative impairments in skilled motor performance and is sensitive to detect chronic deficits at chronic post-ischemic time intervals. The ladder rung walking task [J. Neurosci. Methods 115 (2002) 169] is a motor test that assesses skilled walking and measures both forelimb and hindlimb placing, stepping and inter-limb co-ordination. In this study we tested the effect of two different technical variants of endothelin-1 application on infarct volume and motor skills (1) application via pre-implanted guiding cannula in awake animals and (2) via direct injection under halothane anaesthesia. We showed that the ladder rung walking task is sensitive in the assessment of loss of fine motor function after induction of relatively small lesions. In animals with implanted cannulas we found a smaller infarct area and an increase in placement errors prior to ischemia animals with eMCAO under anaesthesia showed a long lasting impairment of the contralateral forelimb up to 4 weeks post-eMCAO.

Topics: Anesthetics, Inhalation; Animals; Behavior, Animal; Brain Infarction; Brain Ischemia; Chronic Disease; Disease Models, Animal; Endothelin-1; Halothane; Hindlimb; Male; Physical Conditioning, Animal; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Somatosensory Cortex; Statistics, Nonparametric; Time Factors; Wakefulness; Walking

The plasma expression levels of ET-1, TXB2, 6-keto-PGF1alpha, vWF at different time after cerebral ischemia were assayed for observing the effects of baicalin, jasminoidin, cholalic acid, hydrolysis fluid of nacre and the combined prescription (CP) on cerebral vasoconstriction and endothelial cells in MCAO rats.. The plasma levels of ET-1, TXB2, 6-keto-PGF1alpha in MCAO rats were detected by the method of RIA and the plasma expressions of vWF were observed by ELISA.. The levels of ET-1, TXB2/6-keto-PGF1alpha and vWF all increased at different time after cerebral ischemia, so do TXB2 at 12 hours after ischemia. The expression of 6-keto-PGF1alpha significantly reduced at different time point after ischemia in MCAO rat. There were no significant changes after medicine treating 12 hours except baicalin's increasing 6-keto-PGF1alpha level. Jasminoidin and CP significantly reduced the expression of ET-1 at 24 hours after ischemia, so do all effective components except CP on expression of TXB2 at 12 hours after ischemia. The expression of TXB2 was significantly decreased by baicalin and CP at 24 hours after cerebral ischemia. Both baicalin and cholalic acid significantly increased the expression of 6-keto-PGF1alpha at 12 hours after ischemia while cholalic acid and hydrolysis fluid of nacre increased its level after ischemia for 24 hours. TXB2/6-keto-PGF1alpha ratio was reduced distinctively by baicalin, jasminoidin, cholalic acid, CP at the point of 12 hours, while decreased by baicalin and CP, and increased by jasmionoidin at the point of 24 hours. On the other hand, baicalin, hydrolysis fluid of nacre significantly reduced and jasminoidin increased the expression of vWF at the point of 12 hours. At the point of 24 hours, expression of vWF reduced by hydrolysis fluid of nacre and increased by baicalin.. The higher plasma expression of ET-1, TXA2 in plasma aggravated cerebral vasoconstriction and damaged endothelial cells. At the same time, the effective components of "Qing Kai Ling" inhibit the expression of ET-1 , TXA2 and reduce both TXB2/6-keto-PGF1alpha ratio and level of vWF. As a result, they relax cerebral microvessel and protect endothelial cells by different pathway at different target points.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Brain Ischemia; Cholic Acid; Drugs, Chinese Herbal; Endothelial Cells; Endothelin-1; Endothelium, Vascular; Flavanones; Male; Rats; Rats, Sprague-Dawley; Thromboxane B2; Time Factors; von Willebrand Factor

Previous studies have established the usefulness of endothelin-1 (ET-1) for the production of focal cerebral ischemia. The present study assessed the behavioral effects of focal ET-1-induced lesions of the sensorimotor cortex (SMC) in adult rats as well as cellular and structural changes in the contralateral homotopic motor cortex at early (2 days) and later (14 days) post-lesion time points. ET-1 lesions resulted in somatosensory and postural-motor impairments in the contralateral (to the lesion) forelimb as assessed on a battery of sensitive measures of sensorimotor function. The lesions also resulted in the development of a hyper-reliance on the ipsilateral forelimb for postural-support behaviors. In comparison to sham-operated rats, in layer V of the motor cortex opposite the lesions, there were time- and laminar-dependent increases in the surface density of dendritic processes immunoreactive for microtubule-associated protein 2, in the optical density of N-methyl-D-asparate receptor (NMDA) subunit 1 immunoreactivity, and in the numerical density of cells immunolabeled for Fos, the protein product of the immediate early gene c-fos. These findings corroborate and extend previous findings of the effects of electrolytic lesions of the SMC. It is likely that compensatory forelimb behavioral changes and transcallosal degeneration play important roles in these changes in the cortex opposite the lesion, similar to previously reported effects of electrolytic SMC lesions.

Topics: Animals; Brain Ischemia; Corpus Callosum; Endothelin-1; Functional Laterality; Immunohistochemistry; Male; Microtubule-Associated Proteins; Motor Cortex; Movement Disorders; Nerve Degeneration; Neuronal Plasticity; Proto-Oncogene Proteins c-fos; Pyramidal Cells; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Somatosensory Cortex; Somatosensory Disorders; Time Factors

