Global Cerebral Ischemic Injury Research Articles

Impaired Cerebral Mitochondrial Oxidative Phosphorylation Function in a Rat Model of Ventricular Fibrillation and Cardiopulmonary Resuscitation

Postcardiac arrest brain injury significantly contributes to mortality and morbidity in patients suffering from cardiac arrest (CA). Evidence that shows that mitochondrial dysfunction appears to be a key factor in tissue damage after ischemia/reperfusion is accumulating. However, limited data are available regarding the cerebral mitochondrial dysfunction during CA and cardiopulmonary resuscitation (CPR) and its relationship to the alterations of high-energy phosphate. Here, we sought to identify alterations of mitochondrial morphology and oxidative phosphorylation function as well as high-energy phosphates during CA and CPR in a rat model of ventricular fibrillation (VF). We found that impairment of mitochondrial respiration and partial depletion of adenosine triphosphate (ATP) and phosphocreatine (PCr) developed in the cerebral cortex and hippocampus following a prolonged cardiac arrest. Optimal CPR might ameliorate the deranged phosphorus metabolism and preserve mitochondrial function. No obvious ultrastructural abnormalities of mitochondria have been found during CA. We conclude that CA causes cerebral mitochondrial dysfunction along with decay of high-energy phosphates, which would be mitigated with CPR. This study may broaden our understanding of the pathogenic processes underlying global cerebral ischemic injury and provide a potential therapeutic strategy that aimed at preserving cerebral mitochondrial function during CA.

Delta opioid receptor agonist BW373U86 attenuates post-resuscitation brain injury in a rat model of asphyxial cardiac arrest

ObjectiveThe aim of this study was to investigate whether the DOR agonist BW373U86 conferred neuroprotection following ACA when given after resuscitation and to determine the long-term effects of chronic BW373U86 treatment on ACA-elicited brain injury. MethodsAnimals were divided into acute and chronic treatment groups. Each group consisted of four sub-groups, including Sham, ACA, BW373U86 (BW373U86+ACA), and Naltrindole groups (Naltrindole and BW373U86+ACA). The DOR antagonist Naltrindole was used to confirm the possible receptor-dependent effects of BW373U86. ACA was induced by 8min of asphyxiation followed by resuscitation. All drugs were administered either immediately after the restoration of spontaneous circulation (ROSC) in acute-treatment groups or over 6 consecutive days in chronic-treatment groups. Alterations of cAMP response element-binding protein (CREB) and phosphorylated CREB (pCREB) were analyzed by western blot and immunohistochemistry. Neurological functions were assessed by neurological deficit score (NDS) and Morris Water Maze performance. Neurodegeneration was monitored by immunofluorescence and Nissl staining. ResultsACA induced massive neuron loss and serious neurological function deficits. BW373U86 significantly reduced both of these negative effects and increased CREB and pCREB expression in the hippocampus; these effects were reversed with acute Naltrindole treatment. The protective effects of BW373U86 persisted until 28d post-ROSC with chronic treatment, but these effects were not reversed by Naltrindole. ConclusionsBW373U86 attenuates global cerebral ischemic injury induced by ACA through both DOR-dependent and DOR-independent mechanisms. CREB might be an important molecule in mediating these neuroprotective effects.

S100B protein in serum is elevated after global cerebral ischemic injury.

BACKGROUND:S100B protein in patients with cardiac arrest, hemorrhagic shock and other causes of global cerebral ischemic injury will be dramatically increased. Ischemic brain injury may elevate the level of serum S100B protein and the severity of brain damage.METHODS:This article is a critical and descriptive review on S100B protein in serum after ischemic brain injury. We searched Pubmed database with key words or terms such as “S100B protein”, “cardiac arrest”, “hemorrhagic shock” and “ischemia reperfusion injury” appeared in the last five years.RESULTS:S100B protein in patients with cardiac arrest, hemorrhagic shock and other causes of ischemic brain injury will be dramatically increased. Ischemic brain injury elevated the level of serum S100B protein, and the severity of brain damage.CONCLUSION:The level of S100B protein in serum is elevated after ischemic brain injury, but its mechanism is unclear.

