Mechanisms Of Ischemic Brain Injury Research Articles

Fatty Acid-Binding Proteins: Their Roles in Ischemic Stroke and Potential as Drug Targets.

Stroke is among the leading causes of death and disability worldwide. However, despite long-term research yielding numerous candidate neuroprotective drugs, there remains a lack of effective neuroprotective therapies for ischemic stroke patients. Among the factors contributing to this deficiency could be that single-target therapy is insufficient in addressing the complex and extensive mechanistic basis of ischemic brain injury. In this context, lipids serve as an essential component of multiple biological processes and play important roles in the pathogenesis of numerous common neurological diseases. Moreover, in recent years, fatty acid-binding proteins (FABPs), a family of lipid chaperone proteins, have been discovered to be involved in the onset or development of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. However, comparatively little attention has focused on the roles played by FABPs in ischemic stroke. We have recently demonstrated that neural tissue-associated FABPs are involved in the pathological mechanism of ischemic brain injury in mice. Here, we review the literature published in the past decade that has reported on the associations between FABPs and ischemia and summarize the relevant regulatory mechanisms of FABPs implicated in ischemic injury. We also propose candidate FABPs that could serve as potential therapeutic targets for ischemic stroke.

Comparison of Effects of Metformin, Phenformin, and Inhibitors of Mitochondrial Complex I on Mitochondrial Permeability Transition and Ischemic Brain Injury.

Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides—metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5–7-day (premature) and 2–3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced—in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.

Stroke Research in China over the Past Decade: Analysis of NSFC Funding.

We analyzed the projects and published studies funded by NSFC (National Natural Science Foundation of China) in the field of stoke. Further more, we searched the published papers supported by NSFC in the last decade using the keywords of "stroke" or "cerebrovascular disease" and analyzed the results according to the topics associated with the mechanisms of ischemic brain injury, novel techniques and new drugs, medical imaging, translational medicine, and traditional Chinese medicine.

Dynamics of pial vessels reactivity after brief cerebral ischemia

Cerebral blood vessel reactivity is one of the main determinants of final outcome of brain ischemia. Most of studies on the vascular mechanisms of ischemic brain injury, however, focus on the acute changes within ischemic period or several hours after it. Dilatatory capacity of cerebral arterioles (perfusion reservoir) is considered as an important factor of brain perfusion elevation in critical situations.The aim of the present study was to examine the pial vessel reactivity in response to hypercapnia in rats, subjected to transient global cerebral ischemia, at 7, 14 and 21 days after ischemia. Materials and methods. Transient global cerebral ischemia was induced in anesthetized Wistar rats by bilateral common carotid artery occlusion for 12 min with simultaneous controlled hypotension to 45±3 mm Hg, followed by blood reinfusion and recovery from anesthesia. Three different groups of rats were re-anesthetized at 7, 14 or 21 days after ischemia and subjected to microvascular reactivity studies using in vivo video microscopy. Hypercapnia was caused by i.v. injection of acetazolamide. The changes in diameter of pial arteries and veins in response to hypercapnia were measured. Results and discussion. Global cerebral ischemia led to marked decrease in pial vessels (both arteries and veins) reactivity in response to hypercapnia, caused by i.v. injection of acetazolamide. In intact rats, i.v. injection of acetazolamide led to pial arteries dilation and pial veins constriction; in animals subjected to ischemia-reperfuion. the numbers of dilated large arteries and constricted small veins were much less, as well as the extent of arterial dilation. Reactivity changes were observed in all time points studied. Conclusions. Thus, transient global cerebral ischemia cause marked and long lasting (3 weeks) decrease in pial vessel reactivity in response to hypercapnia.

Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury.