In this study, we investigated the acute hemodynamic effects of an infusion of the endothelin-1 (ET-1)-A-selective receptor antagonists BQ-610 and BQ-123 in heatstroke rats with circulatory shock and cerebral ischemia. Heatstroke was induced by putting the anesthetized adult Sprague-Dawley rats into an ambient temperature of 42 degrees C. The moment in which the mean arterial pressure dropped irreversibly from the peak for an extent of 25 mmHg was taken as the onset of heatstroke. The interval between initiation of heat exposure and heatstroke onset was found to be about 80 min for rats treated with vehicle solution. When the animals were exposed to 42 degrees C for 80 min, hyperthermia, arterial hypotension, decrement of cardiac output (due to decreased stroke volume and decreased total peripheral resistance), increment of plasma ET-1 and tumor necrosis factor-alpha, and increment of cerebral ischemia and injury markers were manifested. Prior antagonism of ET-1 A receptors with BQ-610 (0.5 mg/kg, i.v.) or BQ-123 (1 mg/kg, i.v.), but not ET-1B receptors with BQ-788 (0.5 mg/kg, i.v.), 60 min before the initiation of heat exposure, appreciably alleviated hyperthermia, arterial hypotension, decreased cardiac output, increment of tumor necrosis factor-alpha, and increment of cerebral ischemia (e.g., glutamate and lactate/pyruvate ratio) and injury (e.g., glycerol) markers exhibited during heatstroke. The data indicates that ET-1A receptor antagonism may maintain appropriate levels of mean arterial pressure and cerebral circulation during heatstroke by reducing production of tumor necrosis factor-alpha.

Topics: Animals; Brain Ischemia; Endothelin-1; Heat Stroke; Male; Oligopeptides; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Shock; Time Factors

Gamma-hydroxybutyrate (GHB) and its lactone, gamma-butyrolactone (GBL) have been previously shown to produce a protective effect in animal models of cerebral ischaemia/hypoxia, as well as in human conditions of head injury-induced coma. The aim of the present research was to study the effect of GHB in experimental conditions of focal cerebral damage, either induced by ischaemia or excitotoxicity. Under general anaesthesia, rats were injected into the right striatum with either endothelin-1 (ET-1, 0.43 nmol) or kainic acid (7.5 nmol) in a volume of 1 microl. Sham-lesioned rats received 1 microl of the solvent. Both ET-1- and kainic acid-lesioned rats were randomly assigned to one of the following intraperitoneal (i.p.) treatments: (i) and (ii) GHB, 100 or 300 mg kg(-1) 2 h after the lesion, followed by 50 or 100 mg kg(-1), respectively, every 12 h; (iii) saline, 2 ml kg(-1), same schedule. Sham animals were treated with saline, 2 ml kg(-1), same schedule. Treatments lasted for 10 days. The higher dose of GHB produced a significant protection against the ET-1-induced impairments in sensory-motor orientation and coordinated limb use (evaluated 24 and 42 days after the lesion) and in place learning and memory (Morris test, performed 19 and 39 days after the lesion). The same dose regimen reduced the circling behaviour induced by apomorphine in kainate-lesioned rats (10 days after the lesion), and limited or prevented at all the histological damage produced either by ET-1 or by kainic acid (evaluated 43 or 10 days after the lesion, respectively). These results show that GHB limits both histological and functional consequences of a focal ischaemic or excitotoxic insult of the brain, in rats, even if the treatment is started 2 h after the lesion.

Topics: Animals; Apomorphine; Brain; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin-1; Kainic Acid; Learning; Male; Memory Disorders; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Sodium Oxybate

The relationship between glutamate and dopamine release, apoptosis and ischaemic damage was studied following induction of transient focal cerebral ischaemia under normothermic (37 degrees C) and postischaemic (resuscitative) mild hypothermic (34 degrees C for 2 h) conditions in sevoflurane anaesthetized male Wistar rats. Focal ischaemia was induced by infusing endothelin-1 adjacent to the middle cerebral artery. In vivo microdialysis was used to sample glutamate and dopamine from striatum and parietal cortex of the ipsilateral hemisphere. The volume of ischaemic damage and the degree of apoptosis were determined 24 h after the insult. In both striatum and cortex of the normothermic group an initial increase in extracellular glutamate and dopamine levels following endothelin-1 infusion was observed. Striatal glutamate levels remained enhanced (250% of baseline) throughout the experiment, while the other neurotransmitter levels returned to baseline values. Hypothermia significantly attenuated the endothelin-1 induced glutamate release in the striatum. It also reduced apoptosis and infarct volume in the cortex. These results indicate that: (i) postischaemic mild hypothermia exerts its neuroprotective effect by inhibiting apoptosis in the ischaemic penumbral region; and (ii) this effect is not associated with an attenuation of glutamate or dopamine release in the cortex.

Topics: Animals; Apoptosis; Brain Ischemia; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Dopamine; Endothelin-1; Extracellular Space; Glutamic Acid; Hypothermia, Induced; Male; Microdialysis; Rats; Rats, Wistar

Delayed cerebral vasospasm has a major impact on the outcome of subarachnoid hemorrhage. Two important candidates to cause the arterial spasm are the red blood cell product oxyhemoglobin and the vasoconstrictor endothelin-1, although oxyhemoglobin alone is not sufficient to induce cerebral ischemia and endothelin-1 leads to ischemia only at relatively high concentrations. In this study, we demonstrated that the combination of oxyhemoglobin and endothelin-1 triggered spreading neuronal activation in rat cortex in vivo. In contrast with the expected transient increase of regional cerebral blood flow during spreading depression, however, cerebral blood flow decreased profoundly and was long-lasting, paralleled by delayed repolarization of the steady (direct current) potential. These changes are characteristic of cortical spreading ischemia. Replacing oxyhemoglobin for the nitric oxide synthase inhibitor Nomega-nitro-L-arginine mimicked these effects, implicating nitric oxide scavenging functions of oxyhemoglobin. Furthermore, the effect of endothelin-1 was related to a reduction of Na(+)-/K(+)-ATPase activity rather than solely to its vasoconstrictive properties. In conclusion, the threshold concentration of endothelin-1 that induces cerebral ischemia is profoundly reduced via a complex interaction between the neuronal/astroglial network and the cortical microcirculation if nitric oxide availability declines. The results may have implications for the understanding of subarachnoid hemorrhage-related cortical lesions.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Cerebrovascular Circulation; Cortical Spreading Depression; Endothelin-1; Free Radical Scavengers; Hemoglobins; Male; Membrane Potentials; Neurons; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Subarachnoid Space

