796 SECTION VI Pediatric Critical Care Neurologic protectneuronsbyabsorbingextracellularpotassium Capillary(blood brainbarrier[BBB])leakageresults in interstitial (vasogenic)edema Incr[.]
796 S E C T I O N V I Pediatric Critical Care: Neurologic protect neurons by absorbing extracellular potassium Capillary (blood-brain barrier [BBB]) leakage results in interstitial (vasogenic) edema Increased concentrations of at least four oxyradical species that break down membranes and proteins, worsen the microcirculation, and possibly also damage the nucleus may 111 be formed: Superoxide anion (O ˙2 nFe11, ) leading to hydroxyl radical (•OH) (via the iron-catalyzed Fe Haber-Weiss/Fenton reaction); free lipid radicals (FLR) and peroxynitrite (OONO2-) O ˙2 may be formed from several sources: (1) directly from eicosanoid metabolism; (2) by the previously described XO system; (3) via quinone-mediated reactions within and outside the electron transport chain (from mitochondria [M]); and (4) by activation of NADPH-oxidase in accumulated neutrophils in the microvasculature or after diapedesis into tissue Increased O ˙2 leads to increased hydrogen peroxide (H2O2) production as a result of intracellular action of superoxide dismutase (SOD) [H2O2] is controlled by intracellular catalase Increased O ˙2 further leads to increased •OH, because of conversion of H2O2 to •OH via the Haber-Weiss/Fenton reaction, with iron liberated from mitochondria This reaction is promoted by acidosis •OH and OONO2 damage cellular lipids, proteins, and nucleic acids Also, AA is metabolized by the cyclooxygenase pathways to prostaglandins (PGs), including thromboxane A2, or by the lipoxygenase pathway to produce leukotrienes (LTs), and by the cytochrome P-450 pathway These products can act as neurotransmitters and signal transducers in neuronal and glial cells and can activate thrombotic and inflammatory pathways in the microcirculation Inflammatory reactions after ischemia have been shown to occur in extracerebral organs, focal brain ischemia, or brain trauma To date, they have not been demonstrated after temporary complete global brain ischemia Neuronal injury can signal interleukin-1 and other cytokines to be produced and trigger endogenous activation of microglia, with additional injury (QA, quinolinic acid) In addition, tissue or endothelial injury—particularly associated with necrosis—can signal the endothelium to produce adhesion molecules (intercellular [ICAM], e-selectin [e-sel], p-selectin [p-sel]), cytokines, chemokines, and other mediators, triggering local involvement of systemic inflammatory cells in an interaction between blood and damaged tissue Reoxygenation restores [ATP] through oxidative phosphorylation, which may result in massive uptake of [Ca211]i into mitochondria, which are swollen from increased osmolality Thus, mitochondria loaded with bound [Ca211] may self-destruct by rupturing and releasing additional free radicals Increased [Ca211]i by itself and by triggering free radical reactions may result in lipid peroxidation, leaky membranes, and cell death Neuronal damage can be caused, in part, by increased EAA (excitotoxicity) During reperfusion, [Ca211]i and increased EAAs normalize Their contribution to ultimate death of neurons is more likely through the cascades that they have triggered during ischemia During ischemia and subsequent reperfusion, loading of cells and calcium maldistribution in cells are believed to be the key trigger common to the development of cell death This calcium loading signals a wide variety of pathologic processes Proteases, lipases, and nucleases are activated, which may contribute to activation of genes or gene products (i.e., caspases [Casp] or P53) critical to the development of programmed cell death (PCD, i.e., apoptosis, autophagy, or regulated necrosis), or inactivation of genes or gene products normally inhibiting this process Activation of nNOS by calcium can lead to production of nitric oxide (NO), which can combine with superoxide to generate peroxynitrite (OONO2) OONO2 and •OH both can lead to DNA injury and PCD or protein and membrane peroxidation and necrosis, respectively Nerve growth factor (NGF) nuclear immediate early response genes (IERG) such as heat shock protein, free radical scavengers (FRSs), adenosine, and other endogenous defenses (ED) may modulate the damage (Courtesy P Safar, MD, and P Kochanek, MD, with input from N Bircher, MD, and J Severinghaus, MD.) Reperfusion Injury Clinical Pathophysiology Reoxygenation and reperfusion are essential to recovery of any organ after ischemia Experimental evidence suggests, however, that certain aspects of reperfusion result in tissue injury.87,88 Reperfusion injury is a complex series of