Cyclooxygenase-2 (COX-2) is an important contributor to ischemic brain injury. Identification of the downstream mediators of COX-2 toxicity may allow the development of targeted therapies. Of particular interest is the cyclopentenone family of prostaglandin metabolites. Cyclopentenone prostaglandins (CyPGs) are highly reactive molecules that form covalent bonds with cellular thiols. Protein disulfide isomerase (PDI) is an important molecule for the restoration of denatured proteins following ischemia. Because PDI has several thiols, including thiols within the active thioredoxin-like domain, we hypothesized that PDI is a target of CyPGs and that CyPG binding of PDI is detrimental. CyPG-PDI binding was detected invitro via immunoprecipitation and MS. CyPG-PDI binding decreased PDI enzymatic activity in recombinant PDI treated with CyPG, and PDI immunoprecipitated from neuronal culture treated with CyPG or anoxia. Toxic effects of binding were demonstrated in experiments showing that: (a) pharmacologic inhibition of PDI increased cell death in anoxic neurons, (b) PDI overexpression protected neurons exposed to anoxia and SH-SY5Y cells exposed to CyPG, and (c) PDI overexpression in SH-SY5Y cells attenuated ubiquitination of proteins and decreased activation of pro-apoptotic caspases. In conclusion, CyPG production and subsequent binding of PDI is a novel and potentially important mechanism of ischemic brain injury. We show that CyPGs bind to PDI, cyclopentenones inhibit PDI activity, and CyPG-PDI binding is associated with increased neuronal susceptibility to anoxia. Additional studies are necessary to determine the relative role of CyPG-dependent inhibition of PDI activity in ischemia and other neurodegenerative disorders.

Consistent delayed unilateral neuronal death after modified transient focal cerebral ischemia in mice that mimics neuronal injury after transient global cerebral ischemia.

Numerous studies have attempted to reveal the pathophysiology of ischemic neuronal injury using a representative transient global cerebral ischemia (tGCI) model in rodents; however, most of them have used gerbil or rat models. Recent advances in transgene and gene-knockout technology have enabled the precise molecular mechanisms of ischemic brain injury to be investigated. Because the predominant species for the study of genetic mutations is the mouse, a representative mouse model of tGCI is of particular importance. However, simple mouse models of tGCI are less reproducible; therefore, a more complex process or longer duration of ischemia, which causes a high mortality rate, has been used in previous tGCI models in mice. In this study, the authors aimed to overcome these problems and attempted to produce consistent unilateral delayed hippocampal CA1 neuronal death in mice. C57BL/6 mice were subjected to short-term unilateral cerebral ischemia using a 4-mm silicone-coated intraluminal suture to obstruct the origin of the posterior cerebral artery (PCA), and regional cerebral blood flow (rCBF) of the PCA territory was measured using laser speckle flowmetry. The mice were randomly assigned to groups of different ischemic durations and histologically evaluated at different time points after ischemia. The survival rate and neurological score of the group that experienced 15 minutes of ischemia were also evaluated. Consistent neuronal death was observed in the medial CA1 subregion 4 days after 15 minutes of ischemia in the group of mice with a reduction in rCBF of < 65% in the PCA territory during ischemia. Morphologically degenerated cells were mostly positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and cleaved caspase 3 staining 4 days after ischemia. The survival rates of the mice 24 hours (n = 24), 4 days (n = 15), and 7 days (n = 7) after being subjected to 15 minutes of ischemia were 95.8%, 100%, and 100%, respectively, and the mice had slight motor deficits. The authors established a model of delayed unilateral hippocampal neuronal death in C57BL/6 mice by inducing ischemia in the PCA territory using an intraluminal suture method and established inclusion criteria for PCAterritory rCBF monitored by laser speckle flowmetry. This model may be useful for investigating the precise molecular mechanisms of ischemic brain injury.

Collateral circulation prevents masticatory muscle impairment in rat middle cerebral artery occlusion model.