This study assessed the behavioral and dendritic structural effects of combining subdural motor cortical electrical stimulation with motor skills training following unilateral sensorimotor cortex lesions in adult male rats. Rats were pre-operatively trained on a skilled forelimb reaching task, the Montoya staircase test, and then received endothelin-1 induced ischemic lesions of the sensorimotor cortex. Ten to 14 days later, electrodes were implanted over the peri-lesion cortical surface. Rats subsequently began 10 days of rehabilitative training on the reaching task in 1 of 3 conditions: 1. 50 Hz stimulation during training, 2. 250 Hz stimulation during training or 3. no stimulation. No significant difference in performance was found between the 250 Hz and no stimulation groups. The 50 Hz stimulation group had significantly greater rates of improvement with the impaired forelimb in comparison to 250 Hz and no stimulation groups combined. Fifty Hz stimulated animals also had a significant increase in the surface density of dendritic processes immunoreactive for the cytoskeletal protein, microtubule-associated protein 2, in the peri-lesion cortex compared to the other groups. These results support the efficacy of combining rehabilitative training with cortical electrical stimulation to improve functional outcome and cortical neuronal structural plasticity following sensorimotor cortical damage.

Topics: Analysis of Variance; Animals; Behavior, Animal; Body Weight; Brain Ischemia; Cell Count; Cerebral Cortex; Corpus Striatum; Dendrites; Disease Models, Animal; Dose-Response Relationship, Radiation; Electric Stimulation; Electrodes; Endothelin-1; Forelimb; Immunohistochemistry; Male; Microtubule-Associated Proteins; Motor Skills; Neuronal Plasticity; Rats; Rats, Long-Evans; Recovery of Function; Sensory Thresholds; Staining and Labeling

Axon injury following cerebral ischemia has received little scientific attention compared to the abundance of information dealing with the pathophysiology of grey matter ischemia. There are differences in the initial response of grey and white matter to ischemia in vitro. In this study we investigate whether the vasoactive peptide, endothelin-1, can generate a focal ischemic lesion in the white matter and compare the findings with endothelin-1-induced lesions in the grey matter. Using a minimally invasive technique to microinject endothelin-1 into selected brain regions, we observed an acute reduction in local MRI perfusion in the injected hemisphere after 1 hour. Twenty-four hours after microinjection of 10 pmoles of endothelin-1, we observed a loss of neurons in the grey matter. At 72 hours, neutrophils were absent and a macrophage/microglia response and astrocyte gliosis were detected. No breakdown in the blood-brain barrier was detected. After injection of 10 pmoles endothelin-1 into the cortical white matter, we observed prolific amyloid precursor protein-positive immunostaining (indicative of axonal disruption) and an increase in tau-1 immunostaining in oligodendrocytes at 6 hours. Similar to the grey matter lesions, no neutrophils were present, a macrophage/microglia response did not occur until 72 hours and there was no disruption in the blood-brain barrier. Focal injections of endothelin-1 into specific areas of the rat CNS represent a model to investigate therapeutic approaches to white matter ischemia.

Topics: Animals; Brain; Brain Ischemia; Central Nervous System; Endothelin-1; Male; Rats; Rats, Wistar

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

To study the protective effect of Ligustrazine on IR after local cerebral ischemia of rat.. Models of rat IR after local cerebral ischemia were prepared by electrocagulation of the middle cerebral artery, and changes of serum insulin, tomer necrosis factor-alpha (TNF-alpha), plasma endothelin-1 (ET-1), nitric oxide (NO) and nitric oxide synthase (NOS) were observed 2 weeks after the ischemia.. Ligustrazine could significantly reduce serum insulin (P < 0.01), the content of plasma ET-1 (P < 0.01) and serum TNF-alpha (P < 0.01), the activity of brain tissue NO and NOS (P < 0.01). The drug also increased insulin sensitivity indexes (ISI).. The protective effects of Ligustrazion on IR cerebral ischemia may be related to decreasing ET-1 content in plasma, TNF-alpha content in serum, NO content and NOS activities in tissue.

Topics: Animals; Brain Ischemia; Endothelin-1; Female; Infarction, Middle Cerebral Artery; Insulin; Insulin Resistance; Male; Neuroprotective Agents; Pyrazines; Random Allocation; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Elevated levels of endothelin-1 (ET-1) have been reported in cerebral ischemia. A role for ET may prove more important if the vascular receptors were changed. We addressed whether there is any change in ET receptor expression in cerebral ischemia.. The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method. The basilar artery and both MCAs were removed after 46 hours of recirculation. The contractile responses to ET-1, a combined ET(A) and ET(B) receptor agonist, and sarafotoxin 6c (S6c), a selective ET(B) receptor agonist, were examined in vitro, and ET receptor mRNA was quantified by real-time polymerase chain reaction.. S6c, which had no contractile effect per se on fresh or sham-operated rat cerebral arteries, induced a marked contraction in the occluded MCA (E(max) [maximum contraction, calculated as percentage of the contractile capacity of 63.5 mmol/L K+]=68+/-68%; P<0.0001), while there was no difference in the responses to ET-1 after cerebral ischemia. Real-time polymerase chain reaction revealed a significant upregulation of both the ET(A) and ET(B) receptors (both P<0.05) in the occluded MCA compared with the nonoccluded MCA from the same rats.. Focal cerebral ischemia in rat induces increased transcription of both ET(A) and ET(B) receptors, which results in the appearance of a contractile response to the ET(B) receptor agonist S6c. These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia.