Protective Effect of Crocin against Cerebral Ischemia in a Dose-dependent Manner in a Rat Model of Ischemic Stroke

Crocin is a water-soluble carotenoid isolated from the Crocus sativus L (saffron) stigma. It has previously been reported that it has protective effects against renal, cardiac, and global cerebral ischemic injury. However its therapeutic effects remain to be clarified regarding ischemic reperfusion injuries, brain edema, and activity of antioxidant enzymes in a transient model of focal cerebral ischemia. Transient focal cerebral ischemia was induced by 60-minute middle cerebral artery occlusion (MCAO), followed by 23-hour reperfusion. Crocin at doses of 15, 30, 60, and 120 mg/kg intraperitoneally were injected at the start of ischemia. Infarct volume and neurologic outcome were evaluated 24 hours after MCAO. For the therapeutic time window measurement, crocin (60 mg/kg) was given 1, 3, and 6hours after ischemia; 24 hours later brain edema and antioxidant enzyme activity were assessed. The results indicated that treatment with crocin at doses of 30, 60, and 120 mg/kg significantly decreased infarct volume by 64%, 74%, and 73%, respectively. Administration of crocin (60 mg/kg) 1 hour before, at the start, or 1hour after ischemia reduced brain edema by 48%, 52%, and 51%, respectively. Moreover, crocin (60 mg/kg) significantly reduced malondialdehyde (MDA) content and increased activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the ischemic cortex (P< .001). Our findings indicate that crocin has protective effects against ischemic reperfusion injury and cerebral edema in a rat model of stroke. These effects of crocin may have been exerted primarily by suppression of the production of free radicals and increased antioxidant enzyme activity.

Hypoxic preconditioning attenuates global cerebral ischemic injury following asphyxial cardiac arrest through regulation of delta opioid receptor system

This study was designed to investigate whether delta opioid receptor (DOR) is involved in the neuroprotective effect induced by hypoxic preconditioning (HPC) in the asphyxial cardiac arrest (CA) rat model. Twenty-four hours after the end of 7-day HPC, the rats were subjected to 8-min asphyxiation and resuscitated with a standardized method. In the asphyxial CA rat model, HPC improved the neurological deficit score (NDS), inhibited neuronal apoptosis, and increased the number of viable hippocampal CA1 neurons at 24 h, 72 h, or 7 days after restoration of spontaneous circulation (ROSC); however, the above-mentioned neuroprotection of HPC was attenuated by naltrindole (a selective DOR antagonist). The expression of hypoxia-inducible factor-1α (HIF-1α) and DOR, and the content of leucine enkephalin (L-ENK) in the brain were also investigated after the end of 7-day HPC. HPC upregulated the neuronal expression of HIF-1α and DOR, and synchronously elevated the content of L-ENK in the rat brain. HIF-1α siRNA was used to further elucidate the relationship between HIF-1α and DOR in the HPC-treated brain. Knockdown of HIF-1α by siRNA markedly abrogated the HPC induced upregulation of HIF-1α and DOR. The present study demonstrates that the expression of DOR in the rat brain is upregulated by HIF-1α following exposure to 7-day HPC, at the same time, HPC also increases the production of endogenous DOR ligand L-ENK in the brain. DOR activation after HPC results in prolonged neuroprotection against subsequent global cerebral ischemic injury, suggesting a new mechanism of HPC-induced neuroprotection on global cerebral ischemia following CA and resuscitation.

Delayed ischemic postconditioning protects hippocampal CA1 neurons by preserving mitochondrial integrity via Akt/GSK3β signaling

Delayed ischemic postconditioning (Post C), which involves a brief ischemia followed by reperfusion 2 days after 8–10 min global cerebral ischemia (GCI), has been shown to exert a remarkable protection of the vulnerable hippocampal CA1 region of the brain and attenuation of behavioral deficits, although the mechanisms remain poorly understood. The purpose of the current study was to explore the effect of Post C upon mitochondrial integrity, cytochrome c release and Bax translocation as a potential key mechanism for Post C protection of the critical hippocampal CA1 region neurons. The results of the study revealed that ischemic Post C (3 min) administered 2 days after 8-min GCI exerted a robust preservation from GCI injury, as evidenced by the increase of NeuN-positive and the decrease of TUNEL-positive cells, as well as morphological features of mitochondrial integrity in the hippocampal CA1 region. We also found that Post C significantly blocked inner mitochondrial membrane potential depolarization, as shown by JC-1 staining, and attenuates cytochrome c release and Bax translocation induced by GCI. Pre-treatment of the PI3K inhibitor LY294002, 20 min prior to Post C, significantly attenuated Post C-induced elevation of p-Akt and p-GSK3β, as well as prevented Post C enhancement of mitochondrial integrity and Post C neuroprotection. The results suggest that phosphorylation of Akt and subsequent inactivation of GSK3β signaling is critical in mediating Post C beneficial effects upon mitochondrial integrity, function and neuroprotection following GCI injury.