The rat suture middle cerebral artery occlusion (MCAO) is a frequently used animal model for investigating the mechanisms of ischemic brain injury. During suture MCAO, transection of the external carotid artery (ECA) potentially restrains blood flow and impairs masticatory muscle and other ECA-supported territories, consequently influencing post-operation animal survival. This study was aimed at investigating the effect of ECA transection on the hemodynamic alterations using a novel synchrotron radiation (SR) angiography technique and magnetic resonance imaging in live animals. Fifteen male adult Sprague-Dawley rats were used in this study. Animals underwent MCAO, in which the ECA was transected. SR angiography was performed before and after MCAO. Rats then underwent magnetic resonance imaging (MRI) to detect the tissue lesion both intra- and extra-cranially. Animals with SR angiography without other manipulations were used as control. High-resolution cerebrovascular morphology was analyzed using a novel technique of SR angiography. The masticatory muscle lesion was further examined by hematoxylin and eosin staining. MRI and histological results showed that there was no masticatory muscle lesion at 1, 7 and 28 days following MCAO with ECA transection. In normal condition, the ECA and its branch external maxillary artery were clearly detected. Following ECA transection, the external maxillary artery was still observed and the blood supply appeared from the anastomotic branch from the pterygopalatine artery. SR angiography further revealed the inter-relationship of hemisphere extra- and intra-cranial vasculature in the rat following MCAO. Transection of the ECA did not impair masticatory muscles in rat suture MCAO. Interrupted blood flow could be compensated by the collateral circulation from the pterygopalatine artery.

Mitochondrial dynamics: cell-type and hippocampal region specific changes following global cerebral ischemia.

Mitochondria are organelles that undergo continuous cycles of fission and fusion. This dynamic nature of mitochondria is important for cell physiology. Transgenic mouse models that express mitochondria targeted fluorescence protein, in either neurons or astrocytes, were used to examine the role of alterations in mitochondrial morphology in mechanisms of ischemic brain injury. The animals were subjected to global cerebral ischemia and allowed to recover before their brains were perfusion fixed and processed for histology and confocal microscopy. After capturing z-stack images from different hippocampal sub-regions, mitochondrial organelles were 3D reconstructed using volocity software and then their morphological parameters were calculated. The data shows cell-type specific alterations in mitochondrial dynamics following ischemia. Fission is activated in all hippocampal areas at 2h recovery with mitochondria in CA1 becoming progressively more fragmented during the 24h recovery period. Mitochondria in CA3 and dentate gyrus neurons started to re-fuse after 24h of recirculation; this was even more pronounced 3days after ischemia. Astrocytic mitochondria underwent transient fission 2h after ischemic insult and regained their normal shape at 24h recovery. Surprisingly, no positive correlation was found between increased nitrotyrosine levels and mitochondrial fission, particularly in ischemia resistant CA3 and dentate gyrus neurons. Our data suggest that ischemia resistant neurons are able to shift their mitochondrial dynamics toward fusion after extensive fragmentation. The re-fusion ability of fragmented mitochondria is most likely a vital feature for cell survival.

English

Introduction Although cardiothoracic surgeons are aware of the deleterious effects of prolonged exposure to cardiopulmonary bypass on brain function and structure, how to best protect the brain during the sensitive time of interruption of normal cerebral blood flow remains controversial. Short communication The complex functional anatomy of the brain on the one hand, and the multifactorial mechanisms of neural injury on the other hand, has resulted in a significant gap in translating the current basic science understanding of the molecular mechanisms of ischemic brain injury to effective clinical strategies and outcome assessment. Here, the goal is to address the current understanding of the mechanisms underlying brain injury after cardiopulmonary bypass and relevant strategies of neuroprotection. Conclusion By identifying the key players in neuronal injury following hypothermic circulatory arrest, their potential as therapeutic targets can be evaluated in novel neuroprotective strategies.

Biological Sex and Mechanisms of Ischemic Brain Injury

Cerebrovascular disease is a leading cause of death-from-disease and of disability worldwide, affecting some 15 million people. The incidence of stroke or "brain attack" is unlikely to recede for a decade at minimum by most predictions, despite large public health initiatives in stroke prevention. It has been well established that stroke is also one of the most strikingly sex-specific diseases in its epidemiology, and in some cases, in patient outcomes. For example, women sustain lower rates of ischemic stroke relative to men, even beyond their menopausal years. In contrast, outcomes are worse in women in many clinical studies. The biological basis for this sexual dimorphism is a compelling story, and both hormone-dependent and hormone-independent factors are involved, the latter of which is the subject of this brief review. Understanding the molecular and cell-based mechanisms underlying sex differences in ischemic brain injury is an important step toward personalized medicine and effective therapeutic interventions in patients of both sexes.

