Insulin induces dephosphorylation of eukaryotic initiation factor 2alpha and restores protein synthesis in vulnerable hippocampal neurons after transient brain ischemia.

Brain reperfusion causes prompt, severe, and prolonged protein synthesis suppression and increased phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha(P)] in hippocampal CA1 and hilar neurons. The authors hypothesized that eIF2alpha(P) dephosphorylation would lead to recovery of protein synthesis. Here the effects of insulin, which activates phosphatases, were examined by immunostaining for eIF2alpha(P) and autoradiography of in vivo 35S amino acid incorporation. Rats resuscitated from a 10-minute cardiac arrest were given 0, 2, 10 or 20 U/kg of intravenous insulin, underwent reperfusion for 90 minutes, and were perfusion fixed. Thirty minutes before perfusion fixation, control and resuscitated animals received 500 microCi/kg of 35S methionine/cysteine. Alternate 30-microm brain sections were autoradiographed or immunostained for eIF2alpha(P). Controls had abundant protein synthesis and no eIF2alpha(P) in hippocampal neurons. Untreated reperfused neurons in the CA1, hilus, and dentate gyrus had intense staining for eIF2alpha(P) and reduced protein synthesis; there was little improvement with treatment with 2 or 10 U/kg of insulin. However, with 20 U/kg of insulin, these neurons recovered protein synthesis and were free of eIF2alpha(P). These results show that the suppression of protein synthesis in the reperfused brain is reversible; they support a causal association between eIF2alpha(P) and inhibition of protein synthesis, and suggest a mechanism for the neuroprotective effects of insulin.

Calpain mediates eukaryotic initiation factor 4G degradation during global brain ischemia.

Global brain ischemia and reperfusion result in the degradation of the eukaryotic initiation factor (eIF) 4G, which plays a critical role in the attachment of the mRNA to the ribosome. Because eIF-4G is a substrate of calpain, these studies were undertaken to examine whether calpain I activation during global brain ischemia contributes to the degradation of eIF-4G in vivo. Immunoblots with antibodies against calpain I and eIF-4G were prepared from rat brain postmitochondrial supernatant incubated at 37 degrees C with and without the addition of calcium and the calpain inhibitors calpastatin or MDL-28,170. Addition of calcium alone resulted in calpain I activation (as measured by autolysis of the 80-kDa subunit) and degradation of eIF-4G; this effect was blocked by either 1 micromol/L calpastatin or 10 micromol/L MDL-28,170. In rabbits subjected to 20 minutes of cardiac arrest, immunoblots of brain postmitochondrial supernatants showed that the percentage of autolyzed calpain I increased from 1.9% +/- 1.1% to 15.8% +/- 5.0% and that this was accompanied by a 68% loss of eIF-4G. MDL-28,170 pretreatment (30 mg/kg) decreased ischemia-induced calpain I autolysis 40% and almost completely blocked eIF-4G degradation. We conclude that calpain I degrades eIF-4G during global brain ischemia.

Effect of brain ischemia and reperfusion on the localization of phosphorylated eukaryotic initiation factor 2 alpha.

Postischemic brain reperfusion is associated with a substantial and long-lasting reduction of protein synthesis in selectively vulnerable neurons. Because the overall translation initiation rate is typically regulated by altering the phosphorylation of serine 51 on the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha), we used an antibody specific to phosphorylated eIF-2 alpha [eIF-2(alpha P)] to study the regional and cellular distribution of eIF-2(alpha P) in normal, ischemic, and reperfused rat brains. Western blots of brain postmitochondrial supernatants revealed that approximately 1% of all eIF-2 alpha is phosphorylated in controls, eIF-2(alpha P) is not reduced by up to 30 minutes of ischemia, and eIF-2(alpha P) is increased approximately 20-fold after 10 and 90 minutes of reperfusion. Immunohistochemistry shows localization of eIF-2(alpha P) to astrocytes in normal brains, a massive increase in eIF-2(alpha P) in the cytoplasm of neurons within the first 10 minutes of reperfusion, accumulation of eIF-2(alpha P) in the nuclei of selectively vulnerable neurons after 1 hour of reperfusion, and morphology suggesting pyknosis or apoptosis in neuronal nuclei that continue to display eIF-2(alpha P) after 4 hours of reperfusion. These observations, together with the fact that eIF-2(alpha P) inhibits translation initiation, make a compelling case that eIF-2(alpha P) is responsible for reperfusion-induced inhibition of protein synthesis in vulnerable neurons.

