Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats.

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30 min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.

Down-regulation of interleukin 7 mRNA by hypoxia is calcium dependent.

OBJECTIVE: The discovery of IL-7R(alpha) polymorphisms implicated in the pathogenesis of multiple sclerosis has highlighted the importance of interleukin 7 (IL-7) in central nervous system diseases. Hypoxia affects neurological disease states in part by modulating expression of many early and late response genes. The present work used cultured PC12 cells to investigate the effect of hypoxia on IL-7 expression. METHOD: PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium. RNA was isolated and reverse transcriptase-polymerase chain reaction (RT-PCR) was run to quantify messenger RNA (mRNA) change. Western blots were used to assess IL-7 protein change in the medium. Extracellular free Ca(2+) was removed by using Ca(2+)-free DMEM/F12 with 1 mM ethylene glycol tetraacetic acid for 45 minutes before the start of hypoxia. RESULTS: Exposure of PC12 cells to 1% oxygen for 6 hours decreased IL-7 mRNA by 77% using RT-PCR (p<0.01). Exposure to 1% oxygen for 24 hours decreased IL-7 protein in the medium by 21% (p<0.05). As hypoxia duration increased (2, 4, 6 and 24 hours) or oxygen concentrations decreased (10%, 5% and 1%), IL-7 mRNA expression progressively decreased. Removal of extracellular free Ca(2+) completely prevented these hypoxia-induced decreases of IL-7 mRNA. DISCUSSION: Since IL-7 exhibits trophic properties in developing brain, down-regulation of IL-7 by hypoxia may contribute to hypoxia-induced injury to neural cells.

Modeling intracerebral hemorrhage: glutamate, nuclear factor-kappa B signaling and cytokines.

A significant amount of new information has been generated in animal models of intracerebral hemorrhage during the past several years. These include findings on the pathophysiological, biochemical and molecular processes that underlie the development of brain tissue injury after intracerebral hemorrhage as well as potential new treatments. We review these various findings that include glutamate receptor activation, oxidative stress development, intracellular signaling through the transcription factor, nuclear factor-kappaB, and markedly upregulated cytokine gene expression. We also briefly review the surgical treatment for intracerebral hemorrhage and list the pharmacological treatment studies that have recently appeared.

A novel duplex ELISA method for quantitation of plasma proteins in areas of brain edema.

We have developed a novel type of duplex enzyme-linked immunosorbent assay (ELISA) for the quantitation of the major plasma proteins, IgG and albumin, in edematous brain tissue. We test this duplex ELISA on our porcine intracerebral hemorrhage (ICH) model and show that it is as accurate and sensitive as independent single ELISAs. This method is useful as a marker of edema in brain tissue and the same design can be applied to other proteins and sample types.

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis.

Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T

Brain genomics of intracerebral hemorrhage.

After intracerebral hemorrhage (ICH), many changes of gene transcription occur that may be important because they will contribute to understanding mechanisms of injury and recovery. Therefore, gene expression was assessed using Affymetrix microarrays in the striatum and the overlying cortex at 24 h after intracranial infusions of blood into the striatum of adult rats. Intracerebral hemorrhage regulated 369 of 8,740 transcripts as compared with saline-injected controls, with 104 regulated genes shared by the striatum and cortex. There were 108 upregulated and 126 downregulated genes in striatum, and 170 upregulated and 69 downregulated genes in the cortex. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed upregulation of IL-1-beta, Lipcortin 1 (annexin) and metallothionein 1,2, and downregulation of potassium voltage-gated channel, shaker-related subfamily, beta member 2 (Kcnab2). Of the functional groups of genes modulated by ICH, many metabolism and signal-transduction-related genes decreased in striatum but increased in adjacent cortex. In contrast, most enzyme, cytokine, chemokine, and immune response genes were upregulated in both striatum and in the cortex after ICH, likely in response to foreign proteins from the blood. A number of these genes may contribute to brain edema and cellular apoptosis caused by ICH. In addition, downregulation of growth factor pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway could also contribute to perihematoma cell death/apoptosis. Intracerebral hemorrhage-related downregulation of GABA-related genes and potassium channels might contribute to perihematoma cellular excitability and increased risk of post-ICH seizures. These genomic responses to ICH potentially provide new therapeutic targets for treatment.

Gene expression in blood changes rapidly in neutrophils and monocytes after ischemic stroke in humans: a microarray study.