Can Genes Modify Stroke Outcome and By What Mechanisms?

The approach to neuroprotection in stroke has relied mainly on exogenously administered drugs derived from research probing the cellular mechanisms of ischemic brain injury. This approach has had limited translational impact. Over 1000 putative neuroprotective agents have been developed, and over 100 agents have advanced to clinical trials. None, however, have shown clinical efficacy.1 By contrast, a gene-based approach seeks to harness endogenous neuroprotective programs for stroke therapy. Experimentally, ischemic brain injury can be attenuated, and stroke outcome improved, by gene expression modification in the brain. The brain and other organs have highly conserved, endogenous neuroprotective programs, the induction of which reduces ischemic injury. The neuroprotective programs can be induced in the model of ischemic tolerance: brief exposure to sublethal ischemia produces tolerance to a subsequent, severe ischemic challenge. A number of laboratories have taken this approach with differing mechanistic foci, which have been reviewed elsewhere.2–5 The protection induced by tolerance is substantial, gene-based, and dependent on new protein synthesis. The neuroprotection of tolerance has been demonstrated in experimental cardiopulmonary bypass surgery6 and in stroke in the primate.7 A clinical counterpart likely exists in human brain: patients with prodromal transient ischemic attacks have milder strokes.8–11 Two small clinical trials have shown potential benefits of preconditioning, and 5 additional trials are underway.12 Thus, there is considerable therapeutic potential in understanding gene expression changes in tolerance and in dissecting the biological mechanisms that regulate them. This is an evolving story deriving mainly from the authors' and our collaborators' laboratories. ### Individual Genes Over the last several decades, research has identified numerous genes whose regulation may affect the outcome of stroke. Because cell death following harmful ischemia is mediated by programmed cell death-like mechanisms, many studies of neuroprotection have focused on apoptotic mediators as potential effectors …

Simple model of forebrain ischemia in mouse

The availability of genetically engineered mice allows the unraveling of the role of specific proteins in mechanisms of ischemic brain injury. Due to the high variability of their vascular anatomy, mouse models of global cerebral ischemia are rather complex. In the present study, we describe a simple model of mouse forebrain ischemia where the bilateral common carotid artery occlusion (BCCO) is combined with isoflurane-induced hypotension. The forebrain ischemia was induced by BCCO that was preceded by increase of the isoflurane level from 1.5% to 5% in the respiratory gases. This caused a decrease of the mean arterial blood pressure (MABP) to about 30mmHg and the cerebral blood flow dropped to 5% of the control after the BCCO. During the 10min ischemic period both MABP and CBF remained stable and the reperfusion was induced by reducing the isoflurane level to 0% followed by removal of the carotid clamps. Mice were allowed 1, 2, 3 or 5 days survival followed by histologic analysis. The number of CA1 uninjured neurons was assessed utilizing a stereological approach. Neurodegeneration was observed at 2 days after the onset of reperfusion. At 3 days of recovery, about 40% of neurons survived and the cell death did not further increase at 5 days. Degenerative neurons were also detected in the striatum and sporadically in the cortex. This study demonstrates the feasibility of using the described model in mice that can be utilized to examine the effect of new neuroprotective compounds or use transgenic animals to test new hypothesis.

K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15 min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20 min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.

Experimental Model Considerations for the Study of Protein-Energy Malnutrition Co-Existing with Ischemic Brain Injury

Protein-energy malnutrition (PEM) affects ~16% of patients at admission for stroke. We previously modeled this in a gerbil global cerebral ischemia model and found that PEM impairs functional outcome and influences mechanisms of ischemic brain injury and recovery. Since this model is no longer reliable, we investigated the utility of the rat 2-vessel occlusion (2-VO) with hypotension model of global ischemia for further study of this clinical problem. Male, Sprague-Dawley rats were exposed to either control diet (18% protein) or PEM induced by feeding a low protein diet (2% protein) for 7d prior to either global ischemia or sham surgery. PEM did not significantly alter the hippocampal CA1 neuron death (p = 0.195 by 2-factor ANOVA) or the increase in dendritic injury caused by exposure to global ischemia. Unexpectedly, however, a strong trend was evident for PEM to decrease the consistency of hippocampal damage, as shown by an increased incidence of unilateral or no hippocampal damage (p=0.069 by chi-square analysis). Although PEM caused significant changes to baseline arterial blood pH, pO(2), pCO(2), and fasting glucose (p<0.05), none of these variables (nor hematocrit) correlated significantly with CA1 cell counts in the malnourished group exposed to 2-VO (p>0.269). Intra-ischemic tympanic temperature and blood pressure were strictly and equally controlled between ischemic groups. We conclude that co-existing PEM confounded the consistency of hippocampal injury in the 2-VO model. Although the mechanisms responsible were not identified, this model of brain ischemia should not be used for studying this co-morbidity factor.

