R. David Andrew

Seizure and acute osmotic change: Clinical and neurophysiological aspects

There are a number of clinical situations where overhydration may occur. If the reduction in plasma osmolality is acute, passive water influx swells brain cells, shrinking the extracellular space around them. It is during this time that susceptibility to generalized tonic-clonic seizure dramatically increases. Common clinical examples include hastened rehydration therapy, the dialysis disequilibrium syndrome, compulsive polydipsia, the syndrome of inappropriate ADH secretion (SIADH) and post-TURP syndrome. Treatments that tend to restore normal cellular volume (dehydration, mannitol infusion) help protect against this form of seizure. Support for a correlation between plasma osmolality and seizure susceptibility is scattered amongst the literature of several medical disciplines and spans almost 70 years. However a cellular basis to explain how overhydration might promote epileptiform activity has been examined only recently. The neocortical and hippocampal brain slice preparations permit an examination of how acute osmotic change alters cortical excitability independent of vascular damage, brain compression or other factors secondary to brain swelling. Electrophysiological evidence indicates that hyposmolality promotes epileptiform activity by strengthening both excitatory synaptic communication in neocortex and field effects among the entire cortical population. Moreover there is little evidence that associated hyponatremia in itself leads to increased CNS excitability. Such findings help in understanding how rapid lowering of plasma osmolality in clinical situations can promote the hyperexcitability associated with generalized tonic-clonic seizure.

Real-time passive volume responses of astrocytes to acute osmotic and ischemic stress in cortical slices and in vivo revealed by two-photon microscopy.

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 (−40 mOsm) and shrank during dehydration (+40 or +80 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+]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 (Andrew et al., 2007 ) 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.

Seizure susceptibility and the osmotic state.

In some unknown manner, water uptake by brain cells (hyposmolality) promotes generalized seizure in humans and experimental animals, whereas cell dehydration (hyperosmolality) protects against it. We have replicated both scenarios in slices of hippocampus undergoing electrographic seizures. Surprisingly, a shift in osmolality does not change the excitability of individual neurons but rather, it alters the degree to which neurons interact. Hyposmolality enhances both excitatory synaptic transmission in neocortex and field (ephaptic) effects, the latter arising when cortical cells fire as a population. We propose that these increased excitatory interactions promote the synchrony that characterizes epileptiform activity.

Glutamate Does Not Mediate Acute Neuronal Damage after Spreading Depression Induced by O2/Glucose Deprivation in the Hippocampal Slice

This study argues that, in contrast to accepted excitotoxicity theory, O2/glucose deprivation damages neurons acutely by eliciting ischemic spreading depression (SD), a process not blocked by glutamate antagonists. In live rat hippocampal slices, the initiation, propagation, and resolution of SD can be imaged by monitoring wide-band changes in light transmittance (i.e., intrinsic optical signals). Oxygen/glucose deprivation for 10 minutes at 37.5°C evokes a propagating wave of elevated light transmittance across the slice, representing the SD front. Within minutes, CA1 neurons in regions undergoing SD display irreversible damage in the form of field potential inactivation, swollen cell bodies, and extensively beaded dendrites, the latter revealed by single-cell injection of lucifer yellow. Importantly, glutamate receptor antagonists do not block SD induced by O2/glucose deprivation, nor do they prevent the resultant dendritic beading of CA1 neurons. However, CA1 neurons are spared if SD is suppressed by reducing the temperature to 35°C during O2/glucose deprivation. This supports previous electrophysiologic evidence in vivo that SD during ischemia promotes acute neuronal damage and that glutamate antagonists are not protective of the metabolically stressed tissue. The authors propose that the inhibition of ischemic SD should be targeted as an important therapeutic strategy against stroke damage.

Evidence against Volume Regulation by Cortical Brain Cells during Acute Osmotic Stress

The cell bodies of neurons and glia examined in culture respond to severe osmotic stress (100 to 200 mOsm) by passive volume change that is followed within several minutes by volume regulation, even in the face of maintained osmotic change. However, in clinical situations, the brain does not experience such precipitous and severe changes in brain hydration. In this study we examined if there is evidence from the hippocampal slice preparation supporting the type of volume regulation observed in cultured brain cells. Within the CA1 region we imaged changes in light transmittance (LT), recorded the evoked field potential, and monitored tissue resistance (all measures of cell volume change) during the first hour of osmotic stress to search for evidence of volume regulation. During superfusion of hypo-osmotic aCSF (-40 mOsm), LT increased 24 to 28% in the dendritic regions of CA1 neurons. The LT reached a plateau which was maintained throughout a 45-min application interval, more than enough time to reveal a regulatory volume decrease. Upon return to control saline, LT immediately returned to baseline and settled there. Hypo-osmolality reversibly increased the relative tissue resistance (RREL) measured across the CA1 region with a time course identical to the increase in LT. Conversely, hyperosmotic aCSF (mannitol, +40 mOsm) decreased both RREL by 8% and LT by 15.5% with no indication of a regulatory volume increase. The CA1 cell body layer showed only slight hypo-osmotic swelling whereas exposure to the glutamate agonist quinolinic acid caused pronounced swelling in this region. Even when osmolality was decreased by 120 mOsm for 20 min, dendritic regions responded passively with no regulatory volume decrease. However, when aCSF Cl- was substituted, the CA1 dendritic regions displayed immediate swelling followed by a dramatic volume reduction under normosmotic conditions, indicating that such behavior can be evoked by extreme aCSF dilution. We conclude that in the brain slice preparation, the cortical cells do not exhibit classic volume regulation in response to sudden physiological changes in osmolality. Moreover it is the dendritic region, not the cell body region, that displays dynamic volume change during osmotic challenge.

Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation.

During ischaemia neurons depolarize and release the neurotransmitter l‐glutamate, which accumulates extracellularly and binds to postsynaptic receptors. This initiates a sequence of events thought to culminate in immediate and delayed neuronal death. However, there is growing evidence that during ischaemia the development of spreading depression (SD) can be an important determinant of the degree and extent of ischaemic damage. In contrast, SD without metabolic compromise (as occurs in migraine aura) causes no discernible damage to brain tissue. SD is a profound depolarization of neurons and glia that propagates like a wave across brain tissue. Brain cell swelling, an early event of both the excitotoxic process and of SD, can be assessed by imaging associated intrinsic optical signals (IOSs). We demonstrate here that IOS imaging clearly demarcates the ignition site and migration of SD across the submerged hippocampal slice of the rat. If SD is induced by elevating [K+]o, the tissue fully recovers, but in slices that are metabolically compromised at 37.5 °C by oxygen/glucose deprivation (OGD) or by ouabain exposure, cellular damage develops only where SD has propagated. Specifically, the evoked CA1 field potential is permanently lost, the cell bodies of involved neurons swell and their dendritic regions increase in opacity. In contrast to OGD, bath application of l‐glutamate (6–10 mm) at 37.5 °C evokes a non‐propagating LT increase in CA1 that reverses without obvious cellular damage. Moreover, application of 2–20 mm glutamate or various glutamate agonists fail to evoke SD in the submerged hippocampal slice. We propose that SD and OGD together (but not alone) constitute a ‘one‐two punch’, causing acute neuronal death in the slice that is not replicated by elevated glutamate. These findings support the proposal that SD generation during stroke promotes and extends acute ischaemic damage.

Interpretation of Intrinsic Optical Signals and Calcein Fluorescence during Acute Excitotoxic Insult in the Hippocampal Slice

Immediate (acute) neuronal damage in response to overstimulation of glutamate receptors results from toxic exposure to food poisons acting as glutamate analogues. Glutamate agonist application evokes dramatic intrinsic optical signals (IOSs) in the rat hippocampal slice preparation, particularly in the CA1 region. Theoretically IOSs are generated by alterations to neuronal and glial structure that change light transmittance (LT) in live brain tissue. To better understand such signals, IOSs evoked by the glutamate agonist N-methyl-D-aspartate were imaged in the rat hippocampal slice. We correlated these excitotoxic signals with: (1) biophysical principles governing light transport, (2) tissue volume changes as measured using a free intracellular fluorophore (calcein), (3) dendritic morphology visualized by dye injection, and (4) standard histopathology. In theory LT elevation evoked during acute excitotoxic swelling is generated by change to subcellular structure that reduces light scattering during cell swelling. However, in responsive dendritic regions, initial LT elevation caused by cell swelling was overridden by the formation of dendritic beads, a conformation that increased light scattering (thereby reducing LT) even as the calcein signal demonstrated that the tissue continued to swell. Thus IOS imaging reveals acute somatic and dendritic damage during excitotoxic stress that can be monitored across slices of brain tissue in real time.

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.

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.

Interferon-α inhibits long-term potentiation and unmasks a long-term depression in the rat hippocampus

Interferons (IFN) appear to have various neuromodulatory actions. Here, we characterized the actions of IFN-alpha on the electrophysiological properties of CA1 hippocampal neurons using intracellular recordings. Superfusion of this cytokine did not alter the resting membrane potential, cell input resistance, action potentials, nor GABA-mediated fast synaptic potentials. IFN-alpha inhibited glutamate-mediated excitatory postsynaptic potentials (gEPSPs) and reversed or prevented long-term potentiation (LTP) induced by high-frequency tetanic stimulation. IFN-alpha reduced gEPSP amplitude far below its control value. Only a short-term potentiation (STP) was observed when either IFN-alpha or D-2-amino-5-phosphonovalerato (APV; NMDA receptor antagonist) were present during tetanic stimulation. After this STP in presence of APV, IFN-alpha had no effect on gEPSPs. APV had no effect on LTP when applied after tetanic stimulation and did also not prevent IFN-alpha effect on LTP. Genistein (a tyrosine kinase inhibitor) or heat inactivation prevented IFN-alpha effects. IFN-alpha also decreased the depolarization induced by local application of glutamate but did not modify those induced by NMDA. Similarly, IFN-alpha reversed the potentiation (induced by tetanic stimulation) of glutamate-induced depolarizations. IFN-alpha did not affect long-term depression (LTD) induced by low-frequency tetanic stimulation. In conclusion, IFN-alpha-induced inhibition of LTP is, at least in part, mediated by a postsynaptic effect, by tyrosine kinase activity, and by non-NMDA glutamate receptors. Inhibition of LTP by IFN-alpha unmasks LTD which is induced by the same high-frequency tetanic stimulation.