interactions between parenchyma and microcirculatory elements resulting in detrimental effects that negate some fraction of the benefits of reperfusion The magnitude of reperfusion injury varies with the organ in question; the duration and type of hypoxic-ischemic insult; and the timing, duration, and magnitude of reperfusion.89,90 In the case of cardiac arrest, ischemia and reperfusion are global events In the brain, early reperfusion (5–15 minutes) after asphyxia results in significant hyperemia.91,92 In many organs and in the brain after focal insults, progressive microcirculatory failure is thought to be an important aspect of reperfusion injury.93 Four key mechanisms hypothesized to be important to reperfusion injury in the brain are (1) excitotoxicity and calcium accumulation, (2) protease activation, (3) oxygen radical formation, and (4) membrane phospholipid hydrolysis and mediator formation See ExpertConsult.com for additional details Cerebral Blood Flow and Metabolism After Resuscitation Detailed regional and temporal CBF patterns in the early postresuscitation period have been ascertained from animal models, as patients’ clinical instability does not allow early and serial CBF assessment with current state-of-the-art techniques such as arterial spin label magnetic resonance imaging (ASL-MRI) The pioneering studies in which global CBF was measured in animal models of global ischemia164 showed that after 15 minutes of global brain ischemia in dogs, CBF transiently increased to levels well above baseline for 15 minutes, followed by progressive reduction to a level below normal for the remainder of the monitoring period (90 minutes) This pattern of early transient postischemic hyperemia and subsequent delayed postischemic hypoperfusion has been observed in many global cerebral ischemia models, including both VF and asphyxial arrest.92,165–167 The levels of hyperemia and subsequent hypoperfusion vary in relation to the duration of the insult.168 Although these phases of increased and decreased CBF characterize the net global effect, regional CBF is e796.e1 Neuronal Death Pathophysiology Necrosis, which is characterized by denaturing and coagulation of cellular proteins, is the basic pattern of pathologic cell death that results from a progressive reduction in the cellular content of ATP.57 Necrosis involves progressive derangements in energy and substrate metabolism that are followed by a series of morphologic alterations, including swelling of cells and organelles, subsurface cellular blebbing, amorphous deposits in mitochondria, condensation of nuclear chromatin, and, finally, breaks in plasma and organellar membranes.57 Although necrotic cell death was traditionally felt to be entirely irreversible, studies showing that some degree of necrotic cell death responds to treatment after hypoxiaischemia implicate regulated or programmed necrosis, also termed necroptosis.58,59 Cell death after hypoxic-ischemic insults can also occur by apoptosis.60 Development of apoptosis usually requires new protein synthesis and the activation of endonucleases Two distinct types of characteristic cleavage of deoxyribonucleic acid (DNA) have been described The most well-described, caspase-dependent apoptosis, involves cleavage by caspase-activated deoxyribonuclease at linkage regions between nucleosomes to form fragments of double-stranded DNA.61 This produces a pattern of DNA cleavage observed on Southern blot analysis, termed DNA laddering In contrast, caspase-independent apoptosis results in large-scale DNA fragmentation induced by the mitochondrial flavoprotein apoptosis-inducing factor (AIF).62,63 Selective vulnerable cell death in brain regions such as the CA1 region of the hippocampus after transient global brain ischemia appears to occur by apoptosis.64 Thalamic-delayed neuronal death was caused by Fas-mediated apoptosis in a model of neonatal hypoxia-ischemia65 and durable electrophysiologic disturbances are observed in thalami after asphyxia cardiac arrest in juvenile rats.66 Li and colleagues67 reported that apoptosis in the postischemic brain is not limited to scattered neuronal death in what have been traditionally deemed to be selectively vulnerable regions, but it is seen even in penumbral regions around evolving cerebral infarcts (see eFig 65.1) Finally, the proportion of apoptosis in the developing brain after ischemia appears to be sex dependent, as females but not males respond to antiapoptotic agents after neonatal hypoxia-ischemia.68 Cultures of embryonic neurons from male versus female rats exhibit differential vulnerability to various stresses,69 underscoring the concept of sexual dimorphism in the development of brain injury though observational studies fail