Ischemic brain and peripheral white blood cells release cytokines, chemokines and other molecules that activate the peripheral white blood cells after stroke. To assess gene expression in these peripheral white blood cells, whole blood was examined using oligonucleotide microarrays in 15 patients at 2.4+/-0.5, 5 and 24 h after onset of ischemic stroke and compared with control blood samples. The 2.4-h blood samples were drawn before patients were treated either with tissue-type plasminogen activator (tPA) alone or with tPA plus Eptifibatide (the Combination approach to Lysis utilizing Eptifibatide And Recombinant tPA trial). Most genes induced in whole blood at 2 to 3 h were also induced at 5 and 24 h. Separate studies showed that the genes induced at 2 to 24 h after stroke were expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes. These genes included: matrix metalloproteinase 9; S100 calcium-binding proteins P, A12 and A9; coagulation factor V; arginase I; carbonic anhydrase IV; lymphocyte antigen 96 (cluster of differentiation (CD)96); monocarboxylic acid transporter (6); ets-2 (erythroblastosis virus E26 oncogene homolog 2); homeobox gene Hox 1.11; cytoskeleton-associated protein 4; N-formylpeptide receptor; ribonuclease-2; N-acetylneuraminate pyruvate lyase; BCL6; glycogen phosphorylase. The fold change of these genes varied from 1.6 to 6.8 and these 18 genes correctly classified 10/15 patients at 2.4 h, 13/15 patients at 5 h and 15/15 patients at 24 h after stroke. These data provide insights into the inflammatory responses after stroke in humans, and should be helpful in diagnosis, understanding etiology and pathogenesis, and guiding acute treatment and development of new treatments for stroke.

Plasma proteins in edematous white matter after intracerebral hemorrhage confound immunoblots: an ELISA to quantify contamination.

Intracerebral hemorrhage (ICH) and traumatic brain injury can induce brain tissue edema (i.e., interstitial and/or vasogenic), containing high concentrations of plasma proteins. To understand biochemical processes in edema development following these insults, it would be useful to examine alterations in various proteins (e.g., transcription factors, signaling). However, determining altered protein responses in edematous brain tissue using standard immunoblotting techniques is problematic due to contaminating plasma proteins. To solve this problem, we developed an enzyme-linked immunosorbent assay (ELISA) method to quantify the two major plasma proteins, albumin and immunoglobulin G (IgG), that comprise about 80% of the total plasma proteins. We tested our method on edematous white matter samples from our porcine ICH model. To induce ICH, we infused autologous arterial whole blood (3 mL) into frontal hemispheric white matter of pentobarbital- anesthetized pigs ( approximately 20 kg) over 15 min. We froze brains in situ at various times up to 24 h post- ICH and sampled white matter adjacent and contralateral to hematomas. We prepared cytoplasmic extracts that we subjected to ELISA and immunoblotting analyses. Our results demonstrate that this ELISA method is accurate, reproducible, and enables the concentrations of albumin and IgG in edematous brain tissue samples to be accurately determined. By using this correction method, equal amounts of cellular protein can be loaded onto gels during immunoblotting procedures. This method is applicable to edematous tissue samples in brain injury models in which high plasma protein concentrations result from interstitial or vasogenic edema development.

Bilirubin production and oxidation in CSF of patients with cerebral vasospasm after subarachnoid hemorrhage.

Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) remains a significant cause of mortality and morbidity; however, the etiology is, as yet, unknown, despite intensive research efforts. Research in this laboratory indicates that bilirubin and oxidative stress may be responsible by leading to formation of bilirubin oxidation products (BOXes), so we investigated changes in bilirubin concentration and oxidative stress in vitro, and in cerebral spinal fluid (CSF) from SAH patients. Non-SAH CSF, a source of heme oxygenase I (HO-1), and blood were incubated, and in vitro bilirubin production measured. Cerebrospinal fluid from SAH patients was collected, categorized using stimulation of vascular smooth muscle metabolism in vitro, and information obtained regarding occurrence of vasospasm in the patients. Cerebral spinal fluid was analyzed for hemoglobin, total protein and bilirubin, BOXes, malonyldialdehyde and peroxidized lipids (indicators of an oxidizing environment), and HO-1 concentration. The formation of bilirubin in vitro requires that CSF is present, as well as whole, non-anti-coagulated blood. Bilirubin, BOXes, HO-1, and peroxidized lipid content were significantly higher in CSF from SAH patients with vasospasm, compared with nonvasospasm SAH CSF, and correlated with occurrence of vasospasm. We conclude that vasospasm may be more likely in patients with elevated BOXes. The conditions necessary for the formation of BOXes are indeed present in CSF from SAH patients with vasospasm, but not CSF from SAH patients without vasospasm.