The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats

Aims The therapeutic effect of pinocembrin, together with the therapeutic time window, in the transient global cerebral ischemia/reperfusion (I/R) rats was investigated. Main methods Adult male Sprague–Dawley rats were subjected to global cerebral ischemia for 20 min by four-vessel occlusion. Pinocembrin (1 and 5 mg/kg) was administrated intravenously 30 min before ischemia and 30 min, 2 h, 6 h after reperfusion, respectively. Neurological scores, brain edema and histological examination by Nissl staining were employed to assess the neuronal injury after ischemia and the neuroprotection by pinocembrin. The activities of superoxide dismutase (SOD), myeloperoxidase (MPO) and the content of malondialdehyde (MDA) in brain tissue were tested by colorimetric assays. Alterations of neurotransmitters were determined by a high performance liquid chromatography–electrochemical method. Key findings Pinocembrin significantly ameliorated neurological deficits and brain edema, and alleviated the degree of hippocampal neuronal loss at 24 h after global cerebral I/R with a broad therapeutic time window. It was found that treatment with pinocembrin reduced the compensatory increase of SOD activity and decreased the MDA level and MPO activity in a dose-dependent manner. The metabolic balance between excitatory and inhibitory amino acids was modulated by pinocembrin treatment. Significance These findings suggest that pinocembrin provides neuroprotection against global cerebral ischemic injury with a wide therapeutic time window, which may be attributed to its antioxidative, antiinflammatory and antiexcitotoxic effects.

Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats

Objective:This study was undertaken to evaluate the neuroprotective activity of Wedelia calendulacea against cerebral ischemia/reperfusion induced oxidative stress in the rats.Materials and Methods:The global cerebral ischemia was induced in male albino Wistar rats by occluding the bilateral carotid arteries for 30 min followed by 1 h and 4 h reperfusion. At various times of reperfusion, the histopathological changes and the levels of malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione–s–transferase (GST), and hydrogen peroxide (H2O2) activity and brain water content were measured.Results:The ischemic changes were preceded by increase in concentration of MDA, hydrogen peroxide and followed by decreased GPx, GR, and GST activity. Treatment with W. calendulacea significantly attenuated ischemia–induced oxidative stress. W. calendulacea administration markedly reversed and restored to near normal level in the groups pre-treated with methanolic extract (250 and 500 mg/kg, given orally in single and double dose/day for 10 days) in dose-dependent way. Similarly, W. calendulacea reversed the brain water content in the ischemia reperfusion animals. The neurodegenaration also conformed by the histopathological changes in the cerebral-ischemic animals.Conclusion:The findings from the present investigation reveal that W. calendulacea protects neurons from global cerebral–ischemic injury in rat by attenuating oxidative stress.

Behavioral improvement after transplantation of neural precursors derived from embryonic stem cells into the globally ischemic brain of adolescent rats

We intended to determine whether transplanted neural precursors, derived from mouse embryonic stem (ES) cells, can migrate and differentiate into mature neurons and glial cells in damaged brains and improve functional deficits caused by global cerebral ischemic injury in adolescent rats. Global ischemia was induced using the four-vessel occlusion method. ES cells that display enhanced expression of yellow fluorescent protein were co-cultured in N2 supplemented media with PA6 cells that had stromal derived inducing activity. Neural precursor cells were directly transplanted bilaterally into hippocampal C3 areas 2 weeks after induction of global ischemia. Assessments of the Morris water-maze test at eight weeks and, the Open field activity levels at two, four, six and eight weeks after transplantation were carried out according to standard methods. From neural precursors, we were able to generate neural lineages, including neurons and glial cells in vitro. Eight weeks following transplantation, cellular migration as well as generation of neural cells including neurons, astrocytes, and oligodendrocytes developed from the grafted ES cell-derived neural precursors were observed. Cell-transplanted animals exhibited enhanced functional recovery on sensorimotor and behavioral tests, compared to vehicle-treated control animals. Therefore, transplantation of mouse ES cell-derived neural precursor cells shows promise for improving recovery after global ischemia in adolescent rats.

