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.

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.

The physiological conditions that swell mammalian neurons are clinically important but contentious. Distinguishing the neuronal component of brain swelling requires viewing intact neuronal cell bodies, dendrites, and axons and measuring their changing volume in real time. Cultured or dissociated neuronal somata swell within minutes under acutely overhydrated conditions and shrink when strongly dehydrated. But paradoxically, most central nervous system (CNS) neurons do not express aquaporins, the membrane channels that conduct osmotically driven water. Using 2-photon laser scanning microscopy (2PLSM), we monitored neuronal volume under osmotic stress in real time. Specifically, the volume of pyramidal neurons in cerebral cortex and axon terminals comprising cerebellar mossy fibers was measured deep within live brain slices. The expected swelling or shrinking of the gray matter was confirmed by recording altered light transmittance and by indirectly measuring extracellular resistance over a wide osmotic range of -80 to +80 milliOsmoles (mOsm). Neurons expressing green fluorescent protein were then imaged with 2PLSM between -40 and +80 mOsm over 20 min. Surprisingly, pyramidal somata, dendrites, and spines steadfastly maintained their volume, as did the cerebellar axon terminals. This precluded a need for the neurons to acutely regulate volume, preserved their intrinsic electrophysiological stability, and confirmed that these CNS nerve cells lack functional aquaporins. Thus, whereas water easily permeates the aquaporin-rich endothelia and glia driving osmotic brain swelling, neurons tenatiously maintain their volume. However, these same neurons then swell dramatically upon oxygen/glucose deprivation or [K+]0 elevation, so prolonged depolarization (as during stroke or seizure) apparently swells neurons by opening nonaquaporin channels to water.

The brain swells over the several minutes that follow stroke onset or acute hypo-osmotic stress because cells take up water. Measuring the volume responses of single neurons and glia has necessarily been confined to isolated or cultured cells. Two-photon laser scanning microscopy enables real-time visualization of cells functioning deep within living neocortex in vivo or in brain slices under physiologically relevant osmotic and ischemic stress. Astrocytes and their processes expressing green fluorescent protein in murine cortical slices swelled in response to 20 min of overhydration (240 mOsm) and shrank during dehydration (140 or 180 mOsm) at 32-34°C. Minute-by-minute monitoring revealed no detectable volume regulation during these osmotic challenges, particularly during the first 5 min. Astrocytes also rapidly swelled in response to elevated [K 1 ] o for 3 min or oxygen/glucose deprivation (OGD) for 10 min. Post-OGD, astroglial volume recovered quickly when slices were re-supplied with oxygen and glucose, while neurons remained swollen with beaded dendrites. In vivo, rapid astroglial swelling was confirmed within 6 min following intraperitoneal water injection or during the 6-12 min following cardiac arrest. While the astrocytic processes were clearly swollen, the extent of the astroglial arbor remained unchanged. Thus, in contrast to osmo-resistant pyramidal neurons that lack known aquaporins, astrocytes passively respond to acute osmotic stress, reflecting functional aquaporins in their plasma membrane. Unlike neurons, astrocytes better recover from brief ischemic insult in cortical slices, probably because their aquaporins facilitate water efflux. V V C

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Intracellular recordings from mammalian neuroendocrine cells showed that steady, injected currents can modify and block periodic spike bursts previously associated with increased neurohormone release. Spike afterpotentials could sum to form plateau potentials, which generated bursts and did not depend on axonal conduction or chemical synapses. Therefore, bursting involves a spike-dependent, positive-feedback mechanism endogenous to single neuroendocrine cells.

An important but poorly understood event associated with ischemia is anoxic depolarization (AD), a sudden and profound depolarization of neurons and glia in cortical and subcortical gray matter. Leao first measured the AD as a wave of electrical silence moving across the cerebral cortex in 1947 and noted its similarity to spreading depression (SD). SD is harmless when coursing through normoxic cortical tissue as during migraine aura. However for 3-4 h following focal ischemia, the additional metabolic stress arising from recurring SD in the penumbra expands the ischemic core, so SD blockade is potentially beneficial therapeutically. In the present study, we measured intrinsic optical signals (IOSs) to monitor anoxic depolarization in submerged rat neocortical slices during O2/glucose deprivation (OGD). After approximately 6 min of OGD, the AD was imaged as a focal increase in light transmittance which then propagated across neocortical gray at approximately 2 mm/min. Although the slice was globally stressed, the AD always initiated focally, sometimes at multiple sites. Its propagation was coincident with a transient negative shift in the extracellular potential, the electrical signature of AD. Acute damage to neocortex (measured as a delayed decrease in LT and as a loss of the evoked field potential) followed only where the AD had propagated, so it is the combined metabolic demands of AD and OGD that acutely damages all layers of the neocortex. Glutamate receptor antagonists (2 mM kynurenate or 25 microM AP-5/10 microM CNQX) did not block AD initiation, slow its propagation or prevent post-AD damage. This study shows that acute ischemic damage is greatly exacerberated by AD during metabolic stress and that glutamate receptor antagonists are not protective. Using this slice model, therapeutically tolerable drugs that block the AD and SD can be investigated.

Spreading depression (SD) was analyzed in hippocampal and neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or imaging of intracellular calcium dynamics. The goal was to determine the roles of intracellular calcium (Ca2+int) waves in the generation and propagation of SD. Imaging of intrinsic optical signals in the hippocampus showed that ouabain consistently induced SD, which characteristically started in the CA1 region, propagated at 15-35 micrometer/sec, and traversed across the hippocampal fissure to the dentate gyrus. In the dendritic regions of both CA1 and the dentate gyrus, SD caused a transient increase in light transmittance, characterized by both a rapid onset and a rapid recovery. In contrast, in the cell body regions the transmittance increase was prolonged. Simultaneous imaging of intracellular calcium and intrinsic optical signals revealed that a slow Ca2+int increase preceded any change in transmittance. Additionally, a wave of increased Ca2+int typically propagated many seconds ahead of the change in transmittance. These calcium increases were also observed in individual astrocytes injected with calcium orange, indicating that Ca2+int waves were normally associated with SD. However, when hippocampal slices were incubated in calcium-free/EGTA external solutions, SD was still observed, although Ca2+int waves were completely abolished. Under these conditions SD had a comparable peak increase in transmittance but a slower onset and a faster recovery. These results demonstrate that although there are calcium dynamics associated with SD, these increases are not necessary for the initiation or propagation of spreading depression.