Mechanisms Of Ischemic Brain Damage Research Articles

Priming and Combined Strategies for the Application of Mesenchymal Stem Cells in Ischemic Stroke: A Promising Approach.

Ischemic stroke (IS) is a leading cause of death and disability worldwide. Tissue plasminogen activator (tPA) administration and mechanical thrombectomy are the main treatments but have a narrow time window. Mesenchymal stem cells (MSCs), which are easily scalable in vitro and lack ethical concerns, possess the potential to differentiate into various types of cells and secrete a great number of growth factors for neuroprotection and regeneration. Moreover, MSCs have low immunogenicity and tumorigenic properties, showing safety and preliminary efficacy both in preclinical studies and clinical trials of IS. However, it is unlikely that MSC treatment alone will be sufficient to maximize recovery due to the low survival rate of transplanted cells and various mechanisms of ischemic brain damage in the different stages of IS. Preconditioning was used to facilitate the homing, survival, and secretion ability of the grafted MSCs in the ischemic region, while combination therapies are alternatives that can maximize the treatment effects, focusing on multiple therapeutic targets to promote stroke recovery. In this case, the combination therapy can yield a synergistic effect. In this review, we summarize the type of MSCs, preconditioning methods, and combined strategies as well as their therapeutic mechanism in the treatment of IS to accelerate the transformation from basic research to clinical application.

Photochemicallly Induced Thrombosis as a Model of Ischemic Stroke

Better understanding of ischemic brain injury mechanisms is important for the development and improvement of diagnostic and therapeutic modalities for management of ischemic stroke. As experimental studies are on demand, there’s a need for relevant models of focal brain lesions. Photochemically induced thrombosis remains one of the most popular models of ischemic stroke.The purpose of the reviewis to consider the pathogenesis and applicational relevance of the photochemical thrombosis in ischemic stroke modeling.Material and methods.The information was searched using PubMed and Google Scholar databases and keywords «photothrombotic stroke» without language restrictions. 74 papers out of more than 600 sources were found the most relevant for the purpose of this review and selected for the analysis. Of these, more than 50% have been published in the last five years. The criterion for excluding a source was an inconsistency with the objectives of the review and low information content.Results.We outlined a variety of features in modeling photothrombotic stroke, analyzed the advantages and disadvantages of the model, presented data on current method’s modifications, as well as approaches to evaluation of brain lesions in ischemic stroke induced by photothrombosis, and summarized information about the mechanisms of brain damage induced in this model.Conclusion.Several advantages of the photothrombotic stroke model, such as low invasiveness, high reproducibility, inherent control of brain infarction volume and low mortality, determine its active use in experimental studies of ischemic stroke. Pathological processes in the brain modeled by photochemical thrombosis are similar to the processes occurring in acute ischemic cerebral circulation events. Therefore, this model provides insights into cellular and molecular mechanisms of ischemic brain damage, and can be used for developing novel therapeutic approaches for management of ischemic stroke.

Современные нейрохимические и молекулярно-генетические аспекты патогенеза неонатальных судорог