Topics: Animals; Basilar Artery; Brain Ischemia; Disease Models, Animal; Endothelin-1; In Vitro Techniques; Infarction, Middle Cerebral Artery; Male; Middle Cerebral Artery; Peptide Elongation Factor 1; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Up-Regulation; Vasoconstriction; Vasoconstrictor Agents; Viper Venoms

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

We tested the hypothesis that endothelin-1 (ET-1), a cerebrovasoconstrictive peptide, would alter microvascular oxygen balance during focal cerebral ischemia. In this study, male Wistar rats were placed in control (n=9) and ET-1-treated (n=9) groups. Cortical ischemia was induced by middle cerebral artery (MCA) occlusion in isoflurane (1.4%) anesthetized rats. Forty minutes after MCA occlusion, 10(-7) M ET-1 or saline was applied to the ischemic cortex (IC) for a period of 20 min; the fluid was changed every 5 min. After 1 h of ischemia, regional cerebral blood flow (rCBF) was determined using a 14C-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined microspectrophotometrically. The cerebral blood flow (45% control, 45% ET-l) and oxygen consumption (24% control, 44% ET-1) of the IC were significantly lower than the contralateral cortex. ET-1 of 10(-7) M did not cause a statistically significant alteration in regional cerebral blood flow or oxygen consumption of the IC, but did increase the average venous O(2) saturation of the IC from 50 +/- 1% to 55 +/- 2% (p<0.04). A significant (p<0.05) increase was observed in O(2) supply/consumption ratio in the ET-1-treated IC (2.79 +/- 0.26 ml O(2)/min/100 g in ET-1-treated IC vs. 2.41 +/- 0.12 ml O(2)/min/100 g in the control IC) compared to the control IC. ET-1 also significantly lowered the frequency of small veins with less than 50% O(2) saturation in the IC (39 out of 70 veins in IC vs. 17 out of 70 veins in ET-1-treated IC). Thus, the exogenous application of 10(-7) M ET-1 improved microvascular oxygen supply/consumption balance during focal cerebral ischemia.

Topics: Animals; Blood Gas Analysis; Blood Pressure; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Heart Rate; Infarction, Middle Cerebral Artery; Male; Oxygen; Oxygen Consumption; Rats; Telencephalon

This unit presents models that are both used to study ischemic mechanisms and to test for neuroprotective agents or agents that enhance recovery from stroke. The Tamura model is one of the best characterized focal ischemia models in which the middle cerebral artery is occluded by electrocoagulation. Also described is the intraluminal monofilament model, the spontaneously hypertensive rat (SHR), and the newer endothelin-1 model. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.

Topics: Animals; Benzoxazines; Brain; Brain Ischemia; Coloring Agents; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelin-1; Gerbillinae; Infarction, Middle Cerebral Artery; Ligation; Male; Motor Activity; Neuroprotective Agents; Oxazines; Postural Balance; Rats; Species Specificity; Staining and Labeling; Stereotaxic Techniques; Suture Techniques

This study evaluates the effects of N-(2-[bis (4-fluorophenyl)methoxy]ethyl)-1-butanamine hydrochloride (LY393613), a novel neuronal (N/P/Q-type) Ca(2+) channel blocker, in ischaemia. For comparison, two commonly used L-type Ca(2+) channel blockers; nimodipine and verapamil were also evaluated. Ischaemia was induced in freely moving rats by micro-injection of endothelin-1 near the middle cerebral artery. In vivo microdialysis, laser Doppler flowmetry and histology were used to monitor ischaemia. Administration of LY393613, before and after the insult, attenuated the ischaemia-induced glutamate release, but not the dopamine release. Both nimodipine and verapamil failed to affect transmitter releases significantly, when administered post-occlusion. None of the compounds tested, produced any significant change in striatal blood flow. Histology showed that ischaemic damage was significantly less in LY393613 pre-treated rats. In conclusion, LY393613, a neuronal N/P/Q-Ca(2+) channel blocker, can attenuate ischaemic brain damage. The protective mechanism appears to be mainly the attenuation of the ischaemia-induced glutamate release, rather than its effect on cerebral hemodynamics.

Topics: Amines; Animals; Brain Ischemia; Butanes; Calcium Channel Blockers; Corpus Striatum; Dopamine; Endothelin-1; Glutamic Acid; Laser-Doppler Flowmetry; Male; Microdialysis; Nimodipine; Rats; Rats, Wistar; Time Factors; Verapamil

Brain tissue damage due to ischemia/reperfusion has been shown to be caused, in part, by activated macrophages infiltrating into the post-ischemic brain. Using the Middle Cerebral Artery Occlusion (MCAO) mouse model, this study demonstrated that, in vivo, both endothelin-1 (Et-1), a potent vasoconstrictor, and the macrophage chemokine, monocyte chemoattractant factor-1 (MCP-1) are induced in ischemia. Further studies, using human brain-derived endothelial cells (CNS-EC), showed that in vitro, Et-1 can directly stimulate MCP-1 mRNA expression and MCP-1 protein; and this Et-1-induced MCP-1 production is mediated by the ET(A) receptor. Inflammatory cytokines, tumor necrosis factor alpha and interleukin-1beta, functioned additively and synergistically, respectively, with Et-1 to increase this MCP-1 production. Partial elucidation of the signal transduction pathways involved in Et-1-induced MCP-1 production demonstrated that protein kinase C-, but not cAMP-dependent pathways are involved. These data demonstrate that Et-1, functioning as an inflammatory peptide, increased levels of MCP-1, suggesting a mechanism for chemokine regulation during ischemia/reperfusion injury.

