C. William Shuttleworth

Zinc: new clues to diverse roles in brain ischemia

Cerebral ischemia is a leading cause of morbidity and mortality, reflecting the extraordinary sensitivity of the brain to a brief loss of blood flow. A significant goal has been to identify pathways of neuronal injury that are selectively activated after stroke and may be amenable to drug therapy. An important advance was made nearly 25 years ago when Ca(2+) overload was implicated as a critical link between glutamate excitotoxicity and ischemic neurodegeneration. However, early hope for effective therapies faded as glutamate-targeted trials repeatedly failed to demonstrate efficacy in humans. In a review in 2000 in this journal, we described new evidence linking a related cation, zinc (Zn(2+)), to neuronal injury, emphasizing sources and mechanisms of Zn(2+) toxicity. The current review highlights progress over the last decade, emphasizing mechanisms through which Zn(2+) ions (from multiple sources) participate together with Ca(2+) in different stages of cascades of ischemic injury.

NAD(P)H Fluorescence Transients after Synaptic Activity in Brain Slices: Predominant Role of Mitochondrial Function

Excitatory stimulation in hippocampal slices results in biphasic NAD(P)H fluorescence transients. Previous studies using differing stimulus protocols agreed that the oxidation phase is a consequence of mitochondrial metabolism, but the reduction phase has been attributed to (1) mitochondrial nicotinamide adenine dinucleotide (NADH) generation or (2) astrocytic glycolysis triggered by glutamate uptake. In an attempt to reconcile these two views, the present study examined NAD(P)H signals evoked by a wide range of stimulus durations (40 ms to 20secs). A combination of ionotropic glutamate receptor (iGluR) antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (APV)) virtually abolished responses to brief stimuli (40 to 200 ms, 50 Hz), but a significant fraction of the signal elicited by extended stimulation (20 secs, 32 Hz) was resistant to CNQX/APV. Glycolysis was inhibited by removal of glucose and addition of 2-deoxyglucose (2DG) (10 mmol/l) or iodoacetic acid (IAA, 1 mmol/l). Pyruvate was provided as an alternative substrate for oxidative phosphorylation and the A1 receptor antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) included to prevent decreases in synaptic efficacy. If sufficient pyruvate was supplied, responses to brief and extended stimuli were unaffected by glycolytic inhibition and not significantly reduced by an inhibitor of glucose uptake (3-O-methyl glucose, 3 mmol/l). When timed to arrive at the peak of overshoots generated by extended synaptic stimulation, brief pyruvate applications (10 mmol/l, 2mins) had little effect on evoked NAD(P)H increases. Flavoprotein autofluorescence transients after extended stimuli matched (with inverted sign) NAD(P)H responses. Responses to extended stimuli were not reduced by a nonselective inhibitor of glutamate uptake DL-Threo-β-benzyloxyaspartic acid (TBOA). These results suggest that NAD(P)H transients report mitochondrial dynamics, rather than recruitment of glycolytic metabolism, over a wide range of stimulus intensities.

Use of NAD(P)H and flavoprotein autofluorescence transients to probe neuron and astrocyte responses to synaptic activation.

Synaptic stimulation in brain slices is accompanied by changes in tissue autofluorescence, which are a consequence of changes in tissue metabolism. Autofluorescence excited by ultraviolet light has been most extensively studied, and is due to reduced pyridine nucleotides (NADH and NADPH, collectively termed NAD(P)H). Stimulation generates a characteristic compound NAD(P)H response, comprising an initial fluorescence decrease and then an overshooting increase that slowly recovers to baseline levels. Evoked NAD(P)H transients are relatively easy to record, do not require the addition of exogenous indicators and have good signal-noise ratios. These characteristics make NAD(P)H imaging methods very useful for tracking the spread of neuronal activity in complex brain tissues, however the cellular basis of synaptically-evoked autofluorescence transients has been the subject of recent debate. Of particular importance is the question of whether signals are due primarily to changes in neuronal mitochondrial function, and/or whether astrocyte metabolism triggered by glutamate uptake may be a significant contributor to the overshooting NAD(P)H fluorescence increases. This mini-review addresses the subcellular origins of NAD(P)H autofluorescence and the evidence for mitochondrial and glycolytic contributions to compound transients. It is concluded that there is no direct evidence for a contribution to NAD(P)H signals from glycolysis in astrocytes following synaptic glutamate uptake. In contrast, multiple lines of evidence, including from complimentary flavoprotein autofluorescence signals, imply that mitochondrial NADH dynamics in neurons dominate compound evoked NAD(P)H transients. These signals are thus appropriate for studies of mitochondrial function and dysfunction in brain slices, in addition to providing robust maps of postsynaptic neuronal activation following physiological activation.