Towards a pharmacological neuro-protectant: can anesthesia deliver?

Transient global cerebral ischemic injury (tGCI) continues to be a significant cause of death and disability. Global cerebral ischemia usually results from complex medical conditions, including cardiac arrest, circulatory shock, and birth asphyxia or iatrogenic interventions, such as circulatory arrest for cardiovascular surgery. The pathophysiology of global cerebral ischemia is complex. Experimental studies have identified a number of potential therapeutic treatments. Some of these treatments have targeted the initial metabolic events following brain ischemia, including energy failure, calcium ion flux, free radical generation, and the attenuation of excitotoxicity. These harmful mechanisms have been targeted with pharmacological interventions in experimental models. However, none of these promising pharmacological therapies have been successfully translated into clinical use. Such ‘‘easy to administer’’ therapies could be stocked in crash carts for administration to patients once the heart has been restarted. By contrast, physiological therapies, such as therapeutic hypothermia, have gained success in both experimental and clinical settings. This type of therapy is difficult to administer and involves extensive infrastructure, resources, and personnel. However, even therapeutic hypothermia may not be applicable across all human populations. Pre-conditioning is a strategy that involves increasing the brain’s tolerance to ischemia by exposing the brain to minor ischemic insults or pharmacological agents prior to the occurrence of a major ischemic event. While anesthetic preconditioning has shown initial promise in protecting the brain from ischemic events in pre-clinical studies, to date, there is no convincing clinical epidemiology supporting this practice. The pre-clinical science regarding the potential of anesthetic preconditioning is far from clear. This is illustrated in a recent article by Codaccioni et al., who report a transient but non-sustained improvement in neurocognitive testing in a rodent model of focal brain ischemia. In reality, this may simply be reflective of and/or specific to the particular model employed in their experiments. Any potential neuroprotective effects of anesthetic preconditioning depend on the anesthetic agent used to precondition, the species studied, the model of ischemia, the age and sex of the animal, the duration of preconditioning, and the interval between preconditioning and ischemic insult. In other words, it is not clear whether this will translate into a universally applicable therapy for tGCI. Anesthetic preconditioning is one of several strategies that prepare the brain to anticipate or react to the ischemic insult. Examples of other strategies used to precondition the brain include toxins, temperature, pharmaceuticals, and ischemia itself, with the latter showing most promise. The common theme of these strategies is to unbalance several key cell signal transcriptional programs in favour of cell survival, rather than any single metabolic pathway or target protein. Neuronal death after tGCI is complex. Characteristic lesions in vulnerable regions of the brain (such as the hippocampus and the striatum) appear from 12 h to 3 days after resuscitation and continue to evolve in the following weeks to months. Most cells that die do so by programmed cell death (PCD), as opposed to necrosis. This type of neuronal death has several features of apoptosis, with the cysteine-dependent aspartate-directed proteases family (the caspases) featuring strongly as executioners of PCD. Caspases normally exist in an inactivated state (pro-caspases), but under stressful conditions, cleavage (activation) D. R. Doherty, MBBCh (&) Department of Anesthesiology, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON K1H 8L1, Canada e-mail: ddoherty@cheo.on.ca