Topics: Animals; Brain; Brain Ischemia; Cells, Cultured; Chemokine CCL2; Cyclic AMP-Dependent Protein Kinases; Cytokines; Endothelin-1; Endothelium; Humans; Male; Mice; Mice, Inbred C57BL; Protein Kinase C; Receptor, Endothelin A; Receptors, Endothelin; RNA, Messenger; Up-Regulation

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

Hyperthermia during or after stroke is known to worsen neuronal damage. Paradoxically, when hyperthermia precedes stroke, it can protect against a subsequent ischemic insult. Other stressors including restraint also have a similar pre-conditioning effect. In the present study, we report the unanticipated finding that conscious rats, restrained for the purpose of intravenous infusion, had markedly reduced neuronal and functional deficits after middle cerebral artery occlusion compared with unrestrained rats. Restrained rats had significantly higher body temperature prior to stroke than unrestrained rats. The findings suggest restraint leading to mild hyperthermia may be sufficient to induce adaptive processes which protect against subsequent ischemia.

Topics: Adaptation, Physiological; Animals; Body Temperature; Brain Ischemia; Cerebral Cortex; Cerebral Infarction; Corpus Striatum; Endothelin-1; Hyperthermia, Induced; Male; Motor Activity; Rats; Rats, Wistar; Restraint, Physical; Stroke

The vasoconstrictive peptide endothelin-1 (ET-1) has been used previously to transiently occlude the middle cerebral artery (MCA) in rats. However, the duration of the resulting reduction in cerebral blood flow (CBF) and the reperfusion characteristics are poorly understood. In this study perfusion and T(2)-weighted MRI were used together with histology to characterize the cerebral perfusion dynamics and lesion development following ET-1 injection. Twenty-two rats received an intracerebral injection of ET-1 adjacent to the MCA. CBF was reduced to 30-50% of control levels, and a significant reduction persisted for 16 h in the cortex and 7 h in the striatum. The lesion size measured by T(2)-weighted imaging at 48 h correlated with the final infarct size measured by histology at 7 d. The sustained reduction in CBF and the gradual development of the ischemic lesion resemble human stroke evolution, suggesting that this model may be useful for evaluating therapeutic agents, particularly when treatment is delayed.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Endothelin-1; Magnetic Resonance Imaging; Male; Rats; Rats, Sprague-Dawley

The purpose of this study was to determine whether the transport of small hydrophilic molecules across the blood-brain barrier (BBB) during focal cerebral ischemia could be altered by a topical application of endothelin-1 (ET-1) in the ischemic cortex (IC). Forty minutes after middle cerebral artery (MCA) occlusion, patches of 10 nM ET-1 (low-endothelin group), 100 nM ET-1 (high-endothelin group), or normal saline (control group) were placed on the IC of rats for a 20-min period. One hour after MCA occlusion, transfer coefficient (Ki) of [14C-alpha-]aminoisobutyric acid (14C-AIB) or regional cerebral blood flow (rCBF) was determined. Vital signs were not significantly different among the experimental groups. In the control group (n=8), the Ki of the IC was significantly higher than that of the contralateral cortex (CC; 11.9+/-5.8 vs 5.0+/-1.9 microl/g per minute). In the low-endothelin group (n=8), the Ki of the IC was still significantly higher than that of the CC (9.4+/-5.2 vs 5.3+/-2.5 microl/g per minute). However, in the High-endothelin group (n=8), the Ki of the IC was not different from that of the CC (6.9+/-2.1 vs 5.6+/-2.3 microl/g per minute) and 42% lower than that of the control group. The rCBF was not affected by 100 nM of ET-1 [control (n=6): IC 53+/-18 ml/100 g per minute, CC 94+/-23 ml/100 g per minute; high-endothelin (n=6): IC 49+/-15 ml/100 g per minute, CC 98+/-24 ml/100 g per minute]. Our data suggest that the application of endothelin-1 in the IC could reduce the transfer coefficient of small hydrophilic molecules across the BBB during focal ischemia.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Capillary Permeability; Cerebral Cortex; Endothelin-1; Infarction, Middle Cerebral Artery; Ligation; Male; Rats; Rats, Long-Evans

Endothelins (ETs) are potent vasoconstrictors and may play a role in the pathophysiology of several diseases. Limited and controversial data exist on their role in human ischemic stroke. We planned a prospective, observational, and longitudinal clinical study to test whether ET-1 levels increase in various phases of ischemic stroke and whether the ET-1 levels correlate with neurological scores, stroke etiology, stroke risk factors, or final outcome.. We measured plasma ET-1 levels with a sandwich-enzyme immunoassay method in 101 consecutive patients with ischemic stroke on admission and 1 week, 1 month, and 3 months after stroke and in 101 sex- and age-matched control subjects. At each sampling, the patients underwent a complete neurological evaluation. All stroke risk factors were recorded, an array of laboratory tests were performed, and the subtype of ischemic stroke was determined. The patients were contacted 3 years later for prognostic determination.. ET-1 levels in patients (2.4+/-1.3 pg/mL on admission, 2.2+/-1.4 pg/mL at 1 week, 2.1+/-1.4 pg/mL at 1 month, and 2.1+/-1.2 pg/mL at 3 months) were not different from those of the control subjects (2.2+/-0.9 pg/mL) at any time point. No correlation was found between the ET-1 levels and stroke etiology, stroke risk factors, stroke recurrence risk, age, sex, or neurological scores, except that ET-1 levels correlated with the use of warfarin and with body mass index.. Plasma ET-1 levels were normal in patients with ischemic stroke. Our findings cannot exclude a role of ETs in the pathophysiology of ischemic stroke because plasma levels might not accurately reflect intracerebral concentrations, but they also do not support the occurrence of a major plasma ET-1 level increase at any phase of stroke. Our patient population is the largest ever reported in whom ET-1 levels were measured, but it consisted of mild and moderately ill patients with stroke due to the study design, of which the aim was long-term observation, which excludes severely ill patients.