The continuum of spreading depolarizations in acute cortical lesion development: Examining Leão's legacy.

A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leão’s historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum of spreading mass depolarizations, a concept that is central to understanding their pathologic effects. Within minutes of acute severe ischemia, the onset of persistent depolarization triggers the breakdown of ion homeostasis and development of cytotoxic edema. These persistent changes are diagnosed as diffusion restriction in magnetic resonance imaging and define the ischemic core. In delayed lesion growth, transient spreading depolarizations arise spontaneously in the ischemic penumbra and induce further persistent depolarization and excitotoxic damage, progressively expanding the ischemic core. The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage.

Direct Visualization of Trapped Erythrocytes in Rat Brain after Focal Ischemia and Reperfusion

Partial microcirculatory stasis after cerebral ischemia and reperfusion is a potential factor in delayed cell death. Sometimes described as the “no-reflow” phenomenon, limitations in current detection techniques have left the extent and spatial distribution of the phenomenon undetermined, which has led to some doubt as to its actual existence. The authors describe a new method, based on erythrocyte autofluorescence, that allows the erythrocytes trapped in the microvasculature, and thus blocking recirculation, to be directly visualized. Using this method, the authors have examined the spatial and temporal characteristics of this phenomenon in the rat intraluminal model of cerebral ischemia and reperfusion. Up to 15% of the capillaries in the ischemic penumbra remained occluded at least 2 hours after reperfusion. The amount of capillary bed showing trapped erythrocytes was more severe in the ischemic penumbra region than in the ischemic core. These results indicate that the no-reflow phenomenon may contribute to the developing damage in ischemic penumbra region, leading to additional injury after reperfusion.

Recording, analysis, and interpretation of spreading depolarizations in neurointensive care: Review and recommendations of the COSBID research group

Spreading depolarizations (SD) are waves of abrupt, near-complete breakdown of neuronal transmembrane ion gradients, are the largest possible pathophysiologic disruption of viable cerebral gray matter, and are a crucial mechanism of lesion development. Spreading depolarizations are increasingly recorded during multimodal neuromonitoring in neurocritical care as a causal biomarker providing a diagnostic summary measure of metabolic failure and excitotoxic injury. Focal ischemia causes spreading depolarization within minutes. Further spreading depolarizations arise for hours to days due to energy supply-demand mismatch in viable tissue. Spreading depolarizations exacerbate neuronal injury through prolonged ionic breakdown and spreading depolarization-related hypoperfusion (spreading ischemia). Local duration of the depolarization indicates local tissue energy status and risk of injury. Regional electrocorticographic monitoring affords even remote detection of injury because spreading depolarizations propagate widely from ischemic or metabolically stressed zones; characteristic patterns, including temporal clusters of spreading depolarizations and persistent depression of spontaneous cortical activity, can be recognized and quantified. Here, we describe the experimental basis for interpreting these patterns and illustrate their translation to human disease. We further provide consensus recommendations for electrocorticographic methods to record, classify, and score spreading depolarizations and associated spreading depressions. These methods offer distinct advantages over other neuromonitoring modalities and allow for future refinement through less invasive and more automated approaches.