to identify sex as a predictor of outcome following pediatric cardiac arrest.70 Autophagy is a homeostatic process that recycles cell resources during periods of nutrient stress.71 Autophagy is upregulated after experimental brain ischemia,72,73 which can be considered profound nutrient stress.74 Studies show that blocking autophagy after hypoxia-ischemia can be protective or detrimental.54,75,76 As such, autophagy’s role after acute brain injury is controversial, and it may depend on the stage of brain development and sex of the patient, animal, or cell.74,77 Mouse pups lacking Atg7 (a necessary component for autophagy) or rat pups treated with an inhibitor of autophagy (3-methyladenine) are protected from focal hypoxia-ischemia.72,73 Knockdown of Atg7 using small interfering ribonucleic acid (siRNA) was shown to reduce autophagy and Purkinje neuron death in juvenile rats after asphyxia cardiac arrest, with the beneficial effects more prominent in female versus male rat pups.78 The proportion of neuronal death that occurs via apoptosis, necrosis, autophagy, or other pathways after cerebral ischemia remains undetermined.79,80 Although neurons may appear histologically normal in the days after reperfusion, electron microscopy reveals changes present within hours.80 Moreover, it remains possible that treatments inhibiting apoptosis, for example, may simply convert cell death to necrosis or autophagy, or another pathway.81,82 Although speculative, it is possible that after cardiac arrest and resuscitation, a continuum exists in neurons from recovery to necrosis83 that depends on the duration of the insult, the local milieu, and the given brain region.84–86 e796.e2 Anoxia, Ischemia, Reperfusion Pathophysiology Excitotoxicity and Calcium Accumulation Glutamate and aspartate are the major excitatory amino acid neurotransmitters in the mammalian central nervous system (CNS), but both also have neurotoxic properties Pioneering studies by Rothman94 demonstrated in vitro that hypoxia-induced neuronal death is mediated by synaptic activity Inhibition of synaptic glutamate release or blockade of glutamate receptors prevented hypoxia-induced neuronal injury Glutamate is the major neurotransmitter in the selectively vulnerable zones and accumulates extracellularly at supraphysiologic levels in these regions after hypoxic or ischemic insults.40 In other regions asphyxia induces significant increases in dopamine, serotonin, norepinephrine, and g-aminobutyric acid, which are larger than the increases in glutamate in relative, though not absolute, concentrations.23 Glutamate is released at the presynaptic terminal in response to neuronal stimulation and acts by binding to postsynaptic dendritic receptors Two main classes of excitatory neurotransmitter receptors have been identified One class consists of the ligandgated ion channels (“ionotropic” receptors) and includes N-methylD-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or quisqualate, and kainate receptor subtypes Toxicity caused by NMDA receptor (NMDAR) activation is usually rapid, whereas AMPA or kainate receptor–mediated cell death is somewhat slower to develop.95 Activation of synaptic NMDARs is neuroprotective whereas activation of extrasynaptic NMDARs by glutamate spillover in ischemia has the opposite effect The other class of excitatory neurotransmitter receptors includes the metabotropic receptors, which are coupled with G proteins and modulate intracellular second messengers such as calcium, cyclic nucleotides, and inositol triphosphate.96 When activated, the ionotropic glutamate receptors open sodium channels and may also initiate membrane depolarization and spreading depression.97 With ionotropic receptor activation, rapid excitatory amino acid–mediated calcium accumulation occurs In the face of ischemia, this calcium accumulation is exacerbated by cellular energy failure, which disables the sodium-potassium adenosine triphosphatase (Na1/K1-ATPase) membrane pump and results in further calcium accumulation.40 Calcium influx causes death of neurons in culture under anoxic conditions or in the presence of glutamate.40 The intracellular accumulation of calcium (1) activates proteases, lipases, and endonucleases, resulting in the breakdown of membrane phospholipids; (2) activates neuronal nitric oxide synthase (nNOS), resulting in nitric oxide (NO) production and, in the presence of superoxide, peroxynitrite formation; (3) damages mitochondria; (4) disrupts nucleic acid sequences; and (5) ultimately mediates cell death via necrosis, programmed necrosis, apoptosis, or autophagy (see Fig 65.2) The disturbance of the finely regulated intracellular calcium homeostasis is now recognized as a possible final common pathway