Soluble Epoxide Hydrolase Inhibition: Targeting Multiple Mechanisms of Ischemic Brain Injury with a Single Agent

Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolic conversion and degradation of P450 eicosanoids called epoxyeicosatrienoic acids (EETs). Genetic variations in the sEH gene, designated EPHX2, are associated with ischemic stroke risk. In experimental studies, sEH inhibition and gene deletion reduce infarct size after focal cerebral ischemia in mice. Although the precise mechanism of protection afforded by sEH inhibition remains under investigation, EETs exhibit a wide array of potentially beneficial actions in stroke, including vasodilation, neuroprotection, promotion of angiogenesis and suppression of platelet aggregation, oxidative stress and post-ischemic inflammation. Herein we argue that by capitalizing on this broad protective profile, sEH inhibition represents a prototype "combination therapy" targeting multiple mechanisms of stroke injury with a single agent.

Current Status of Neuroprotection for Cerebral Ischemia

Abundant preclinical studies have identified multiple mechanisms of ischemic brain injury and have provided proof of principle that strategies designed to counter these mechanisms can protect the ischemic brain. This review article emphasizes the translation of these strategies from the laboratory to clinical trials. It is a disappointing fact that many agents have been brought to clinical trial despite only modest or inconsistent preclinical evidence of neuroprotective efficacy. Preclinical investigations require rigorous attention to a variety of variables that may influence outcome. The widely touted STAIR criteria represent constructive guidelines for preclinical testing but, as experience has shown, do not increase the likelihood of translational success. Of the approximately 160 clinical trials of neuroprotection for ischemic stroke conducted as of late 2007, only approximately 40 represent larger-phase completed trials, and fully one half of the latter utilized a window to treatment of >6 hours, despite strong preclinical evidence that this delay exceeds the likely therapeutic window of efficacy in acute stroke. Other shortcomings of these trials include the use of agents lacking robust, consistent preclinical efficacy; inability to achieve adequate dosing in humans; and suboptimal clinical and statistical design features. Taken together, these factors identify areas of needed improvement for future trials.

Introduction to the neurovascular unit

For many years, a neurocentric view toward mechanisms of ischemic brain injury and repair has significantly contributed to our knowledge of the molecular pathways of excitotoxicity, oxidative stress and apoptosis. However, translation of these laboratory results into clinically effective stroke treatments remains a major challenge. To address this issue and the prediction that strokes will increase in our aging population, the U.S. Congress requested the National Institute of Neurological Disorders and Stroke (NINDS) to develop a stroke research action plan through the Stroke Progress Review Group (SPRG). The SPRG identified research on the cellular and functional interactions among the capillaries, glia, and neurons of the brain, termed the unit (NVU), as a top priority. The NVU strategy looks beyond the single cell for a more integrative answer to ischemic brain damage which may be closer to modeling the clinical reality. The NVU construct is intended to facilitate a better understanding of the integration of cerebrovascular and neurobiological mechanisms in the development of the healthy brain, in the maintenance of function in the aging brain, and in neurological disorders and stroke. A major goal is to improve our knowledge of cell-cell communication within the NVU and how vascular function may contribute to the initiation and/or progression of neurological diseases over the lifespan. The NVU concept focuses attention on the interactions among cells of the brain and blood vessels and includes the endothelium, extracellular matrix, glia, pericytes and neurons. After ischemia, perturbations in neurovascular functional integrity initiate several cascades of injury. Neuronal cell death ultimately underlies ischemic brain injury and the NVU concept suggests that proximal triggers in endothelium play an important upstream role. For example, signals such as oxidative stress, together with neutrophil and/or platelet interactions with activated endothelium, upregulate matrix metalloproteinases, plasminogen activators and other proteases, which degrade matrix and lead to blood–brain barrier leakage. Disruption of cell-matrix homeostasis might also trigger cell death pathways in both vascular and parenchymal compartments. Refocusing research on how endothelial injury affects brain tissue damage applies not only to stroke, but may also contribute to understanding neurodegenerative disorders, like vascular dementia, Alzheimer's disease, MS and ALS, in which vascular changes are observed. Thrombolysis trials firmly establish the idea that timely reperfusion can salvage the ischemic brain. The efficacy of hypothermia so far confirms that multiple molecular cascades are indeed operational in human brain and that neuroprotection is an achievable goal. Ultimately, new approaches for targeting the NVU, based on biological considerations, could be developed to improve potential combination or multi-targeted treatments for stroke and other neurological disorders.