Brain nuclear DNA survives cardiac arrest and reperfusion.

Iron-mediated peroxidation of brain lipids is known to occur during reperfusion following cardiac arrest. Since in vitro damage to DNA is caused by similar iron-dependent peroxidation, we tested whether free radical damage to genomic DNA also develops during reperfusion following cardiac arrest and resuscitation. Genomic DNA was isolated from the cerebral cortex in (i) normal dogs, (ii) dogs subjected to a 20-min cardiac arrest, and (iii) dogs resuscitated from a 20-min cardiac arrest and then allowed to reperfuse for 2 or 8 h. DNA strand nicks were evaluated by in vitro labeling of newly created 3' and 5' termini. DNA base damage was evaluated utilizing reaction with piperidine prior to labeling of 5' termini. The 3' DNA termini were labeled before and after digestion with exonuclease III, and the 5' DNA termini were labeled before and after treatment with piperidine. In vitro experiments with genomic DNA damaged by oxygen radicals verified that these labeling methods identified radical damage. In the experimental animal groups, terminal incorporation and electrophoretic mobility of brain nuclear DNA are not significantly changed either by 20 min of complete brain ischemia or during the first 8 h of reperfusion. We conclude that genomic DNA is not extensively damaged during cardiac arrest and early reperfusion, and therefore such DNA damage does not appear to be an important early aspect of the neurologic injury that accompanies cardiac arrest and resuscitation.

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

Thymine glycols and pyrimidine dimers in brain DNA during post-ischemic reperfusion.

Free-radical reactions, known to occur in the reperfused brain, damage DNA in vitro. We therefore examined the hypothesis that thymine glycols and thymine dimers, which are known to block transcription and are formed by free radical mechanisms, are formed in brain DNA during reoxygenation following ischemia. Such biochemical lesions could account for the failure of protein synthesis that occurs following an ischemic insult. Large dogs were anesthetized, instrumented, and divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without resuscitation; (3) 20-min cardiac arrest, resuscitation and 2 h reperfusion; and (4) 20-min cardiac arrest, resuscitation and 8 h reperfusion. Genomic DNA was isolated from the cerebral cortex. Thymine glycols were labeled by reduction with [3H]NaBH4. Pyrimidine dimers were determined by ELISA using antibody prepared against ultraviolet irradiated DNA. The data was evaluated by Kruskal-Wallis ANOVA with alpha = 0.05. The rabbit antibodies detected the thymine dimer content in 10 pg UV irradiated DNA but did not react with normal DNA. Borohydride labeling qualitatively detected thymine glycols generated by treatment of DNA with osmium tetroxide. There was no difference between the DNAs from the experimental groups in the content of thymine glycols or pyrimidine dimers (P = 0.608 and P = 0.219, respectively). We conclude that significant quantities of thymine glycols and thymine dimers are not formed in brain DNA during post-ischemic reperfusion. Therefore, the inhibition of brain protein synthesis during reperfusion, observed by other investigators, is unlikely to be caused by interruption of transcription by these species.

The effect of EMHP on post-cardiac arrest survival of rats.

1-Ethyl-2-methyl-3-hydroxy-pyrid-4-one (EMHP), a low molecular weight iron chelator that is soluble in hydrocarbon solvents and presumably in lipids, was studied for in vitro inhibition of radical-mediated peroxidation of DNA. We also investigated the acute toxicity of EMHP by administering 40, 100, and 300 mg/kg intravenously to Wistar rats, and we then examined the in vivo effect of the 40 mg/kg dose following a 10-min cardiac arrest and resuscitation in rats. EMHP prevented iron-dependent radical-mediated DNA breaks of the supercoiled plasmid Bluescribe by the Fenton reagent (400 microM iron, 30 microM H2O2) when present at EMHP/Fe ratios of 16:1 and 32:1. The 300-mg/kg dose was lethal in 3 of 5 normal rats, and the 100-mg/kg dose was associated with excessive mortality post-resuscitation. The 40-mg/kg dose was well tolerated post-resuscitation, but it did not improve either 3-day survival or neurologic outcome.

