Rapid tau protein dephosphorylation and differential rephosphorylation during cardiac arrest-induced cerebral ischemia and reperfusion.

The effects of cerebral ischemia/reperfusion on phosphorylation of microtubule-associated tau proteins were assessed in a canine model of cardiac arrest. As tau proteins are phosphorylated by kinases involved in different transduction signal pathways, their phosphorylation state is an excellent marker of neuronal homeostasis and microtubule dynamics. Canine brain tau proteins were characterized by immunoblotting using phosphorylation-dependent antibodies and antisera raised against different amino- and carboxy-terminal tau sequences. The present study reports a complete dephosphorylation of tau proteins during ischemia, which is shown by a higher electrophoretic mobility and the almost (if not total) disappearance of phosphorylation-dependent monoclonal antibody labeling. After 2-hour restoration of spontaneous circulation, a decrease in the electrophoretic mobility was observed, and after 24 hours of reperfusion, a full restoration of the phosphorylation was visualized using phosphorylation-dependent monoclonal antibodies directed against Ser/Thr-Pro sites. However, one particular phosphorylation site involved in tau binding to microtubules, located on Ser262/356, was never fully significantly rephosphorylated, suggesting that microtubule metabolism was still affected after 24 hours of reperfusion. Thus, the sequential and differential recovery of tau phosphorylation after ischemia followed by reperfusion is a useful marker with which to monitor neuronal integrity after brain ischemia.

Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation.

Chronic impairment of aerobic energy metabolism accompanies global cerebral ischemia and reperfusion and likely contributes to delayed neuronal cell death. Reperfusion-dependent inhibition of pyruvate dehydrogenase complex (PDHC) enzyme activity has been described and proposed to be at least partially responsible for this metabolic abnormality. This study tested the hypothesis that global cerebral ischemia and reperfusion results in the loss of pyruvate dehydrogenase immunoreactivity and that such loss is associated with selective neuronal vulnerability to transient ischemia. Following 10 min canine cardiac arrest, resuscitation, and 2 or 24 h of restoration of spontaneous circulation, brains were either perfusion fixed for immunohistochemical analyses or biopsy samples were removed for Western immunoblot analyses of PDHC immunoreactivity. A significant decrease in immunoreactivity was observed in frontal cortex homogenates from both 2 and 24 h reperfused animals compared to samples from nonischemic control animals. These results were supported by confocal microscopic immunohistochemical determinations of pyruvate dehydrogenase immunoreactivity in the neuronal cell bodies located within different layers of the frontal cortex. Loss of immunoreactivity was greatest for pyramidal neurons located in layer V compared to neurons in layers IIIc/IV, which correlates with a greater vulnerability of layer V neurons to delayed death caused by transient global cerebral ischemia.

Lack of a neuroprotective effect from N-acetylcysteine after cardiac arrest and resuscitation in a canine model.

OBJECTIVE: Oxygen free radicals cause brain injury following resuscitation from cardiac arrest. In preclinical trials, some free radical scavenging drugs reduce oxidative neuronal damage after ischemia and reperfusion, but these drugs are generally not yet available for clinical testing or use. N-Acetylcysteine (NAC), a commonly used antidote in acetaminophen poisoning, is also a potent free radical scavenger that can ameliorate oxidative injury following ischemia and reperfusion in neuronal cell culture. We hypothesized that treatment with NAC would improve neurological outcome after cardiac arrest and resuscitation. METHODS: In 16 adult female beagles, 10 min of ventricular fibrillation was followed by 3 min of open-chest CPR, and defibrillation. Immediately following return of spontaneous circulation, animals randomly received either 150 mg/kg NAC (3% solution) (n = 8) or an equivalent volume of normal saline (n = 8). Twenty-three hours later, neurological deficit was scored (0 = normal, 100 = brain death). RESULTS: All animals were successfully resuscitated, and there were no apparent adverse effects to the administration of NAC in post resuscitative animals. There was, however, no significant difference in neurological deficit in the animals receiving NAC (40 +/- 12.9, mean +/- SD) compared to control animals (44 +/- 6.5, P = 0.73). CONCLUSION: No neuroprotective effect was found from the administration of NAC at currently used clinical dosages, to dogs subjected to 10 min of global cerebral ischemia from cardiac arrest and resuscitation.

Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia.

Caspase-9 is critical for cytochrome c (cyto-c)-dependent apoptosis and normal brain development. We determined that this apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca2+ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis.