The article is devoted to the modern aspects of the pathogenesis of neonatal seizures (NS). The main mechanisms of the development of perinatal brain injuries, which are fundamental in the genesis of NS, are considered, the prospects for research devoted to the study of the possibilities of molecular genetic prediction of the individual risk of ischemia and the development of NS are outlined. The mechanisms of ischemic brain damage during the neonatal period and the role of glutamate, the main excitatory neurotransmitter acting on NMDA receptors that regulate the electrical activity of neurons, are described in detail. The review focuses on the involvement of metalloproteinases, primarily MMP-9, which destroys type IV collagen, which is the main component of the basement membrane of the cerebral endothelium, and creates conditions for cell migration across the BBB, thereby causing a cytokine storm and the development of inflammation, leading to NS. The possible role of calcium-dependent proteinases — calpains in the development of NS is analyzed, since in some forms of CNS pathology, uncontrolled hyperactivation of calpains is observed, leading to a disruption of the regulation of neurotransmitter transmission, which can, in turn, become an additional factor for the development of the neurodegenerative process in the brain and the development of seizures. Also are presented the results of studies devoted to the role of ion channels, providing synaptic transmission of excitation from an excited neuron to other cells. It has been shown that molecular defects in ion channels can be one of the factors that create prerequisites for the development of NS. The article shows the prospects for studying the genetic polymorphism of enzymes involved in the pathogenesis of ischemic brain damage, based on ideas about the pathogenesis of cerebral disorders in newborns, which are based on oxidative damage. The concluding part of the article outlines the modern principles of NS therapy, their main goal is to relieve the symptoms of the underlying disease, to ensure the normalization of blood circulation and brain metabolism, to optimize the conditions for the functioning of the preserved brain structures to prevent the formation of severe neurological complications, as well as to maintain optimal parameters of respiration, glucose-electrolyte composition of blood and thermal regime. Based on the data presented, it is concluded that early diagnosis of NS is necessary, which is determined by the fact that seizures in newborns are usually caused by serious damage to the child's brain, and in some cases — by life-threatening conditions, the timely diagnosis of which contributes to the early onset of specific treatment. No conflict of interest was declared by the author. Key words: newborn, cerebral ischemia, neonatal seizures.

CD38 Knockout Mice Show Significant Protection Against Ischemic Brain Damage Despite High Level Poly-ADP-Ribosylation.

Several enzymes in cellular bioenergetics metabolism require NAD+ as an essential cofactor for their activity. NAD+ depletion following ischemic insult can result in cell death and has been associated with over-activation of poly-ADP-ribose polymerase PARP1 as well as an increase in NAD+ consuming enzyme CD38. CD38 is an NAD+ glycohydrolase that plays an important role in inflammatory responses. To determine the contribution of CD38 activity to the mechanisms of post-ischemic brain damage we subjected CD38 knockout (CD38KO) mice and wild-type (WT) mice to transient forebrain ischemia. The CD38KO mice showed a significant amelioration in both histological and neurologic outcome following ischemic insult. Decrease of hippocampal NAD+ levels detected during reperfusion in WT mice was only transient in CD38KO animals, suggesting that CD38 contributes to post-ischemic NAD+ catabolism. Surprisingly, pre-ischemic poly-ADP-ribose (PAR) levels were dramatically higher in CD38KO animals compared to WT animals and exhibited reduction post-ischemia in contrast to the increased levels in WT animals. The high PAR levels in CD38 mice were due to reduced expression levels of poly-ADP-ribose glycohydrolase (PARG). Thus, the absence of CD38 activity can not only directly affect inflammatory response, but also result in unpredicted alterations in the expression levels of enzymes participating in NAD+ metabolism. Although the CD38KO mice showed significant protection against ischemic brain injury, the changes in enzyme activity related to NAD+ metabolism makes the determination of the role of CD38 in mechanisms of ischemic brain damage more complex.

PPAR-γ Ameliorates Neuronal Apoptosis and Ischemic Brain Injury via Suppressing NF-κB-Driven p22phox Transcription.

Peroxisome proliferator-activated receptor-gamma (PPAR-γ), a stress-induced transcription factor, protects neurons against ischemic stroke insult by reducing oxidative stress. NADPH oxidase (NOX) activation, a major driving force in ROS generation in the setting of reoxygenation/reperfusion, constitutes an important pathogenetic mechanism of ischemic brain damage. In the present study, both transient in vitro oxygen-glucose deprivation and in vivo middle cerebral artery (MCA) occlusion-reperfusion experimental paradigms of ischemic neuronal death were used to investigate the interaction between PPAR-γ and NOX. With pharmacological (PPAR-γ antagonist GW9662), loss-of-function (PPAR-γ siRNA), and gain-of-function (Ad-PPAR-γ) approaches, we first demonstrated that 15-deoxy-∆(12,14)-PGJ2 (15d-PGJ2), via selectively attenuating p22phox expression, inhibited NOX activation and the subsequent ROS generation and neuronal death in a PPAR-γ-dependent manner. Secondly, results of promoter analyses and subcellular localization studies further revealed that PPAR-γ, via inhibiting hypoxia-induced NF-κB nuclear translocation, indirectly suppressed NF-κB-driven p22phox transcription. Noteworthily, postischemic p22phox siRNA treatment not only reduced infarct volumes but also improved functional outcome. In summary, we report a novel transrepression mechanism involving PPAR-γ downregulation of p22phox expression to suppress the subsequent NOX activation, ischemic neuronal death, and brain infarct. Identification of a PPAR-γ → NF-κB → p22phox neuroprotective signaling cascade opens a new avenue for protecting the brain against ischemic insult.