Assessment of free radical-induced damage in brain proteins after ischemia and reperfusion.

Brain damage initiated during global ischemia has been shown to be exacerbated by iron-dependent lipid peroxidation during early reperfusion. We hypothesized that other cellular components might be involved in similar free radical reactions. In this study we examined three brain protein fractions and ribosomal RNA for evidence of free radical damage during post-ischemic reperfusion. Global brain ischemia was induced by 20-min cardiac arrest. Dogs were divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without reperfusion; (3) 20-min cardiac arrest and 2 h reperfusion; (4) 20-min cardiac arrest and 8 h reperfusion. Soluble proteins and proteins from ribosomes and synaptosomes were assayed by a dinitrophenylhydrazine method for carbonyl groups, which are characteristic products of protein peroxidation. The ribosomal RNA was also examined by electrophoresis. When proteins from each fraction were peroxidized in vitro by Fenton reagents, carbonyl content increased as [Fe2+] was increased from 0 to 100 microM. However, following reperfusion there was no significant accumulation of carbonyl content in either the soluble (ANOVA P = 0.92) or ribosome (P = 0.10) protein fractions. There was a significant decrease in the carbonyl content of the synaptosome protein fraction after 8 h of reperfusion (P = 0.03). Similarly, although ribosomal RNA fragmentation was observed in ethidium stained agarose gels following in vitro reaction with Fenton reagents, there was no evidence of ribosomal RNA fragmentation or cross-linking following reperfusion. These results suggest that reperfusion free radical reactions do not involve these cellular proteins or ribosomal RNA.

Studies of the protein synthesis system in the brain cortex during global ischemia and reperfusion.

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

A quantitative morphological assessment of the effect of lidoflazine and deferoxamine therapy on global brain ischaemia.

The effect of the combination of two drugs, i.e. lidoflazine (a calcium antagonist), and deferoxamine (an iron chelator) was evaluated following 15 min global brain ischaemia (GBI) and reperfusion in dogs in a randomized blind study. GBI was produced by complete cardiac arrest of 15 min duration. Histopathological analysis performed on in situ fixed brains 40 h post-resuscitation revealed diffuse microhaemorrhages in the control group. These were noted rarely in the treatment group, the mean value of foci of microhaemorrhages/20 low power fields (LPF) being 5.2 in the treatment group versus 28 in the control group (p less than 0.001). Diffuse coagulative necrosis of neurons (ischaemic cell change) in the cerebral cortex, especially lamina 3, hippocampus, striatum, brain stem and cerebellum was present in all cases. Quantitation of the degree of cellular damage obtained by counting the number of anoxic neurons (in consistent regions of the brain) with the use of an image analysis system, revealed no significant difference between the 2 groups. The mean percentages of the ischaemic neurons in the control group in the various areas studied were: parietal cortex, 22.25; hippocampus, 50.37 and cerebellum (Purkinje cells), 66.75; and in the treatment group 25.3, 55.04 and 70.6 respectively. Thus, the lidoflazine-deferoxamine regimen significantly reduced the incidence of microhaemorrhages in the brain, but it did not have any protective effect against anoxic neuronal injury 40 h post-ischaemia in this experimental model of GBI of 15 min duration.

PKR-like endoplasmic reticulum kinase (PERK) activation following brain ischemia is independent of unfolded nascent proteins.