Normoxic ventilation after cardiac arrest reduces oxidation of brain lipids and improves neurological outcome.

BACKGROUND AND PURPOSE: Increasing evidence that oxidative stress contributes to delayed neuronal death after global cerebral ischemia has led to reconsideration of the prolonged use of 100% ventilatory O2 following resuscitation from cardiac arrest. This study determined the temporal course of oxidation of brain fatty acyl groups in a clinically relevant canine model of cardiac arrest and resuscitation and tested the hypothesis that postischemic ventilation with 21% inspired O2, rather than 100% O2, results in reduced levels of oxidized brain lipids and decreased neurological impairment. METHODS: Neurological deficit scoring and high performance liquid chromatography measurement of fatty acyl lipid oxidation were used in an established canine model using 10 minutes of cardiac arrest followed by resuscitation with different ventilatory oxygenation protocols and restoration of spontaneous circulation for 30 minutes to 24 hours. RESULTS: Significant increases in frontal cortex lipid oxidation occurred after 10 minutes of cardiac arrest alone with no reperfusion and after reperfusion for 30 minutes, 2 hours, and 24 hours (relative total 235-nm absorbing peak areas=7.1+/-0.7 SE, 17.3+/-2.7, 14.2+/-3.2, 16.1+/-1.0, and 14.0+/-0.8, respectively; n=4, P<0.05). The predominant oxidized lipids were identified by gas chromatography/mass spectrometry as 13- and 9-hydroxyoctadecadienoic acids (13- and 9-HODE). Animals ventilated on 21% to 30% O2 versus 100% O2 for the first hour after resuscitation exhibited significantly lower levels of total and specific oxidized lipids in the frontal cortex (1.7+/-0.1 versus 3.12+/-0.78 microg 13-HODE/g wet wt cortex., n=4 to 6, P<0.05) and lower neurological deficit scores (45.1+/-3.6 versus 58.3+/-3.8, n=9, P<0.05). CONCLUSIONS: With a clinically relevant canine model of 10 minutes of cardiac arrest, resuscitation with 21% versus 100% inspired O2 resulted in lower levels of oxidized brain lipids and improved neurological outcome measured after 24 hours of reperfusion. This study casts further doubt on the appropriateness of present guidelines that recommend the indiscriminate use of 100% ventilatory O2 for undefined periods during and after resuscitation from cardiac arrest.

Neuroprotective effects of acetyl-L-carnitine after stroke in rats.

STUDY OBJECTIVE: To test the hypothesis that acetyl-L-carnitine (ALCAR) promotes neurologic recovery from experimental focal cerebral ischemia (stroke) in rats. METHODS: We conducted a prospective, randomized, blinded study in which adult male Sprague-Dawley rats were subjected to coagulative occlusion of the distal right middle cerebral artery (MCA) and temporary occlusion of both common carotid arteries (CCAs) for 60 minutes. After the onset of ischemia each rat was given ALCAR (200 mg/kg) or a similar volume of drug vehicle. Neurologic evaluation was performed on postoperative days 1, 2, 3, and 7. Postoperative weight loss was measured at day 7. Infarct volume was measured in separate groups of rats at 24 hours. RESULTS: Neurologic outcomes, as assessed with an 11-point neurologic deficit scoring system, were significantly improved in ALCAR-treated rats on days 1, 2, and 3 (P < .05). Improvement approached significance on day 7. Rats treated with ALCAR also demonstrated significantly less weight loss on day 7 compared with the vehicle-treated controls. We detected no differences, however, in infarct volumes measured between treatment groups. CONCLUSION: Although we noted no differences in infarct volume, postischemic treatment with ALCAR did improve early clinical recovery and prevented significant weight loss in this rat model of focal cerebral ischemia.

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain.

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion.

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to alpha-amino-3-hydroxy-5-methyl- 4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [3H]AMPA, [3H]KA, or [3H] glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20-30% at 30 min postischemia, remaining depressed through 24 h. In cortex, AMPA and metabotropic binding were decreased at several time-points after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.

Distribution of neuronal populations containing neurofilament protein and calcium-binding proteins in the canine neocortex: regional analysis and cell typology.