Neural stem/progenitor cells display a low requirement for oxidative metabolism independent of hypoxia inducible factor‐1alpha expression

Neural stem/progenitor cells (NSPCs) are multipotent cells within the embryonic and adult brain that give rise to both neuronal and glial cell lineages. Maintenance of NSPC multipotency is promoted by low oxygen tension, although the metabolic underpinnings of this trait have not been described. In this study, we investigated the metabolic state of undifferentiated NSPCs in culture, and tested their relative reliance on oxidative versus glycolytic metabolism for survival, as well as their dependence on hypoxia inducible factor‐1alpha (HIF‐1α) expression for maintenance of metabolic phenotype. Unlike primary neurons, NSPCs from embryonic and adult mice survived prolonged hypoxia in culture. In addition, NSPCs displayed greater susceptibility to glycolytic inhibition compared with primary neurons, even in the presence of alternative mitochondrial TCA substrates. NSPCs were also more resistant than neurons to mitochondrial cyanide toxicity, less capable of utilizing galactose as an alternative substrate to glucose, and more susceptible to pharmacological inhibition of the pentose phosphate pathway by 6‐aminonicotinamide. Inducible deletion of exon 1 of the Hif1a gene improved the ability of NSPCs to utilize pyruvate during glycolytic inhibition, but did not alter other parameters of metabolism, including their ability to withstand prolonged hypoxia. Taken together, these data indicate that NSPCs have a relatively low requirement for oxidative metabolism for their survival and that hypoxic resistance is not dependent upon HIF‐1α signaling.

Sustained NMDA receptor activation by spreading depolarizations can initiate excitotoxic injury in metabolically compromised neurons

•  Spreading depolarization (SD) is a profound neuronal and glial depolarization implicated in the progression of acute brain injury. •  In metabolically compromised tissue, SD can trigger irrecoverable injury; however, the underlying cellular mechanisms are not well established. •  We investigated consequences of SD in hippocampal brain slices, using a combination of electrophysiological and single‐cell imaging methods. •  We characterized a brief period of prolonged NMDA receptor (NMDAR) activation after SD onset, which appeared to underlie extended Ca2+ elevations in dendrites. •  Prolonged NMDAR activation was sufficient to cause injury after SD in metabolically compromised neurons, and therefore may contribute to deleterious consequences of SD in pathological conditions.

Localized Loss of Ca2+ Homeostasis in Neuronal Dendrites Is a Downstream Consequence of Metabolic Compromise during Extended NMDA Exposures

Excessive Ca2+ loading is central to most hypotheses of excitotoxic neuronal damage. We examined dendritic Ca2+ signals in single CA1 neurons, injected with fluorescent indicators, after extended exposures to a low concentration of NMDA (5 μm). As shown previously, NMDA produces an initial transient Ca2+ elevation of several micromolar, followed by recovery to submicromolar levels. Then after a delay of ∼20–40 min, a large Ca2+ elevation appears in apical dendrites and propagates to the soma. We show here that this large delayed Ca2+ increase is required for ultimate loss of membrane integrity. However, transient removal of extracellular Ca2+ for varying epochs before and after NMDA exposure does not delay the propagation of these events. In contrast to compound Ca2+ elevations, intracellular Na+ elevations are monophasic and were promptly reversed by the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. MK-801 applied after the transient Ca2+ elevations blocked the delayed propagating Ca2+ increase. Even if applied after the propagating response was visualized, MK-801 restored resting Ca2+ levels. Propagating Ca2+ increases in dendrites were delayed or prevented by (1) reducing extracellular Na+, (2) injecting ATP together with the Ca2+ indicator, or (3) provision of exogenous pyruvate. These results show that extended NMDA exposure initiates degenerative signaling generally in apical dendrites. Although very high Ca2+ levels can report the progression of these responses, Ca2+ itself may not be required for the propagation of degenerative signaling along dendrites. In contrast, metabolic consequences of sustained Na+ elevations may lead to failure of ionic homeostasis in dendrites and precede Ca2+-dependent cellular compromise.

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD‐like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca2+ increases accompany all forms of SD, but persistently elevated Ca2+ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca2+ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca2+ influx via P/Q type channels is important for SD generated by localized application of high K+ solutions. In contrast, SD‐like events recorded in in vitro ischemia models are not usually prevented by Ca2+ removal, but under some conditions, Zn2+ influx via L‐type channels contributes to SD initiation. This review addresses different roles of Ca2+ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn2+ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.