of neuronal death.34,40,56,96 Studies suggest that approaches targeting calcium-calmodulin–dependent protein kinase II may have promise in protecting against neuronal death.98 Protease Activation Protease activation, resulting from increases in intracellular calcium, plays a central role in mediating both necrosis and apoptosis Both calpains and caspases are activated in the brain after cardiac arrest and contribute to injury.99 Calpains are calciumdependent cytosolic cysteine proteases with a homeostatic role in cell cycle regulation and signal transduction, which mediate proteolysis of cytoskeletal proteins and activation of protein kinase C and phospholipases, resulting in necrosis.100 Calpains also proteolyze the sodium/calcium exchanger in neurons during excitotoxicity, creating a positive feedback loop that worsens extrasynaptic NMDA excitotoxicity.101 The caspase family of cysteine proteases may have a more prominent role in the developing versus mature mammalian brain.102,103 After unilateral hypoxic-ischemic brain injury, neonatal rats had increased cytochrome C release and caspase-3 activation versus juvenile and adult rats.77 Regulation of apoptotic machinery also appears to be sex dependent after neonatal hypoxic-ischemic brain injury.104 Comparatively, female rats had more caspase-mediated apoptosis, whereas male rats had more caspase-independent, AIF-mediated apoptosis Oxygen Radical Formation Toxic oxygen radical species produced during postischemic reperfusion have been implicated as important contributors to reperfusion injury and delayed cell death.88 The primary species of interest include superoxide anion, hydrogen peroxide, hydroxyl radical, and the reactive nitrogen species peroxynitrite Very high, pathologic levels of free radical generation occur in the brain early in reperfusion, with resolution within the first hour.105–107 A major source of reactive oxygen species upon reperfusion after cardiac arrest is the mitochondrion.105,108,109 Superoxide generation early in reperfusion is driven by reverse electron transport from complex II to complex I as accumulated succinate is metabolized.110 Mitochondrial oxidative stress after cardiac arrest and TBI results in oxidation of the unique mitochondrial lipid cardiolipin by cytochrome C in the inner mitochondrial membrane.111,112 Inhibition of cardiolipin oxidation using a mitochondrial-targeted nitroxide improved outcome in a preclinical model of pediatric asphyxial cardiac arrest.112 Other potential sources of reperfusion reactive oxygen species include (1) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms (Nox1, Nox2, and Nox4)113; (2) the cyclooxygenase, lipoxygenase, and cytochrome P-450 pathways, in which metabolism of arachidonic acid may produce superoxide anion as an enzymatic by-product88,114; (3) the xanthine oxidase (XO)115,116 pathway, though the importance of XO-mediated free radical generation in humans remains unclear117,118; (4) autoxidation of circulating catecholamines or of neurotransmitter catecholamines may represent another potential source of oxygen radicals; (5) delocalized iron, normally transported in the blood tightly bound to transferrin and stored inside the cell bound to ferritin119,120; and (6) NO increases via NMDAR stimulation and subsequent calcium-mediated activation of nNOS.121 NO in the presence of superoxide produces peroxynitrite.122 However, NO can also serve as a potent antioxidant,123 and beneficial effects of local release of NO and nitrosylation reactions may underlie promising effects of new therapies, such as nitrite therapy or remote ischemic postconditioning.124–126 Free radicals have also been associated directly with an increase release of excitatory amino acids and vice versa.127,128 The brain contains a high concentration of polyunsaturated fatty acids—especially arachidonic acid that, on exposure to oxygen radicals, results in autocatalytic lipid peroxidation.129 Cerebrospinal fluid has low concentrations of iron-binding proteins; therefore, iron released from injured neurons or glia is likely to contribute to these peroxidation reactions Lipid peroxides accumulate in the selectively vulnerable zones during reperfusion after transient forebrain ischemia.120,130 These peroxides only accumulate following reperfusion.131 e796.e3 Oxidative damage impacts brain proteins after reperfusion.131 Pyruvate dehydrogenase, a key mitochondrial matrix enzyme that converts pyruvate into acetyl-coenzyme A, undergoes oxidative protein modification after ischemia that impairs enzyme activity, possibly contributing to neuronal cell death132; oxidative damage to DNA could also play a role.133 Developmental and sex differences exist in terms of the degree of