Neurophysiological experiments in carnivores have revealed the existence of a large number of cortical regions and an organization of sensory systems quite similar to that found in primates. However, the cyto- and chemoarchitecture of the cerebral cortex is relatively poorly known in carnivores. We analyzed the distribution and typology of classes of neurons containing neurofilament protein or the calcium-binding proteins parvalbumin, calbindin, and calretinin in six neocortical regions of the dog. In all these areas, neurofilament protein was present in a subpopulation of medium-to-large size pyramidal neurons predominantly distributed in layers III and V. Parvalbumin was present in a large population of morphologically diverse interneurons. Small ovoid and multipolar neurons were observed throughout the cortical layers, but predominated in layers II and IV. Layers III and V-VI were characterized by the presence of larger and intensely immunoreactive neurons with bitufted or multipolar morphology, and layers V-VI also contained large multipolar neurons. Calbindin was observed in small round and multipolar interneurons in layer II, and typical double bouquet cells in layer III. Layers IV-VI contained isolated double bouquet cells and large multipolar neurons. A few calbindin-immunoreactive pyramidal neurons were also observed in layer V. Calretinin was localized in bipolar and double bouquet cells in layers II and upper III. The lower part of layer III and layers IV-VI contained rare calretinin-immunoreactive neurons. In some areas, layer III displayed a few large isolated multipolar neurons and pyramidal neurons containing calretinin. In addition, the results show that there is a substantial degree of variability in the distribution of these proteins among cortical regions, and that although they are found in morphologically comparable neuronal types in dog, monkeys, and humans, many differences exist in their regional distribution patterns between carnivores and primates.

Distribution of neurofilament protein and calcium-binding proteins parvalbumin, calbindin, and calretinin in the canine hippocampus.

Neurofilament protein and calcium-binding proteins parvalbumin, calbindin, and calretinin are present in morphologically distinct neuronal subpopulations in the mammalian cerebral cortex. Immunohistochemical studies of the hippocampal formation and neocortex have demonstrated that while neurofilament protein and calbindin are localized in subsets of pyramidal neurons, the three calcium-binding proteins are useful markers to differentiate non-overlapping populations of interneurons. To date, most studies have been performed in rodents and primates. In the present analysis, we analyzed the distribution of these proteins in the canine hippocampus. Neurofilament protein was present in large multipolar neurons in the hilus and in pyramidal neurons in the CA3 field, whereas pyramidal neurons in the CA1 field and subiculum were less intensely immunoreactive. Parvalbumin immunoreactivity was observed in large multipolar neurons in the hilus and throughout the CA3-CA1 fields, in a few pyramidal-shaped neurons in the CA1 field and subiculum, and had a distinct neuropil staining pattern in the granule cell layer and stratum pyramidale of the Ammon's horn. Calbindin immunoreactivity displayed a strong labeling of the granule cells and mossy fibers and was also observed in a population of moderately immunoreactive neurons in the CA1 field and subiculum. Calretinin immunoreactivity was relatively weaker overall. The inner molecular layer in the dentate gyrus had a distinct band of labeling, the stratum lacunosum/moleculare contained a punctate neuropil staining, and there were a few small multipolar neurons in the hilus, CA3-CA1 fields, and subiculum. Comparison of the staining patterns observed in the dog hippocampus with those in human, macaque monkeys and rats revealed that although there are some subregional differences among these taxa, the dog may constitute a valuable large animal model for the study of certain neurological conditions that affect humans, in spite of the phylogenetic distance between carnivores and primates.

Distribution of glutamate receptor subunit proteins GluR2(4), GluR5/6/7, and NMDAR1 in the canine and primate cerebral cortex: a comparative immunohistochemical analysis.