Hyperglycemia / hypoglycemia-induced mitochondrial dysfunction and cerebral ischemic damage in diabetics.

Enhancement of ischemic brain damage is one of the most serious complications of diabetes. Studies from various in vivo and in vitro models of cerebral ischemia have led to an understanding of the role of mitochondria and complex interrelated mitochondrial biochemical pathways leading to the aggravation of ischemic neuronal damage. Advancements in the elucidation of the mechanisms of ischemic brain damage in diabetic subjects have revealed a number of key mitochondrial targets that have been hypothesized to participate in enhancement of brain damage. The present review initially discusses the neurobiology of ischemic neuronal injury, with special emphasis on the central role of mitochondria in mediating its pathogenesis and therapeutic targets. Later it further details the potential role of various biochemical mediators and second messengers causing widespread ischemic brain damage among diabetics via mitochondrial pathways. The present review discusses preclinical data which validates the significance of mitochondrial mechanisms in mediating the aggravation of ischemic cerebral injury in diabetes. Exploitation of these targets may provide effective therapeutic agents for the management of diabetes-related aggravation of ischemic neuronal damage.

Deletion of Mitochondrial Uncoupling Protein-2 Increases Ischemic Brain Damage after Transient Focal Ischemia by Altering Gene Expression Patterns and Enhancing Inflammatory Cytokines

Mitochondrial hyperpolarization inhibits the electron transport chain and increases incomplete reduction of oxygen, enabling production of reactive oxygen species (ROS). The consequence is mitochondrial damage that eventually causes cell death. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that dissipate the mitochondrial proton gradient by transporting H(+) across the inner membrane, thereby stabilizing the inner mitochondrial membrane potential and reducing the formation of ROS. The role of UCP2 in neuroprotection is still in debate. This study seeks to clarify the role of UCP2 in transient focal ischemia (tFI) and to further understand the mechanisms of ischemic brain damage. Both wild-type and UCP2-knockout mice were subjected to tFI. Knocking out UCP2 significantly increased the infarct volume to 61% per hemisphere as compared with 18% in wild-type animals. Knocking out UCP2 suppressed antioxidant, cell-cycle, and DNA repair genes, including Sod1 and Sod2, Gstm1, and cyclins. Furthermore, knocking out UCP2 significantly upregulated the protein levels of the inflammatory cytokines, including CTACK, CXCL16, Eotaxin-2, fractalkine, and BLC. It is concluded that knocking out the UCP2 gene exacerbates neuronal death after cerebral ischemia with reperfusion and this detrimental effect is mediated by alteration of antioxidant genes and upregulation of inflammatory mediators.

Effects of monocyte chemoattractant protein 1 on blood-borne cell recruitment after transient focal cerebral ischemia in mice