oxidative stress and amount and function of antioxidant enzymes after brain injury In mice, glutathione and catalase activity in the brain are higher in adult female versus male mice; discrepancies become more exaggerated with age.134 Furthermore, neurons from male rats have less capacity to replenish glutathione levels after cytotoxic stress in vitro and in vivo after asphyxial cardiac arrest.69 Membrane Phospholipid Hydrolysis and Mediator Formation Membrane phospholipids modulate signaling cascades, affecting the development, differentiation, function, and repair of the CNS, functions that become dysregulated with ischemia and oxidative stress.135 Free fatty acids (FFAs) are released from neuronal membranes during ischemia; the amount of FFA released is proportional to the duration of ischemia FFA release continues to change in proportion to duration of ischemia after the completion of energy failure.136 The FFAs released then have potential detrimental effects during the postischemic period by multiple mechanisms: (1) arachidonic acid metabolism via the cyclooxygenase pathway may contribute to oxygen radical production during reperfusion114; (2) FFAs and diacylglycerol directly increase membrane fluidity, inhibit ATPases, increase neurotransmitter release, and uncouple oxidative phosphorylation; (3) enzymatic oxidation of arachidonic acid during reperfusion by cyclooxygenase, lipoxygenase, or cytochrome P-450 produces a large number of bioactive lipids, including prostaglandins, thromboxanes, leukotrienes, and hydroxyl-fatty acids, many of which mediate detrimental effects (see Fig 65.2); and (4) release of lipid mediators from oxidized cardiolipin by phospholipase A2g Studies suggest that the cytochrome P-450 metabolite 20-hydroxyeicosatetraenoic acid may play a role in producing cortical vasoconstriction after cardiac arrest, and inhibitors of the responsible enzyme are being investigated as a possible therapy for cardiac arrest.137 This pathway also appears to play a role in the development of brain edema after cardiac arrest Endogenous Defenses Several endogenous neuroprotectants are produced, induced, or activated after ischemia, and their postulated or proven functions improve cell (specifically neuronal) survival in in vivo and in vitro models The heat shock proteins (HSPs) are induced after ischemia138 and TBI.139 Simon and colleagues138 showed that after global ischemia, the 72 kDa HSP72 is temporally expressed in a pattern that mirrors the pattern of selective vulnerability in the model, seen first in the CA1 region of the hippocampus, followed by CA3, cortex, and thalamus, and, finally, in the dentate granule cells HSP72 is also induced in both gray and white matter of piglets following mild and severe hypoxia.140 The HSPs have generated major interest as potential neuroprotectants because their prior induction by a sublethal stress can afford protection from subsequent injury Transient, subthreshold whole-body hyperthermia reduces subsequent ischemic brain injury in both adult141 and neonatal rats.142 Furthermore, exogenous HSP72 reduces glutamate toxicity in neuronal cell cultures.143 Importantly, overexpression of HSP72 reduces ischemic damage and apoptosis after experimental stroke and global ischemia in vivo.144–146 Another potential mechanism for endogenous neuroprotection is the upregulation of genes that inhibit apoptosis and augment neurogenesis The mammalian gene Bcl-2, a proto-oncogene, can block apoptosis147 and perhaps necrosis as well.148 The Bcl-2 gene is expressed in neurons surviving both focal and global ischemia149,150 and is reduced in degenerating neurons after cardiac arrest in rats.151 Viral transfection of Bcl-2 reduces infarction after focal ischemia,152 and upregulation of Bcl-2 via ceramide administered 30 minutes after hypoxia-ischemia reduced the number of cells with DNA damage in the immature rat brain.153 Forced overexpression of the BCL-2 family member BCL-XL also reduces tissue damage after focal cerebral ischemia in adult rats.154 After TBI in infants and children, cerebrospinal fluid levels of BCL-2 are increased in patients who survive compared with those who die.155 Finally, BCL-2 overexpression promotes neurogenesis in adult mice with and without ischemic injury.156 Adenosine is an endogenous biochemical mediator that may serve a protective role after cerebral ischemia, particularly early after injury It may be produced from ATP breakdown or via the more recently discovered 2,3 cyclic adenosine monophosphate adenosine pathway that has been shown to exist in the brain.157 Adenosine is increased in brain tissue after experimental ischemia158 and in response to hypoxia,159 hypotension,160 and hypoglycemia.161 When bound