The distribution of the AMPA, kainate and NMDA glutamate receptor subunit proteins GluR2(4), GluR5/6/7 and NMDAR1, respectively, were analyzed in the dog hippocampus and neocortex and compared to macaque monkeys and humans. In the dog hippocampus, these glutamate receptor classes exhibited a comparable distribution with few differences in densities of labeled of neurons in the CA1-CA3 fields and in neuropil staining patterns in the dentate gyrus. In particular, the GluR5/6/7 subunit proteins were characterized by a more restricted cellular distribution in the CA1-CA3 fields. In the dog neocortex, the GluR2(4) subunit was found in a higher number of neurons in layers III and V compared to the GluR5/6/7 or NMDAR1 subunits, which were found predominantly in a population of medium-to-large layer V pyramidal neurons. Layers II and VI were consistently densely labeled with all three receptor classes, especially in the case of the GluR5/6/7 and NMDAR1 subunits. All three antibodies used thus far showed an intense labeling of the perikaryon and dendritic segments in the dog cerebral cortex. Apical dendrites could be followed through several layers in some cases, and formed well-stained plexuses in all of the neocortical layers. These patterns were very similar to those observed in the hippocampus and neocortex of both monkey and human, although GluR2(4) and NMDAR1 immunoreactivity was visualized in more heterogeneous populations of cortical neurons in the primates than in dogs. Glutamate is the principal excitatory neurotransmitter in the brain and is involved in the excitotoxic mechanisms occurring in pathologic conditions such as epilepsy and cerebral ischemia. The dog has been shown to represent a reliable large animal model for several neurologic disorders and is used particularly in investigations of the cerebral repercussions of cardiac arrest. The overall similarity of the staining patterns in dogs and primates observed in the present study suggest that the dog model may be highly valuable for the characterization of potential cellular and synaptic shifts in the distribution and expression of specific glutamate receptor subunits, in the context of other biochemical and morphologic effects of global brain ischemia and reperfusion following cardiac arrest.

The role of L-type voltage dependent calcium channels in stimulated [3H]norepinephrine release from canine hippocampal slices following global cerebral ischemia and reperfusion.

The hippocampus is among those brain regions which are selectively vulnerable to ischemic damage. Hippocampal damage due to transient cerebral ischemia is mainly of the delayed, non-necrotic type which may arise after disruption or activation of specific cellular systems, including transmitter release through excitatory amino acid receptors. We investigated the contribution of L-type voltage dependent calcium channels (VDCCs) to glycine (GLY) potentiated N-methyl-D-aspartate (NMDA) receptor- and potassium-stimulated [3H]norepinephrine (NE) release in a canine model of global cerebral ischemia and reperfusion. Tissue was collected from four experimental groups: non-arrested controls (NA), global cerebral ischemia induced by 10 minute cardiac arrest (CA), and CA followed by 30 min or 24 hours reperfusion after restoration of spontaneous circulation. Brain slices prepared from all groups accumulated approximately equivalent amounts of [3H]NE. The sensitivity of [3H]NE release to stimulation by NMDA/GLY or elevated potassium was unchanged after ischemia and reperfusion. About 30% of release stimulated by the addition of 20 mM potassium was inhibited by the NMDA receptor-operated channel antagonist MK801 in all groups except CA in which only 4% of release was inhibited by MK801. The ability of 1 microM nitrendipine (NTP) to block stimulated release indicated that the contribution of the L-type VDCC to potassium or NMDA/GLY-stimulated release was significant only in NA and 24 hour reperfused animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic analysis of L- and N-type voltage-dependent calcium channel binding in canine brain after global cerebral ischemia/reperfusion.

Binding of antagonists to L- and N-type voltage-dependent calcium channels (VDCC) was measured in canine brain following global ischemia and reperfusion. Ischemia was induced by 10 min cardiac arrest, followed by restoration of spontaneous circulation for periods of up to 24 h. Binding of [3H]PN200-110 and [125I]omega-conotoxin GVIA to frozen sections from hippocampus, striatum, parietal cortex and temporal cortex was analyzed using quantitative receptor autoradiography. The binding patterns of the two radioligands were similar in cortex and striatum, but differed in hippocampus. In the latter tissue, [125I]omega-conotoxin GVIA binding was dense over synaptic regions, especially the presynaptic polymorph layer of the dentate gyrus, but was virtually absent over cell body layers. In contrast, [3H]PN200-110 binding was more homogenously distributed, with highest binding in the molecular layer of the dentate gyrus. The binding of [125I]omega-conotoxin GVIA was not different from sham controls at any time point following cardiac arrest. [3H]PN200-110 binding was decreased in each region immediately following ischemia, recovering within 30 min of recirculation. These findings are in contrast to earlier findings of rapid increases in L-type VDCC binding to membrane fractions obtained from cortex and striatum in this model, and suggest that the previously detected increases may be due to a redistribution of channels from subcellular compartments to the plasma membrane during ischemia.

Effectiveness of transnasal butorphanol for the treatment of musculoskeletal pain.