The effects of selective brain hypothermia on intracerebral hemorrhage in rats

Prolonged hypothermia effectively treats global cerebral ischemic injury in animal models as well as in cardiac arrest victims. Furthermore, clinical trials, based upon encouraging animal findings, are underway to assess efficacy in ischemic stroke. Intracerebral hemorrhage (ICH) is a more devastating stroke, but one that shares mechanisms of injury with ischemia. Accordingly, ICH may be amenable to hypothermia treatment. In this study we tested whether selective brain hypothermia improves outcome after an ICH in rats created by infusing 100 μL of autologous whole blood into the striatum. Striatal hypothermia (∼ 32 °C) was induced with a novel method (implanted cooling coil) that does not cause systemic cooling, thereby providing a safer and potentially more effective treatment for stroke than systemic hypothermia. Edema occurred for 4 days after ICH, but it peaked at 3 days (∼ 5%). At this time it was significantly reduced (to ∼ 2%) by cooling starting 1 h after ICH (3 day duration). Next, we determined whether 1 and 12 h delayed cooling treatments (4 day duration) would lessen functional impairment and lesion size. Untreated (normothermic) ICH resulted in significant forelimb use asymmetry, as well as deficits in walking and skilled reaching. These deficits were unaffected by hypothermia, as was the volume of tissue lost (∼ 20 mm 3) at 1 month. Thus, attenuated edema did not result in behavioral or histological benefit. In conclusion, while additional research with alternative cooling protocols and ICH models are required, these findings suggest that while hypothermia lessens edema, it will not be directly neuroprotective after ICH.

Failure of delayed and prolonged hypothermia to favorably affect hemorrhagic stroke in rats

Prolonged hypothermia reduces global and focal cerebral ischemic injury in rodents even when delayed for hours. However, it is not known whether hypothermia can reduce injury following intracerebral hemorrhage (ICH). Accordingly, we studied striatal injury and concomitant motor deficits after 2 days of hypothermia, induced 1 h after creation of an ICH by infusion of bacterial collagenase. Rats were first trained to retrieve food pellets in the Montoya staircase task. They were then implanted with core temperature telemetry probes and later subjected to normothermic ICH or sham operation (vehicle injection). Half self-regulated temperature after surgery; others were cooled to 33 °C (24 h) and then 35 °C (24 h). Hypothermia did not affect behavioral scores of sham animals (89.8% of baseline in staircase test) or histology. Untreated (normothermic) ICH rats lost 23.1 mm 3 of tissue at a 1-month survival, which significantly impaired food pellet retrieval (66.0% retrieval) with the contralateral limb (tested on days 21–25). Contrary to our hypothesis, hypothermia failed to lessen either the reaching impairment (62.8%) or the lesion (22.2 mm 3). While other hemorrhagic insults or complications may be improved with hypothermia, our data suggest that it will not salvage tissue that is quickly lost after ICH. We also assessed walking across a horizontal ladder and spontaneous paw usage in a cylinder test at 1–4 weeks after ICH, but neither test was sufficiently sensitive to this mild insult. This indicates that skilled reaching is more severely disrupted than spontaneous paw usage or walking after a striatal hemorrhage.

Resveratrol protects against global cerebral ischemic injury in gerbils

Increased oxidative stress has been implicated in the mechanisms of delayed neuronal cell death (DND) following cerebral ischemic insult. In this study, we investigated whether resveratrol, a polyphenolic antioxidant enriched in grape, may ameliorate ischemia-induced neuron cell death. Mongolian gerbils were divided into three groups, namely, sham control, ischemia and ischemia treated with resveratrol. Transient global cerebral ischemia was induced by occlusion of both common carotid arteries (CCA) for 5 min. Resveratrol was injected i.p. (30 mg/kg body weight), either during or shortly after CCA occlusion, and again at 24 h after ischemia. Cerebral blood flow was monitored before and during CCA occlusion using a laser Doppler flowmeter. Brain sections were immuno-stained for neurons, astrocytes and microglial cells. A time course study was also carried out to assess the bioavailability of resveratrol in serum, liver and brain using high performance liquid chromatography (HPLC). Morphometric measurements indicated extensive DND in the hippocampal CA1 region 4 days after ischemia and that neuron cell death was marked by the increase in reactive astrocytes and microglial cells. Administration of resveratrol, either during or after CCA occlusion, significantly ( P<0.05) decreased DND as well as glial cell activation. Analysis of resveratrol after i.p. injection indicated its presence in serum, liver and brain with peak activity at 1, 4 and 4 h, respectively. This study demonstrated for the first time that resveratrol, a polyphenolic antioxidant, can cross the blood–brain barrier and exert protective effects against cerebral ischemic injury.

Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice.

We explored susceptibility to injury after global ischemia in SV-129 and C57Black/6 mice, two commonly used-background strains in genetically engineered mice. Mice (n = 84) were subjected to 15, 30, or 75 minutes of bilateral common carotid artery (BCCA) occlusion followed by reperfusion for 72 hours. BCCA occlusion was performed under halothane or chloral hydrate anesthesia, in one experiment, mean arterial blood pressure and regional cerebral blood flow (laser Doppler flowmetry) were matched by controlled exsanguination. Baseline absolute blood flow measurements were obtained in both strains using a tracer, N-isopropyl-[methyl 1,3-14C]-p-iodoamphetamine, indicator fractionation technique (n = 5 per group). Vascular anatomy of the circle of Willis was visualized by intravascular perfusion of carbon black ink (n = 10 per group). Cerebrovascular reactivity was assessed by measuring the diameter of pial vessels (intravital microscopy) to acetylcholine (ACh) superfusion (0.1 to 10 mmol/L) in a closed cranial window preparation (n = 29). Resting blood flow values did not differ between groups in striatum, cerebellum, and brain-stem regions. SV-129 mice were less susceptible than C57Black/6 mice to ischemic injury (0.0 +/- 0.0 versus 1.3 +/- 0.3 damage in hippocampal CA1 region after 30 minutes of ischemia in SV-129 and C57Black/6, respectively; P < .01). Cellular damage (grade 1 to 3 injury) comparable to 30-minute BCCA occlusion was achieved only after 75 minutes of ischemia in SV-129 mice (1.1 +/- 0.3). Ischemic damage was also significantly less in SV-129 mice after blood pressure and flow were matched during ischemia in halothane-anesthetized SV-129 mice (0.5 +/- 0.3 versus 1.4 +/- 0.2, P < .05), or after chloral hydrate anesthesia (0.4 +/- 0.2 versus 1.5 +/- 0.4, P < .05). Hypoplastic posterior communicating arteries were found in all 10 C57Black/6 mice and may explain the greater susceptibility of these mice to injury after BCCA occlusion. More robust vasodilation to ACh in C57Black/6 mice could also indicate genetic differences in responses to vasoactive substances. C57Black/6 mice exhibit enhanced susceptibility to global cerebral ischemic injury, an incompletely formed circle of Willis, and augmented pial vessel dilation to ACh compared with SV-129 mice. Our findings suggest that strain differences may confound results when genetically engineered mice generated from more than a single background strain are used.

REVIEW ■ : Neuroprotection in Brain Ischemia: An Update (Part I

Ischemic brain injury produced by stroke or cardiac arrest is a major cause of human neurological disability. Steady advances in the neurosciences have elucidated pathophysiological mechanisms of brain ischemia and have suggested many therapeutic approaches directed at specific injury mechanisms to achieve neuroprotection of the acutely ischemic brain. The first portion of this two-part review highlights the differentiating features and pathological mechanisms of focal and global cerebral ischemic injury and summarizes a wealth of recent evidence as to how antagonism of excitatory amino acid neurotoxicity, mediated via NMDA as well as non-NMDA receptors, may offer a means of diminishing the extent of ischemic injury. The Neuroscientist 1:95-103, 1995

Does Chronic Hypoxemia Cause Brain Injury?

The study of focal cerebral hypoxic ischemic injury (stroke) has yielded much information about sensorimotor and cognitive function. It has been more difficult to derive neuroanatomic or neurophysiologic insights from global cerebral hypoxic and/or ischemic injury, although the causes of either or both cerebral insults are protean and occur frequently. Recent studies have reported isolated behavioral deficits and particular patterns of cellular injury that follow any one of a number of causes of global hypoxic ischemic cerebral disease (the two forms of injury are often difficult to separate in clinical conditions), and include the following: sudden cardiopulmonary arrest,^1-5sustained systemic hypotension,^6,7cerebral ischemia that follows vasospasm,^8,9and chronic hypoxemic pulmonary disease.¹⁰In this issue of theArchives, Heaton et al (p 1941) have continued their study of patients with COPD. In an earlier publication, they analyzed the prevalence, quality, and severity of See also p 1941.