Monocyte chemoattractant protein 1 (MCP-1) plays an important role in inflammatory reactions following cerebral ischemia. It is known that MCP-1 overexpression leads to increased infarct volume and elevated hematogenous cell recruitment, while MCP-1-deficient mice develop smaller infarcts. It was supposed that MCP-1 dependent macrophage recruitment might be the underlying mechanism of ischemic brain damage but a precise distinction of local microglia and invading macrophages was not performed. In this study we investigated the differential role of MCP-1 on inflammatory cells in MCP-1-deficient mice, using green fluorescent protein (GFP) transgenic bone marrow chimeras. After 30-min of focal cerebral ischemia microglia was rapidly activated and was not different between MCP-1-deficient mice and wild type controls. Activated microglia outnumbered GFP-positive macrophages over the study period. Furthermore, macrophage infiltration was significantly reduced at day 7 in MCP-1-deficient animals (31.2±20.1 cells/mm 2) compared to MCP-1 wild type mice (131.5±66.7 cells/mm 2, P<0.001). Neutrophils were also significantly reduced in MCP-1-deficient mice (62% on day 4% and 87% on day 7; P<0.001). This is the first investigation in cerebral ischemia showing that MCP-1 is necessary for recruiting blood-borne cells to the injury site whereas it does not affect the microglia activation and migration. However, the remarkable predominance of activated microglia and the additional attenuation of invading macrophages suggest that different mechanisms than macrophage recruitment are responsible for the MCP-1-mediated neuroprotective effects after experimental stroke.

RNase Therapy Assessed by Magnetic Resonance Imaging Reduces Cerebral Edema and Infarction Size in Acute Stroke

Ischemic stroke causes cell necrosis with the exposure of extracellular ribonucleic acid (RNA) and other intracellular material. As shown recently, extracellular RNA impaired the blood-brain-barrier and contributed to vasogenic edema-formation. Application of ribonuclease 1 (RNase 1) diminished edema-formation and also reduced lesion volume in experimental stroke. Here we investigate whether reduction of lesion volume is due to the reduction of edema or of other neuroprotective means. Neuroprotective and edema protective effects of RNase 1 pretreatment were assessed using a temporary middle cerebral artery occlusion (MCAO) model in rats. Lesion volume was assessed on magnetic resonance imaging (MRI). T2-relaxation-time and midline-shift as well as brain water content (wet-dry-method) were measured to quantify edema formation. The impact of edema formation on infarct volume was evaluated in craniectomized animals. Exogenous RNase 1 was well tolerated and reduced edema-formation and infarct size (26.7% +/- 10.7% vs. 41.0% +/- 10.3%; p<0.01) at an optimal dose of 42 microg/kg as compared to placebo. Craniectomized animals displayed a comparable edema reduction but no reduction in infarct size. The present study introduces a hitherto unrecognized mechanism of ischemic brain damage and a novel neuroprotective approach towards acute stroke treatment.

Mechanisms of ischemic brain damage

In the United States stroke is the third leading cause of death and the leading cause of disability. Brain injury following stroke results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation and apoptosis. There are very few treatments for stroke and the development of new treatments requires a comprehensive understanding of the diverse mechanisms of ischemic brain damage that are responsible for neuronal death. Here, we discuss the underlying pathophysiology of this devastating disease and reveal the intertwined pathways that are the target of therapeutic intervention.

Mechanisms of Ischemic brain damage

Mechanisms of Ischemic brain damage

Combination therapy in ischemic stroke: synergistic neuroprotective effects of memantine and clenbuterol.

Although excitotoxic overactivation of glutamate receptors has been identified as a major mechanism of ischemic brain damage, glutamate receptor antagonists failed in stroke trials, in most cases because of limited therapeutic windows or severe adverse effects. Therefore, we chose memantine and clenbuterol, both approved safe and efficient in their respective therapeutical categories, and examined combinations of these neuroprotectants for possible therapeutic interactions in ischemic stroke. Combinations of the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (20 mg/kg) with the beta2-adrenoceptor agonist clenbuterol (0.3 to 3 mg/kg) were tested in a mouse model of permanent focal cerebral ischemia. In addition, combinations of memantine (1 to 10 nmol/L) and clenbuterol (1 to 10 nmol/L) were examined in cultured hippocampal neurons exposed to glutamate (500 micromol/L) or staurosporine (200 nmol/L). The infarct size was further reduced by combination therapy as compared with effects of the respective neuroprotectants alone. Of note, in combination with memantine, the therapeutic window of clenbuterol was significantly prolonged up to 2 hours after ischemia. Experiments in postnatal cultures of rat hippocampal neurons exposed to glutamate or staurosporine confirmed that neuroprotection by combinations of memantine and clenbuterol exceeded the effects of the individual compounds. Combinations of memantine with clenbuterol extend the respective therapeutic window and provide synergistic cerebroprotective effects after stroke.