Plasma infusions into porcine cerebral white matter induce early edema, oxidative stress, pro-inflammatory cytokine gene expression and DNA fragmentation: implications for white matter injury with increased blood-brain-barrier permeability.

Plasma infused into porcine cerebral white matter induces both acute interstitial and delayed vasogenic edema. Edematous white matter contains extracellular plasma proteins and rapidly induces oxidative stress as evidenced by increased protein carbonyl formation and heme oxygenase-1 induction. We tested the hypothesis that edematous white matter would also upregulate pro-inflammatory cytokine gene expression and develop DNA damage. We infused autologous plasma into the frontal hemispheric white matter of pentobarbital-anesthetized pigs. We monitored and controlled physiological variables and froze brains in situ at 1, 4 or 24 hrs. We determined edema volumes by computer-assisted morphometry. We measured white matter protein carbonyl formation by immunoblotting, cytokine gene expression by standard RT-PCR methods and DNA fragmentation by agarose gel electrophoresis. White matter edema developed acutely (1 hr) after plasma infusion and increased significantly in volume between 4 and 24 hrs. Protein carbonyl formation also occurred rapidly in edematous white matter with significant elevations (3 to 4-fold) already present at 1 hr. This increase remained through 24 hrs. Pro-inflammatory cytokine gene expression was also rapidly increased at 1 hr post-infusion. Evidence for DNA fragmentation began at 2 to 4 hrs, and a pattern indicative of both ongoing necrosis and apoptosis was robust by 24 hrs. Plasma protein accumulation in white matter induces acute edema development and a cascade of patho-chemical events including oxidative stress, pro-inflammatory cytokine gene expression and DNA damage. These results suggest that in diseases with increased blood-brain barrier (BBB) permeability or following intracerebral hemorrhage or traumatic brain injury, interstitial plasma can rapidly damage white matter.

Local brain hypothermia for neuroprotection in stroke treatment and aneurysm repair.

Hypothermia is well known to provide neuroprotection following various brain insults in experimental animals. Two recently completed clinical trials of whole body hypothermia in out-of-hospital cardiac arrest patients' demonstrated significantly improved survival rates and neurologic outcomes. These results provide new excitement and encouragement for clinical application of hypothermia in cerebrovascular disease. However, the intensive care challenges and adverse events (e.g. prolonged times to target temperatures, shivering and sedation, pneumonia) during the management of hypothermia, dampen enthusiasm for widespread application especially in elderly stroke patients. In this manuscript, we review recent hypothermia trials for stroke. We describe an alternate approach, i.e. local brain cooling, and discuss this new technique with reference to the extensive literature on the marked efficacy of hypothermia. We describe a new technology, the ChillerPad(TM) and ChillerStrip(TM) Systems developed by Seacoast Technologies, Inc. (Portsmouth, NH, USA). The latter device has received FDA approval and will be employed in a trial of local hypothermia for cerebral aneurysm repair. We present our experimental findings that profound local hypothermia does not damage cortical neurons. We also report that local hypothermia protects the blood-brain barrier and markedly reduces vasogenic edema development in an experimental intracerebral hemorrhage model. Lastly, we review potential mechanisms through which hypothermia provides blood-brain barrier protection and reduces edema formation. Clearly, hypothermia has a bright future for cerebrovascular disease treatment if brain cooling can be delivered in a manner that does not compromise the patient or the neurosurgical and intensive care settings. Local brain cooling may be just that new treatment approach.

Glutamate receptor blockade attenuates glucose hypermetabolism in perihematomal brain after experimental intracerebral hemorrhage in rat.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage has no effective treatment. The delayed appearance of edema, apoptosis, and inflammation in perihematomal brain suggests that these events may be targets for therapeutic intervention. To develop successful treatments, we must learn more about the effects of hemorrhage on brain tissue. In this study, we investigated the acute metabolic effects of intrastriatal hemorrhage in rat brain. METHODS: Lysed blood or saline (50 microL each) was injected into the striatum of male Sprague-Dawley rats. The rats recovered for 1 to 72 hours before injection of [14C]-2-deoxyglucose (intraperitoneally) 30 minutes before decapitation. Animals were pretreated with the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonists dizolcilpine maleate (MK-801; 1 mg/kg) or 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX; 30 mg/kg), or saline vehicle. Additional animals received intrastriatal injections of glutamate (1.0 mmol/L), NMDA (1.0 mmol/L), or AMPA (0.1 mmol/L) in the place of blood. Semiquantitative autoradiographs from the brains were analyzed to determine the effects of hemorrhage on relative glucose metabolism. RESULTS: We found an acute phase of increased [14C]-2-deoxyglucose uptake in the perihematomal region that peaks 3 hours after lysed blood injection. Saline injections had no effect on striatal glucose utilization. The increased [14C]-2-deoxyglucose uptake produced by the hemorrhages was blocked by pretreatment with MK-801 and NBQX. Glutamate injections alone had no effect on striatal metabolism, whereas NMDA and AMPA injections increased [14C]-2-deoxyglucose uptake. CONCLUSIONS: The data imply that glutamate activation of NMDA or AMPA receptors increases glucose metabolism in perihematomal brain at early times after intracerebral hemorrhage. This may provide a possible target for the treatment of intracerebral hemorrhage.