Transient global brain ischemia results in an immediate inhibition of protein translation upon reperfusion. During early brain reperfusion protein synthesis is inhibited by alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) phosphorylation by the PKR-like endoplasmic reticulum kinase (PERK). Normally, PERK is held in an inactive, monomeric state by the binding of the endoplasmic reticulum (ER) chaperone GRP78 to the lumenal end of PERK. The prevailing view is that ER stress leads to the accumulation of unfolded proteins in the ER lumen. GRP78 dissociates from PERK to bind these accumulated unfolded proteins, leading to PERK activation, phosphorylation of eIF2alpha, and inhibition of translation. To determine if an increase in unfolded nascent proteins following transient brain ischemia contributes to PERK activation, protein synthesis was blocked by intracerebral injection of anisomycin prior to induction of ischemia. Anisomycin inhibited protein synthesis by over 99% and reduced newly synthesized proteins in the ER to approximately 20% of controls. With an ER nearly devoid of newly synthesized proteins, PERK was still activated and was able to phosphorylate eIF2alpha in CA1 neurons during reperfusion. These data strongly argue that PERK activation is independent of the large increase in unfolded nascent proteins within the ER following transient global brain ischemia.

Brown-Sequard syndrome following heroin injection.

Reported is a case of Brown-Sequard syndrome following attempted heroin injection into the right external jugular vein. A right-sided hemiparalysis with a contralateral sensory loss of touch, pain, proprioception, and temperature developed over several hours to the C3 dermatome level. A myelogram showed a vasculitis pattern in the lower cervical region. Treatment was with high-dose dexamethasone for ten days. After six weeks of inpatient physical therapy, only minimal motor and sensory return was seen. Although this syndrome is usually due to lateral hemisection of the spinal cord by a stab wound or a gunshot wound, in this case we believe it resulted from chemical transection due to the heroin or quinine diluent or both.

Brain injury and repair mechanisms: the potential for pharmacologic therapy in closed-head trauma.

Rotational acceleration from closed-head trauma produces shear-strain brain injury at the interface of gray and white matter. The initial injury is followed by progressive damage involving three key phenomena: progression of subtle focal axonal damage to axonal transection between six and 12 hours after injury, progressive development of tissue microhemorrhages between 12 and 96 hours after injury, and development of tissue and cerebral spinal fluid lactic acidosis that does not appear to be explained by trauma-induced tissue depolarization, activation of phospholipases and the release of free arachidonic acid, radical generation by metabolism of arachidonate, and lipid peroxidation with consequent membrane degradation and partial mitochondrial uncoupling. Because of terminal differentiation, neurons may have a limited membrane repair capability that might be stimulated by growth factors. Other potential therapeutic interventions include calmodulin inhibitors, iron chelators, and free radical scavengers.

Suppression of protein synthesis in the reperfused brain.

BACKGROUND: Brain ischemia and reperfusion produce profound protein synthesis alterations, the extent and persistence of which are dependent on the nature of the ischemia, the brain region, the cell layer within a region, and the particular proteins studied. After transient ischemia, most brain regions recover their protein synthesis capability; however, recovery in the selectively vulnerable areas is poor. It is unknown whether this phenomenon itself provokes or is a consequence of the process of neuronal death. SUMMARY OF REVIEW: Protein synthesis suppression during ischemia is due to energy depletion, but this is quickly reversed upon recirculation. Reperfusion does not appear to damage DNA or transcription mechanisms, although there are changes in the profile of transcripts being made. Similarly, purified ribosomes isolated from reperfused brains can make the normal repertoire of proteins and heat-shock proteins. However, during early reperfusion, newly synthesized messenger RNAs appear to accumulate in the nucleus; this alteration in RNA handling could reflect disruption at any of several steps, including posttranscriptional processing, nuclear pore transport, cytoskeletal binding, or formation of the translation initiation complex. Another mechanism that may be responsible for protein synthesis suppression during late reperfusion is progressive membrane destruction, with consequent shifts in the concentration of ions crucial for ribosomal function. CONCLUSIONS: Protein synthesis suppression after ischemia likely involves a progression of multiple mechanisms during reperfusion. Although the recent work reviewed here offers new insight into the potential mechanisms disrupting protein synthesis, detailed understanding will require further investigation.

Incidence of cocaine-associated rhabdomyolysis.