Mechanisms of ischemic brain injury.

Stroke is the third leading cause of death and the leading cause of long-term disability in the United States. Approximately 80% of all strokes are ischemic and there are limited therapies approved for the treatment of acute ischemic stroke. Understanding the mechanisms of ischemic brain damage is necessary for the development of innovative treatment strategies. In this review, we discuss the hemodynamic and molecular mechanisms of ischemic brain damage and the potential therapeutic strategies, including reperfusion and primary and secondary neuroprotection, and strategies for recovery of function, such as neural plasticity and stem cell transplantation. The effective treatment of ischemic stroke is likely to result from a combination of therapeutic modalities aimed at different mechanisms of ischemic brain damage and delivered at specific times after acute cerebral ischemia.

Mitochondria and ischemic reperfusion damage in the adult and in the developing brain

The developing and the adult brain respond in similar ways to ischemia, but also display clear differences. For example, the relative contributions of necrosis and apoptosis to neuronal death may be different, such that apoptotic mechanisms would be more prevalent in the developing brain. During normal development, more than half of the neurons in some brain regions are removed through apoptosis, and effectors like caspase-3 are highly upregulated in the immature brain. Mitochondria are pivotal regulators of cell death through their role in energy production and calcium homeostasis, their capacity to release apoptogenic proteins and to produce reactive oxygen species. This review will summarize some of the current studies dealing with mitochondria-related mechanisms of ischemic brain damage, with special reference to developmental aspects.

A submersion method to induce hypoxic damage in organotypic hippocampal cultures

Organotypic brain slices cultured on semi-porous membranes is an increasingly popular in vitro preparation for studying mechanisms of ischemic brain damage. To model in vivo hypoxia, cultured brain slices are exposed to anaerobic atmosphere by placing them into a special incubator. This requirement limits the use of in vitro ischemic models to highly specialized laboratories. Here, we describe a simple method that reproduces hypoxic injury, where cultured hippocampal slices are submerged into glucose-free deoxygenated medium for 1 h. The extent and distribution of hippocampal neuronal loss obtained with this treatment resembled that caused by hypoxia in living tissue in situ, i.e. CA1 pyramidal cell layer was most vulnerable and dentate granular cell layer was least susceptible to hypoxia as measured with fluorescence of the viability marker propidium iodide (PI). Electrophysiologic functional impairment determined by field recordings of CA1 pyramidal neurones temporally coincided with the extent of neuronal death. In addition, known neuroprotective treatments, such as hypothermia and phenytoin application ameliorated neuronal damage in a pattern similar to previously published reports. Therefore, the present in vitro model of ischemia is simple, reliable and of low cost. It is well suited for short and long-term studies of the mechanisms of hypoxic brain damage.

分子生物学からみたtherapeutic window

Cerebral ischemia has a relatively simple cause: occlusion of supplying blood vessels. The best treatment, therefore, is to remove the cause. If cerebral blood flow is restored within a short time, the brain is not damaged. The recent advent of thrombolytic agents such as t-PA enabled us to attain this therapeutic goal. This mode of treatment, however, is not always possible, because there is a time window during which therapy must be administered. It is well established that ischemic brain damage depends to a great extent on the duration and degree of flow reduction. If flow reduction is mild or ischemic period lasts only a few hours, patients are likely to recover. What happens when flow reduction is not mild or the duration of ischemia is longer? Ischemia, then, causes irreversible brain damage. To counter this problem, the mechanisms of ischemic brain damage have been investigated for many years, yet they still remain unknown. Glutamate has been postulated as one of the major causes of neuronal vulnerability to ischemia. Indeed, extensive evidence has been accumulated that supports this hypothesis, but anti-glutamatergic drugs have been largely unsuccessful. Recent experiments in this field have been done on the hypothesis that at least a part of ischemic brain damage progresses through apoptosis. When apoptosis is used to mean a cell death process that requires gene expression, it is quite possible that this is the case. Downstream to the main cascade of apoptosis, mitochondria are now believed to play a pivotal role. Loss of mitochondrial membrane potential and leakage of pro-apoptotic substances from the mitochondria are essential steps in apoptosis. On the other hand, loss of ATP and breakdown of mitochondrial energy metabolism are well-known characteristics of classic ischemic necrosis. Investigation of mitochondrial alteration will be the most important goal in elucidating ischemic brain damage in the next several years.