Heme and iron metabolism: role in cerebral hemorrhage.

Heme and iron metabolism are of considerable interest and importance in normal brain function as well as in neurodegeneration and neuropathologically following traumatic injury and hemorrhagic stroke. After a cerebral hemorrhage, large numbers of hemoglobin-containing red blood cells are released into the brain's parenchyma and/or subarachnoid space. After hemolysis and the subsequent release of heme from hemoglobin, several pathways are employed to transport and metabolize this heme and its iron moiety to protect the brain from potential oxidative stress. Required for these processes are various extracellular and intracellular transporters and storage proteins, the heme oxygenase isozymes and metabolic proteins with differing localizations in the various brain-cell types. In the past several years, additional new genes and proteins have been discovered that are involved in the transport and metabolism of heme and iron in brain and other tissues. These discoveries may provide new insights into neurodegenerative diseases like Alzheimer's, Parkinson's, and Friedrich's ataxia that are associated with accumulation of iron in specific brain regions or in specific organelles. The present review will examine the uptake and metabolism of heme and iron in the brain and will relate these processes to blood removal and to the potential mechanisms underlying brain injury following cerebral hemorrhage.

Hematoma removal, heme, and heme oxygenase following hemorrhagic stroke.

The hemorrhagic strokes, intracerebral (ICH) and subarachnoid hemorrhage (SAH), often have poor outcomes. Indeed, the most common hemorrhagic stroke, ICH, has the highest mortality and morbidity rates of any stroke subtype. In this report, we discuss the evidence for the staging of red blood cell removal after ICH and the significance of control of this process. The protective effects of clinically relevant metalloporphyrin heme oxygenase inhibitors in experimental models of ICH and in superficial siderosis are also discussed. We also examine literature paradoxes related to both heme and heme oxygenase in various disorders of the central nervous system. Last, new data are presented that support the concept that heme, although primarily a pro-oxidant, can also have antioxidant properties.

Bilirubin as a cerebrospinal fluid marker of sentinel subarachnoid hemorrhage: a preliminary report in pigs.

OBJECT: A model of subarachnoid hemorrhage (SAH) in pigs was developed to investigate bilirubin concentration in cerebrospinal fluid (CSF) as a potential marker of sentinel SAH. METHODS: Seven male Yorkshire pigs received a 250-microl injection of either whole autologous arterial blood (four animals) or isotonic saline (three animals) into the cisternae magna in an effort to produce volumetrically a model of sentinel SAH and a control injection model, respectively. Cerebrospinal fluid volumes of 100 microl were then collected from both the lumbar cistern and cisternae magna at 1 to 2-hour intervals for a total of 24 hours postinjection. The CSF was then tested for bilirubin. Mean concentrations of bilirubin (+/- standard deviation [SD]) obtained from the lumbar cistern 24 hours following the injection of blood or saline were 4.38 +/- 1.04 microM in the SAH animals and 1.02 +/- 0.05 microM in the controls. At 24 hours postinjection, mean concentrations (+/- SD) of cisternae magna bilirubin were 7.29 +/- 1.33 microM and 1.33 +/- 0.14 microM in the SAH animals and controls, respectively. In the SAH group, both the lumbar cistern and cisternae magna bilirubin concentrations differed significantly from baseline values 12 hours following SAH. CONCLUSIONS: Elevated concentrations of CSF bilirubin can be detected following a low-volume SAH, and the production of bilirubin occurred over a predictable time course. Twelve hours after hemorrhage, an elevated CSF bilirubin concentration was an indicator of hemolysis occurring in the subarachnoid spaces. The presence of bilirubin in CSF is a potential marker for differentiating SAHs from traumatic lumbar punctures in humans.

Minimally invasive therapy for intracerebral hematomas.

The efficacy of surgical treatment of ICH remains unproven and controversial [40]. Although open surgery does not appear to improve the patient's outcome [2], less invasive methods of hematoma evacuation seem to show promising results in improving patient outcome and survival. To date, the only two clinical trials that have demonstrated benefit from surgical treatment over medical therapy for ICH have used minimally invasive techniques [27,38]. Randomized controlled clinical trials comparing minimally invasive surgical techniques versus best medical treatment are needed to determine the best management of ICH.