to A2 receptors, adenosine is a potent cerebrovasodilator and inhibits platelet activation and neutrophil function.162 Bound to A1 receptors, adenosine reduces neuronal metabolism and excitatory amino acid release and stabilizes postsynaptic membranes.162 Thus, the beneficial effects of adenosine after cerebral ischemia include improved regional CBF, reduced local oxygen demand, attenuation of both excitotoxicity and calcium accumulation, and antiinflammatory and rheologic effects Finally, adenosine agonists have been shown to improve survival of selectively vulnerable neurons after ischemia in many studies (reviewed in Rudolphi and coworkers163) CHAPTER 65 Hypoxic-Ischemic Encephalopathy often heterogeneous, particularly during postischemic hypoperfusion, when areas of decreased and increased perfusion may coexist.107,165,169 The ability of antioxidants to blunt reperfusion hyperemia suggests it may be the result of oxidative signaling involving the neurovascular bundle The heterogeneous- and duration-dependent nature of postarrest CBF was characterized using contemporary imaging techniques allowing for regional assessment and a clinically relevant model of pediatric asphyxial cardiac arrest.170 Using ASL-MRI, CBF was measured for the first hours after 8.5, 9, or 12 minutes of asphyxial cardiac arrest in postnatal day 17 rats—approximating a 1- to 4-year-old child in terms of brain development (see Chapter 58) Although the pattern of early global hyperemia followed by hypoperfusion similar to that observed after global ischemia164 was seen after asphyxial arrests lasting 8.5 and minutes, a pattern of global and persistent hypoperfusion was observed as the arrest duration increased to 12 minutes (Fig 65.3) Remarkably, CBF disturbances were also found to be region dependent after asphyxial arrests lasting 8.5 and minutes, with subcortical hyperemia but cortical hypoperfusion (see Fig 65.3) After a 12-minute asphyxial arrest, hyperemia was absent and only hypoperfusion was observed in both cortical and subcortical regions CBF was pressure-passive with epinephrine infusion, perhaps indicating loss of autoregulation after a prolonged arrest This is consistent with descriptions of increased loss of neurovascular coupling with longer global ischemic durations.171 CBF disturbances after reperfusion also depend on the type of cardiac arrest, asphyxial versus VF For the same duration of cardiac arrest, asphyxial cardiac arrest produced marked early hyperemia in both cortex and thalamus, whereas VF cardiac arrest produced modest early hyperemia only in the cortex.92 Thirty minutes postresuscitation, both asphyxial and VF cardiac arrests displayed hypoperfusion, more pronounced in the hippocampus.92 Invasive measurement of brain tissue oxygenation (PbtO2) is a marker of local oxygen extraction fraction and, when coupled to assessment of CBF, is a surrogate marker of cerebral metabolism In the rat pediatric asphyxial cardiac arrest model, postresuscitation cortical PbtO2 values decreased below baseline by 30 minutes and remained low at hours In contrast, significant hyperoxia was observed in the thalamus minutes after ROSC, decreasing to normal values over hours.12 Notably, PbtO2 was a fraction of inspired oxygen (Fio2) dose responsive in both brain regions This suggests that early cortical hypoperfusion after an asphyxial arrest does not represent a coupled blood flow response to reduced Arrest duration (min) 8.5 12 Time after arrest Subcortical hyperemia 15 1h Cortical hypoperfusion 2h • Fig 65.3 Duration 797 and regional dependency of cerebral blood flow (CBF) disturbances acutely after asphyxial cardiac arrest in postnatal day 17 rats CBF data demonstrate that early postresuscitation hyperemia occurs in subcortical regions after an 8.5- and 9-minute but not a 12-minute asphyxial cardiac arrest and that duration-dependent hypoperfusion occurs in cortical regions (Modified from Manole MD, Foley LM, Hitchens TK, et al Magnetic resonance imaging assessment of regional cerebral blood flow after asphyxial cardiac arrest in immature rats J Cereb Blood Flow Metab 2009;29:197–205.) ... deoxyribonuclease at linkage regions between nucleosomes to form fragments of double-stranded DNA.61 This produces a pattern of DNA cleavage observed on Southern blot analysis, termed DNA laddering... histologically normal in the days after reperfusion, electron microscopy reveals changes present within hours.80 Moreover, it remains possible that treatments inhibiting apoptosis, for example, may... activation, rapid excitatory amino acid–mediated calcium accumulation occurs In the face of ischemia, this calcium accumulation is exacerbated by cellular energy failure, which disables the sodium-potassium