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices.

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.

Cellular and Molecular Mechanisms of Ischemic Brain Damage. Advances in Neurology, vol. 71. Edited by Bo K. Siesjö and Tadeusz Wieloch. (Pp. xix + 580; illustrated; £93.75 hardback; ISBN 0 7817 0277 1, ISSN 0091 3952.) Philadelphia: Lippincott‐Raven. 1996.

Journal of AnatomyVolume 190, Issue 3 p. 473-475 Free Access Cellular and Molecular Mechanisms of Ischemic Brain Damage. Advances in Neurology, vol. 71. Edited by Bo K. Siesjö and Tadeusz Wieloch. (Pp. xix + 580; illustrated; £93.75 hardback; ISBN 0 7817 0277 1, ISSN 0091 3952.) Philadelphia: Lippincott-Raven. 1996. D. I. GRAHAM, D. I. GRAHAMSearch for more papers by this author D. I. GRAHAM, D. I. GRAHAMSearch for more papers by this author First published: 19 December 2002 https://doi.org/10.1046/j.1469-7580.1997.190304732.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume190, Issue3April 1997Pages 473-475 RelatedInformation

Bright and dark sides of nitric oxide in ischemic brain injury

There is increasing evidence that nitric oxide (NO), a free radical that can act both as a signaling molecule and a neurotoxin, is involved in the mechanisms of cerebral ischemia. Although early investigations yielded conflicting results, the introduction of more-selective pharmacological tools and the use of molecular approaches for deletion of genes encoding for NO synthase have provided a better understanding of the role of NO in the mechanisms of ischemic brain damage. The evidence reviewed in this article suggests that NO is protective or destructive depending on the stage of evolution of the ischemic process and on the cellular source of NO. Defining the role of NO in cerebral ischemia provides the rationale for new neuroprotective strategies based on modulation of NO production in the post-ischemic brain.

Nitric oxide synthase in cerebral ischemia

The results of our continuing studies on the role of nitric oxide (NO) in cellular mechanisms of ischemic brain damage as well as related reports from other laboratories are summarized in this paper. Repetitive ip administration of NG-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor, protected against neuronal necrosis in the gerbil hippocampal CA1 field after transient forebrain ischemia with a bell-shaped response curve, the optimal dose being 3 mg/kg. Repeated ip administration of L-NNA also mitigated rat brain edema or infarction following permanent and transient middle cerebral artery (MCA) occlusion with a U-shaped response. The significantly ameliorative dose-range and optimal dose were 0.01-1 mg/kg and 0.03 mg/kg, respectively. Studies using a NO-sensitive microelectrode revealed that NO concentration in the affected hemisphere was remarkably increased by 15-45 min and subsequently by 1.5-4 h after MCA occlusion. Restoration of blood flow after 2 h-MCA occlusion resulted in enhanced NO production by 1-2 h after reperfusion. Administration of L-NNA (1 mg/kg, ip) diminished the increments in NO production during ischemia and reperfusion, leading to a remarkable reduction in infarct volume. In brain microvessels obtained from the affected hemisphere, Ca(2+)-dependent constitutive NOS (cNOS) was activated significantly at 15 min, and Ca(2+)-independent inducible NOS (iNOS) was activated invariably at 4 h and 24 h after MCA occlusion. Two hour reperfusion following 2 h-MCA occlusion caused more than fivefold increases in cNOS activity with no apparent alterations in iNOS activity. Thus, we report here based on available evidence that there is good reason to think that NOS activation in brain microvessels may play a role in the cellular mechanisms underlying ischemic brain injury.