A prospective, open-label study of the effectiveness of transnasal butorphanol in the treatment of pain resulting from musculoskeletal injuries. Twenty-eight patients with strains (n = 20), fractures (n = 6), contusions (n = 1), and stab wounds (n = 1) were included. All patients were examined by an attending level emergency medicine physician and deemed to have pain severe enough to warrant parenteral narcotic analgesia. All patients received an initial 1-mg dose of transnasal butorphanol. Subsequent dosing was flexible depending on response to the initial dose. All patients received pain relief from transnasal butorphanol, and only one requested alternative analgesic medication. Fifty-seven percent (n = 16) of patients noticed at least a little relief of pain within 5 minutes of administration and 93% (n = 26) received at least a little relief within 15 minutes. Seventy-one percent of the patients received a 50% reduction of pain within 60 minutes. No serious side effects were noted, but drowsiness occurred in 82% (n = 23) and dizziness in 54% (n = 15) of the patients. One patient discontinued participation in the study because of nausea. In this limited trial transnasal butorphanol proved to be a rapidly effective opioid analgesic. Further controlled studies comparing transnasal butorphanol with standard parenteral narcotics are needed.

Increased activation of L-type voltage-dependent calcium channels is associated with glycine enhancement of N-methyl-D-aspartate-stimulated dopamine release in global cerebral ischemia/reperfusion.

We investigated the relationships among N-methyl-D-aspartate, glycine, L-type voltage-dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200-110 ([3H]isradipine) to L-type voltage-dependent calcium channels, that open as a consequence of N-methyl-D-aspartate-induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage-sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N-methyl-D-aspartate-stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N-methyl-D-aspartate-stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N-methyl-D-aspartate-stimulated release in all treatment groups. The enhancement of N-methyl-D-aspartate-stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30-min reperfused animals. These data suggest that glycine may facilitate opening of the voltage-dependent calcium channels activated by N-methyl-D-aspartate and that this facilitation is blocked by the antagonist nitrendipine.

Postischemic inhibition of cerebral cortex pyruvate dehydrogenase.

Postischemic, mitochondrial respiratory impairment can contribute to prolonged intracellular lactic acidosis, secondary tissue deenergization, and neuronal cell death. Specifically, reperfusion-dependent inhibition of pyruvate dehydrogenase (PDH) may determine the degree to which glucose is metabolized aerobically vs. anaerobically. In this study, the maximal activities of pyruvate and lactate dehydrogenase (LDH) from homogenates of canine frontal cortex were measured following 10 min of cardiac arrest and systemic reperfusion from 30 min to 24 h. Although no change in PDH activity occurred following ischemia alone, a 72% reduction in activity was observed following only 30 min of reperfusion and a 65% inhibition persisted following 24 h of reperfusion. In contrast, no significant alteration in LDH activity was observed in any experimental group relative to nonarrested control animals. A trend toward reversal of PDH inhibition was observed in tissue from animals treated following ischemia with acetyl-L-carnitine, a drug previously reported to inhibit brain protein oxidation, and lower postischemic cortical lactate levels and improve neurological outcome. In vitro experiments indicate that PDH is more sensitive than LDH to enzyme inactivation by oxygen dependent free radical-mediated protein oxidation. This form of inhibition is potentiated by either elevated Ca2+ concentrations or substrate/cofactor depletion. These results suggest that site-specific protein oxidation may be involved in reperfusion-dependent inhibition of brain PDH activity.

Inhibition of postcardiac arrest brain protein oxidation by acetyl-L-carnitine.

Free radical mediated, site-specific protein oxidation has been implicated in the pathophysiology of ischemia/reperfusion brain injury. The purpose of this study was to determine whether this form of molecular damage could be detected in a clinically relevant model employing 10-min cardiac arrest in dogs followed by restoration of spontaneous circulation for up to 24 h. The effects of postischemic acetyl-L-carnitine administration on protein oxidation were also tested due to its previously reported improvement of brain energy metabolism and neurological outcome in this model. Following the experimental period, soluble proteins were extracted from a sample of frontal cortex and reacted with dinitrophenylhydrazine for spectrophotometric measurement of protein carbonyl groups. The most important results of this study were that brain protein carbonyl groups were significantly elevated following 2 and 24 h of reperfusion compared to nonischemic controls, and that postischemic IV administration of acetyl-L-carnitine eliminated the increase in carbonyl groups observed at the 24-h period. These results indicate that brain protein oxidation does occur in a clinically relevant model of complete global cerebral ischemia and reperfusion, and that oxidation is inhibited under treatment conditions that improve neurological outcome.