Topics: Adult; Aged; Aged, 80 and over; Anticoagulants; Body Mass Index; Brain Ischemia; Endothelin-1; Female; Humans; Male; Middle Aged; Nervous System; Prognosis; Reference Values; Risk Factors; Stroke; Warfarin

The aim of this MR study was to determine if vasospasm induced by application of endothelin-1 (ET-1) in the rat brain would model the abnormalities attributed to vasospasm described in patients with subarachnoid haemorrhage (SAH) with reversible neurological deficits. Following application of ET-1 in concentrations of 10(-4) M or 10(-6) M to the middle cerebral artery, there was an immediate drop in pH, an increase in the inorganic phosphate (P(i)) to phosphocreatine (PCr) ratio and elevated lactate. There was gradual recovery to control in the 10(-6) M group, but in the 10(-4) M group there was a loss of approximately 10% in the absolute signal intensities of PCr and adenosine triphosphate (ATP). In a second similarly treated group of animals, the area of the hemisphere with a low apparent diffusion coefficient (ADC) was 27 +/- 6% at 30 min and remained at about 20-21% at 90 min and beyond. Together these data suggest that the regions with persistently low ADC were metabolically compromised, with incomplete recovery of PCr and ATP, and represent irreversibly damaged tissue. This raises the possibility that MR spectroscopy and imaging could be a sensitive indicator of tissue viability. This is a potentially useful model of low flow as seen in clinical vasospasm following SAH.

Topics: Adenosine Triphosphate; Animals; Brain Ischemia; Disease Models, Animal; Endothelin-1; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Male; Rats; Time Factors; Vasospasm, Intracranial

In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.

Topics: Amidines; Animals; Brain Ischemia; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Endothelin-1; Enzyme Inhibitors; Gerbillinae; Humans; Male; Neuroprotective Agents; Nitric Oxide Synthase; Rats; Reperfusion Injury; Time Factors

The aim of the work was to evaluate an influence of CoQ(10) on lactate acidosis, adenosine-5'-triphosphate (ATP) concentrations, oxidized to reduced glutathione ratio and on superoxide dismutase activity in endothelin model of cerebral ischemia in the rat. Light microscopic studies in the central nervous system and morphometric analysis of pyramidal cells in the hippocampus were also performed. Endothelins (ET-1 or ET-3; 20 pmoles) were injected into the right lateral cerebral ventricle (intracerebroventricularly). CoQ(10) was given intraperitoneally (i.p.) just before the operation (i.p. 10 mgkg b. wt.). More severe changes of investigated biochemical parameters were observed in the animals treated with ET-1 in comparison with ET-3. Recovery was noted earlier in the group subjected to ET-3 and CoQ(10) administration, than in the animals subjected to ET-1 and CoQ(10) treatment. Histopathological observations showed sparse foci of a neuronal loss in the cerebral cortex and in the hippocampus only in the ET-1 model of ischemia. Additionally more numerous dark neurons were present in above brain structures following ET-1 administration comparing with ET-3 one. Morphometrical studies demonstrated that CoQ(10) diminished neuronal injury in the hippocampal CA1, CA2 and CA3 zones. Above data indicate on neuroprotective effect of CoQ(10) as a potent antioxidant and oxygen derived free radicals scavenger in the cerebral ischemia.

Topics: Adenosine Triphosphate; Animals; Brain; Brain Ischemia; Brain Stem; Cerebellum; Cerebral Cortex; Cerebral Ventricles; Coenzymes; Disease Models, Animal; Endothelin-1; Functional Laterality; Glutathione; Glutathione Disulfide; Hippocampus; Injections, Intraventricular; Lactates; Male; Neurons; Pyramidal Cells; Rats; Rats, Wistar; Superoxide Dismutase; Ubiquinone

To observe the changes of some neuropeptides and the effect of Fusheng powder (FSP) on neuropeptides in rat's brains in a stable cerebral ischemia and reperfusion (I/L) model.. The models of rat's brain injured were established by repeated cerebral I/R in rats with hyperlipidemia. Radioimmunoassay (RIA) was performed to determine the level of neuropeptides.. After 1 day of I/R, compared with the control group, the contents of endothelin-1 (ET-1), calcitonin gene related peptide (CGRP) and neuropeptide Y (NPY) in the model animals were significantly increased by 24.3%, 33.7% and 51.86% respectively, while the level of somatostatin (SS) decreased by 37.86% (all P < 0.01). Meanwhile after FSP treatment, the contents of neuropeptides were alleviated respectively (P < 0.05, P < 0.01). Apart from the ET, the releases of CGRP, NPY and SS were all recovered in different degree after 7 days of I/R.. There were obvious imbalance of neuropeptides in rat's brains after cerebral I/R and the FSP might antagonize ischemic injury of brain through modulating neuropeptides, which may be one of the therapeutical mechanism in treating cerebral vascular diseases with FSP.

Topics: Animals; Brain; Brain Ischemia; Calcitonin Gene-Related Peptide; Drugs, Chinese Herbal; Endothelin-1; Hyperlipidemias; Male; Neuropeptide Y; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury

To investigate the effects of L-tetrahydropalmatine (L-THP) on energy metabolism, endothelin-1 (ET-1) and NO during acute cerebral ischemia-reperfusion of rats, 24 Wistar rats were randomly divided into four groups, with 6 rats in each group: sham-operation group, simple ischemia group, ischemia-reperfusion group and treatment group (L-THP group). Cerebral ATP, lactate, ET-1 and NO levels were measured in all groups. Our results showed that treatment with L-THP could increase cerebral ATP levels, but decrease cerebral lactate, ET-1 and NO concentrations during ischemia-reperfusion in the treatment group. It is concluded that L-THP could improve cerebral energy metabolism and protect the injured brain tissue, the mechanism of which might be related to suppression of overproduction of ET-1 and NO.