EP1 receptors are responsible for COX-2 mediated neurotoxicity

There is increasing evidence that cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, is involved in the mechanism of ischemic brain injury. However, the reaction products responsible COX-2-mediated neurotoxicity have not been defined. Prostaglandin E2 (PGE2) is the major COX-2 derived prostanoid produced following brain injury and is involved in the mechanisms of glutamate excitotoxicity (Ann. Neurol. 55, 668). PGE2 acts on four receptor subtypes (EP1-4). While activation of EP2 receptors is neuroprotective (J. Neurosci. 24, 257), EP1 receptors mediate injury in other organs (Hypertension 42, 1183). Therefore, in this study we tested the hypothesis that EP1 receptors mediate the neurotoxicity exerted by COX-2.

A Word on Hypothermic Preconditioning

We read the letter by Drs. T. A. Miyamoto and K. J. Miyamoto with interest, and we thank them for their interesting discussion of our study. They raised two issues for discussion. First, they wonder whether hypoxia/anoxia is a component of preconditioning stimuli to induce neuroprotective effects that were observed in our study.1Hypoxia/anoxia has been reported to produce preconditioning effects on the brain.2However, in our study, the brain slices were kept in a buffer that was gassed with 5% CO2–95% O2during the application of hypothermia. Control slices that were also kept in the oxygenated buffer but without having hypothermic preconditioning were used for comparison. In addition, it is generally accepted that hypothermia reduces cerebral metabolic rate and oxygen requirement. Therefore, we do not expect that brain slices during the application of hypothermia require more oxygen and have worse hypoxia (if it happens) than control slices. Therefore, we believe that the neuroprotective effects that we studied in our article are mainly attributed to hypothermic preconditioning. Of course, we cannot exclude the possibility that hypoxia plays a role in neuronal survival or death in our study. However, we hope that the proper controls performed in our study have isolated the hypothermic preconditioning–induced neuroprotection for us to study.Drs. T. A. Miyamoto and K. J. Miyamoto then suggest that intravenous application of taurine may be an alternative method for hypothermic preconditioning to provide neuronal protection against ischemia in clinical practice. This suggestion is interesting and may be practical. However, caution must be executed here because neuroprotection induced by hypothermic preconditioning and taurine are shown only in animal studies,1,3and it is not appropriate to extrapolate these data to humans. Although mild hypothermia that we used in our study is usually safe and easily achievable in clinical practice, more detailed animal studies are needed to show the effectiveness and safety of taurine application for brain ischemia before a clinical trial.4Nevertheless, we should keep our eyes open to any potential neuroprotective agents or methods such as hypothermic preconditioning or taurine application and vigorously test them for potential clinical use.Very few agents or methods have been shown to be effective and practical against brain ischemia in clinical studies in the field of neuroprotection. However, with studies aimed at the mechanisms of ischemic brain injury and neuroprotective methods, it is hoped that more molecular targets and, therefore, more agents with better selectivity to induce neuroprotection will be identified and tested for clinical use.* University of Virginia Health System, Charlottesville, Virginia. zz3c@virginia.edu

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.