Experimental animal models of intracerebral hemorrhage.

Experimental animal ICH models are able to reproduce the overall important pathophysiologic events documented in human ICH, including edema development, markedly reduced metabolism, and tissue pathologic responses. Thus, ICH models serve as an important tool for new understanding of the mechanisms underlying brain injury after an intracerebral bleed. Currently, ongoing studies in several laboratories using these models investigating secondary inflammatory responses as well as intracellular signaling and molecular events are expected to provide therapeutic targets for treating ICH. Future studies should also be directed at one aspect of ICH modeling that has received little attention--potential differences in the hemostatic systems and physical and biochemical properties of clots in animals that might make their susceptibility to aspiration and/or fibrinolytic drugs and rates of rehemorrhage different than in human beings. Also, future efforts should be directed toward the development of a model that mimics the pathophysiologic processes that lead to spontaneous ICH, progression of hemorrhage, and the recurrence of bleeding in human beings. This model would not only provide better understanding of the dynamic events leading to ICH and tissue injury but should also lead to the development of highly effective pharmacologic and surgical treatments.

In vivo testing of a non-invasive prototype device for the continuous monitoring of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a stroke subtype with the highest mortality rate. Hematoma expansion and re-bleeding post-ICH are common and exacerbate the initial cerebral insult. There is a need for continuous monitoring of the neurologic status of patients with an ICH injury. NEW METHOD: A prototype device for non-invasive continuous monitoring of an ICH was developed and tested in vivo using a porcine ICH model. The device consists of receiving and transmitting antennae in the 400-1000 MHz frequency range, placed directly in line with the site of the ICH. The device exploits the differences in the dielectric properties and geometry of tissue media of a healthy brain and a brain with an ICH injury. The power received by the receiving antenna is measured and the percent change in power received immediately after infusion of blood and 30 min after the infusion, allowing for the blood to clot, is calculated. RESULTS: An increase in the received power in the presence of an ICH is observed at 400 MHz, consistent with previous in vitro studies. Frequency sweep experiments show a maximum percent change in received power in the 750-1000 MHz frequency range. COMPARISON WITH EXISTING METHODS: Currently, CT, MRI and catheter angiography (CA) are the main clinical neuroimaging modalities. However, these techniques require specialized equipment and personnel, substantial time, and patient-transportation to a radiology suite to obtain results. Moreover, CA is invasive and uses intra-venous dye or vascular catheters to accomplish the imaging. CONCLUSIONS: The device has the potential to significantly improve neurologic care in the critically ill brain-injured patient.

Do current animal models of intracerebral hemorrhage mirror the human pathology?

Although intracerebral hemorrhage (ICH) has no proven treatment, well-designed studies using animal models of ICH may lead to the development of novel therapies. We briefly review current animal models of ICH. Furthermore, we discuss how these models may be utilized and targeted to facilitate translation of preclinical findings to the clinical arena.

Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain.

A major complication of recanalization therapy after an acute arterial occlusion in brain is hemorrhagic transformation (HT). Although it is known that prolonged ischemia is important in the development of HT, the role of reperfusion in ischemia-reperfusion induced HT is less well studied. To address the effect of reperfusion on HT, we assessed the incidence and severity of hemorrhage in rats after 5 h of middle cerebral artery occlusion (MCAO) followed by 19-hour reperfusion compared to rats with permanent occlusion (PMCAO) at the same 24-hour time point. The incidence and amount of hemorrhage, neurological function, and mortality rates were measured. MCAO (5 h) with 19-hour reperfusion was associated with a significantly higher incidence of cortical hemorrhage compared to PMCAO (81.8% vs 18.2%, p<0.05). Hemorrhage scores were higher in the 5-hour MCAO/reperfusion group compared to PMCAO rats (17.6+/-11.5 vs 2.4+/-5.3 in cortex, 20.4+/-4.6 vs 9.7+/-4.5 in striatum, p<0.01). Neurological function was worse in the ischemia-reperfusion group compared to PMCAO (p<0.05) and mortality rates were insignificantly higher in the 5-hour MCAO/reperfusion group vs PMCAO group (54.5% vs 18.1%; p<0.08). The results suggest that reperfusion after prolonged ischemia is associated with increased hemorrhagic transformation and neurological deterioration as compared to permanent ischemia. Whether pharmacological treatments prior to reperfusion attenuate post-ischemic HT requires further study.