Topics: Animals; Berberine Alkaloids; Brain Ischemia; Case-Control Studies; Endothelin-1; Energy Metabolism; Nitric Oxide; Rats; Rats, Wistar; Reperfusion Injury

Release of inflammatory mediators from leukocytes and endothelial release of vasoactive factors are both important in the pathogenesis of atherosclerosis. To evaluate the concentrations of a specific marker for macrophage activation, neopterin, and the potent endothelial derived vasoconstrictive peptide endothelin-1 (ET-1), during the acute and chronic stages of cerebral ischemia, plasma concentrations of neopterin and ET-1 were measured in 59 patients with acute cerebral infarction or transient ischemic attack (median age 73 years, range 43-93, 27 men) and after a 1-year follow-up in 57/59 (97%) of patients. Plasma neopterin was higher at follow-up (6.3 nmol/L [3.7-21.6] vs 5.6 nmol/L [3.5-17.2]; p < 0.05) than at the acute stage, whereas the plasma ET-1 concentration was unchanged. Plasma concentrations of both neopterin and ET-1 correlated directly with age both in the acute stage (r = 0.42 and r = 0.35, respectively; p < 0.01) and after follow-up (r = 0.34; p < 0.05 and r = 0.27; p = 0.05, respectively). In conclusion, plasma neopterin increased after acute cerebral ischemia, indicating chronic inflammatory activity and continuous macrophage activation in ischemic cerebrovascular diseases.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Brain Ischemia; Endothelin-1; Female; Follow-Up Studies; Humans; Ischemic Attack, Transient; Male; Middle Aged; Neopterin; Time Factors

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.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Dioxanes; Endothelin Receptor Antagonists; Endothelin-1; Male; Microcirculation; Middle Cerebral Artery; Pyridines; Pyrimidines; Rats; Rats, Inbred SHR; Receptor, Endothelin A; Sulfonamides; Tetrazoles

The aim of the work was to evaluate the influence of CoQ10 on superoxide dismutase (SOD) activity levels in the rat model of cerebral ischemia induced by endothelins (ET-1 or ET-3). ETs (20 pmol) were injected into the right lateral cerebral ventricle and immediately CoQ10 was given intraperitoneally (10 mg/kg b.w.). In the brains of experimental animals subjected both to ET-1 and ET-2 administration there was observed a decrease of SOD activity in the brain stem, in the cerebrallum and in the cerebral cortex at all time intervals. ET-1, as compared to ET-3 evoked longer lasting disturbances in SOD activity. In the cerebellum and in the cerebral cortex positive effect of CoQ10 and recovery to the control values was noted after 4 hours in the group subjected to ET-3 injection and after 24 hours in the ET-1 treated animal. Investigated brain areas showed different sensitivity to ETs. Above data may indicate on beneficial effect CoQ10 in the cerebral ischemia via decrease of free radicals concentration.

Topics: Animals; Brain Ischemia; Brain Stem; Cerebellum; Cerebral Cortex; Coenzymes; Disease Models, Animal; Endothelin-1; Endothelin-3; Male; Rats; Rats, Wistar; Superoxide Dismutase; Ubiquinone

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain Ischemia; Calcium; Capillaries; Cattle; Cells, Cultured; Cerebrovascular Circulation; Colforsin; Cyclic AMP; Cycloleucine; Endothelin-1; Endothelium, Vascular; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate; Kainic Acid; Mice; N-Methylaspartate; Neurons; Polymerase Chain Reaction; Rats; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Second Messenger Systems

Topics: Acute Disease; Brain Ischemia; Cerebrovascular Disorders; Endothelin-1; Humans

The effects of ZK-118.182, a stable analogue of PGD2, were evaluated in an endothelin-1-induced cerebral ischemia rabbit model. Ischemia was induced by endothelin-1 injection (0.25 ng bolus) into subcavian artery and ischemic changes were assessed histologically by the number of ischemic neurons in the brain stem. ZK-118.182 (2 micrograms/kg, bolus into subclavian artery) reduced the number of ischemic neurons when injected 20 min after endothelin-1 injection, Iloprost, a stable analogue of PGI2, was also effective in reducing the number of ischemic neurons in a dose of 0.5 microgram/kg (bolus into subclavian artery). The results suggested that ZK-118.182 has a potent antiischemic effect which is comparable to that of iloprost in rabbits.

Topics: Animals; Brain Ischemia; Dinoprost; Disease Models, Animal; Endothelin-1; Iloprost; Neurons; Platelet Aggregation Inhibitors; Rabbits

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1-stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor-alpha (TNF) and IL-1beta functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-beta (TGF-beta), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

Topics: Brain Ischemia; Cells, Cultured; Cerebral Arteries; Cerebral Veins; Chemotaxis, Leukocyte; Culture Media, Conditioned; Drug Synergism; Endothelin-1; Endothelium, Vascular; Gene Expression Regulation; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-10; Interleukin-8; Protein Isoforms; Reperfusion Injury; Risk Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The aim of the work was to evaluate the effect of CoQ10 (10 mg/kg body weight) on the morphological changes in the rat brain after the Et-1 induced cerebral ischemia. Selective necrotic foci and dark neurons were observed in the cerebral cortex, hippocampal CA1, CA2 sectors and dentate gyrus after the administration of Et-1. Around the necrotic foci, glycogen was deposited 24 hours after the ischemic hypoxia. It seems that histopathological changes evoked by Et-1 indicate the complicated mechanism connected with ischemia. After treatment with CoQ10 only sparse neuronal changes were observed. CoQ10, known oxygen-derived free radicals scavenger diminished neuronal damage in the cerebral cortex and in the hippocampus.