STUDY HYPOTHESIS: Rhabdomyolysis is a common complication of cocaine use, and muscle symptoms fail to predict its development. STUDY POPULATION: A prospective, convenience sample of patients presenting to the emergency department of a large inner-city hospital with complaints related to cocaine use were eligible for inclusion. Patients were excluded if they had other potential causes of elevated creatine kinase (CK) levels or rhabdomyolysis. A control group comprised patients who were not cocaine users and satisfied the exclusion criteria. Sixty-eight patients were studied. METHODS: Initial evaluation included determination of the presence of muscle pain or swelling and total CK levels. Patients with a CK level of more than 800 U/L had additional tests, including a urine myoglobin, urine drug screen, and serum phosphorus. Rhabdomyolysis was defined by a serum CK level of more than 1,000 U/L (more than fivefold that of normal). CK levels were compared by two-tailed Student's t test. Muscle symptoms were compared with the development of rhabdomyolysis by Fisher's exact test. RESULTS: The CK level in the cocaine group was 931 +/- 1,785 U/L (mean +/- 1 SD). The CK level in the control group was 242 +/- 168 U/L (P = .028). Of the cocaine users, 24% (eight of 34) had rhabdomyolysis; one developed multiorgan failure and died. No patient in the control group had a CK level of more than 1,000 U/L. Only one cocaine user who developed rhabdomyolysis had muscle symptoms. Three cocaine users had muscle symptoms but did not develop rhabdomyolysis. No patient in the control group had muscle symptoms or developed rhabdomyolysis. Muscle symptoms did not predict the CK level (P = .55). CONCLUSION: This study revealed that 24% of the cocaine users had rhabdomyolysis. Many of the cases of rhabdomyolysis were not predictable from history or physical examination, making laboratory evaluation essential.

Nuclear-envelope nucleoside triphosphatase kinetics and mRNA transport following brain ischemia and reperfusion.

STUDY HYPOTHESIS: We attempted to determine whether the reduced egress of mRNA from brain nuclei following in vivo ischemia and reperfusion is caused by direct damage to the nuclear pore-associated NTPase that impairs the system for nuclear export of polyadenylated, or poly(A)+, mRNA. DESIGN: Prospective animal study. INTERVENTIONS: NTPase activity and poly(A)+ mRNA transport were studied in nuclear envelope vesicles (NEVs) prepared from canine parietal cortex isolated after 20 minutes of ischemia or 20 minutes of ischemia and 2 or 6 hours of reperfusion. RESULTS: Brain NEV NTPase Michaelis-Menten constant (Km) and maximum uptake velocity (Vmax) and the ATP-stimulated poly(A)+ mRNA egress rates were not significantly affected by ischemia and reperfusion. In vitro exposure of the NEVs to the OH. radical-generating system completely abolished NTPase activity. CONCLUSION: We conclude that brain ischemia and reperfusion do not induce direct inhibition of nucleocytoplasmic transport of poly(A)+ mRNA. This suggests that the nuclear membrane is not exposed to significant concentrations of OH. radical during reperfusion.

Global brain ischemia and reperfusion: modifications in eukaryotic initiation factors associated with inhibition of translation initiation.

We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors elF-4E, elF-4G, and elF-2 alpha to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was approximately 0.4 fmol of leucine/min/microgram of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of elF-4E. However, we observed in all 90-min-reperfused samples elF-4G fragments that also bound elF-4E. The amount of elF-2 alpha was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated elF-2 alpha in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated elF-2 alpha was uniformly present after 90 min of reperfusion and represented 24 +/- 3% of the elF-2 alpha in these samples. The serine phosphorylation of elF-2 alpha and partial fragmentation of elF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.

Postischemic tissue injury by iron-mediated free radical lipid peroxidation.

Cell damage initiated during ischemia matures during reperfusion. Mechanisms involved during reperfusion include the effects of arachidonic acid and its oxidative products prostaglandins and leukotrienes, reperfusion tissue calcium overloading, and damage to membranes by lipid peroxidation. Lipid peroxidation occurs by oxygen radical mechanisms that require a metal with more than one ionic state (transitional metal) for catalysis. We have shown that cellular iron is delocalized from the large molecules where it is normally stored to smaller chemical species during postischemic reperfusion. Postischemic lipid peroxidation is inhibited by the iron chelator deferoxamine. Intervention in the reperfusion injury of membranes by chelation of transitional metals is a new and promising therapeutic possibility for protection of the heart and brain.