Topics: Animals; Brain; Brain Ischemia; Cerebral Cortex; Dentate Gyrus; Endothelin-1; Hippocampus; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reactive Oxygen Species; Ubiquinone

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

Endothelin-1 (ET-1) gene expression of rat brain during ischemia and reperfusion as well as the effect of Radix Salviae Miltiorrhizae (RSM) were studied with in situ hybridization. It was found that ET-1 gene expression of cerebral cortex and caudate-putamen was markedly increased both in 24 hours of ischemia and 24 hours of reperfusion groups (P < 0.01, P < 0.05). In RSM-treated rats, although the ET-1 gene expressions of ischemia and reperfusion sides were also increased as compared with contralateral cortex and caudate-putamen, they were significantly lower in RSM-treated rats than those of controls (P < 0.05, P < 0.01 respectively). The present study indicated that RSM can partly inhibit ET-1 gene expression of cerebral cortex and caudate-putamen during ischemia and reperfusion. This may be one of the protective mechanisms of RSM on cerebral ischemia and reperfusion.

Topics: Animals; Brain; Brain Ischemia; Drugs, Chinese Herbal; Endothelin-1; In Situ Hybridization; Male; Plant Extracts; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salvia miltiorrhiza

Tacrolimus (FK506) is a potent immunosuppressant that is presently in clinical use for prevention of allograft rejection. Recently, animal studies reporting significant reductions in the volume of tissue damage associated with cardiac, hepatic, and cerebral ischemia suggest that tacrolimus may also be of use in the clinical management of stroke. In the present study, we examine whether the neuroprotective effects of tacrolimus, as assessed by histological outcome, are accompanied by an amelioration of the skilled motor deficits induced in the rat by middle cerebral artery occlusion (MCAO).. Animals were trained to perform a skilled paw-reaching task before MCAO by perivascular microinjections of endothelin-1. Tacrolimus (1 mg/kg, n = 6) or vehicle (n = 6) was administered by intravenous infusion 1 minute after MCAO. After a 5-day postoperative recovery period, the rats were retested for skilled paw-reaching ability for an additional 9 days.. In vehicle-treated rats, MCAO resulted in a profound bilateral impairment in skilled paw use. Rats treated with tacrolimus, although still impaired, performed significantly better than those treated with vehicle alone (P < .01). Histological analysis, 14 days after occlusion, confirmed the neuroprotective efficacy of tacrolimus with a 66% reduction in the volume of hemispheric brain damage produced by MCAO (P < .01).. The present studies show that tacrolimus not only protects neural tissue from focal cerebral ischemia but also significantly ameliorates the deficits in skilled motor ability produced by this lesion. These data provide further support for the view that tacrolimus may be of use in the treatment of stroke.

Topics: Animals; Brain Ischemia; Cerebral Arteries; Constriction; Endothelin-1; Male; Movement Disorders; Neuroprotective Agents; Psychomotor Performance; Rats; Tacrolimus

To determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest.. Prospective study.. Medical intensive care unit in a university hospital.. 10 comatose patients (Glasgow Coma Score +/- 6)successfully resuscitated from a cardiac arrest occurring outside the hospital.. We measured the pulsatility index (PI) and mean blood flow velocity (MFV) of the middle cerebral artery, the cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate, and cGMP during the first 24 h after cardiac arrest.. The PI decreased significantly from 1.86 +/- 1.02 to 1.05 +/- 0.22 (p = 0.03). The MFV increased significantly from 29 +/- 10 to 62 +/- 25 cm/s (p = 0.003). Cerebral oxygen extraction ratio decreased also from 0.39 +/- 0.13 to 0.24 +/- 0.11 (p = 0.015). Endothelin levels were high but did not change during the study period. Nitrate levels varied widely and showed a slight but significant decrease from 37.1 mumol/l (median; 25th-75th percentiles: 26.8-61.6) to 31.3 mumol/l (22.1-39.6) (p = 0.04). Cyclic guanosine monophosphate levels increased significantly from 2.95 mumol/l (median; 25th-75th percentiles: 2.48-5.43) to 7.5 mumol/l (6.20-14.0) (p = 0.02).. We found evidence of increased cerebrovascular resistance during the first 24 h after cardiac arrest with persistent high endothelin levels, gradually decreasing nitrate levels, and gradually increasing cGMP levels, This suggests that active cerebral vasoconstriction due to an imbalance between local vasodilators and vasoconstrictors plays a role in the delayed hypoperfusion phase.

Topics: Adult; Aged; Aged, 80 and over; Brain Ischemia; Cardiopulmonary Resuscitation; Cerebrovascular Circulation; Coma; Critical Care; Cyclic GMP; Endothelin-1; Female; Heart Arrest; Humans; Male; Middle Aged; Nitrates; Prospective Studies; Vascular Resistance

The experiment was designed to study the association of cerebral ischemia and reperfusion with endothelin-1 (ET-1) gene expression of rat brains and time-dependent changes of ET-1 gene expression during cerebral ischemia.. Thirty-three male SD rats were divided into dot blot hybridization (n = 27) and in situ hybridization groups (n = 6). The focal cerebral ischemia and reperfusion models were made with suture embolism of middle cerebral artery. Dot blot hybridization groups were redivided into control and ischemic subgroups (ischemia for 0.5, 1, 1.5, 3, 6, 12, 24, 48 and 72 h respectively). In situ hybridization groups were redivided into ischemia and reperfusion groups. After 24 h ischemia and 24 h reperfusion, ET-1 gene expressions were investigated with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System.. Dot blot hybridization showed that ET-1 mRNA of cerebral cortex and caudate-putamen was increased at 6 h of ischemia and reached peak at 24 h (3.9 and 3.7 fold respectively), and at 72 h of ischemia it remained at high levels (3.5 and 2.1 fold respectively). In situ hybridization showed that the levels of ET-1 mRNA of cerebral cortex and caudate-putamen were also markedly increased both in 24 h ischemia and 24 h reperfusion groups (P < 0.01, P < 0.05 respectively).. ET-1 gene expression in focal ischemic brain tissue were markedly and progressively increased during cerebral ischemia and reperfusion and down-regulation of ET-1 gene expression may be a new approach to the treatment of ischemic cerebrovascular diseases.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Endothelin-1; Gene Expression; Male; Neostriatum; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger