Anoxic Depolarization Mediates Acute Damage Independent of Glutamate in Neocortical Brain Slices

Abstract

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.

Terminal spreading depolarization and electrical silence in death of human cerebral cortex

“…Note the similarity between the patterns of the terminal SD in the rodent experiment and in the patient ( red arrows in A and C). In both cases, the terminal SD propagated from one electrode to the next corresponding with previous evidence from experiments in animals in vivo and in brain slices 7, 12, 13, 14. In contrast to the spread of the terminal SD, the nonspreading depression (cf asterisks) is seen in the rodent recordings as a silencing of the spontaneous electrical activity (alternating current [AC]–electrocorticography [ECoG]: 0.5–45Hz) that develops simultaneously across the array of the regional 2 electrodes (cf Figs 3B, 4B, 5A, 5C, and 6 for nonspreading depression in patients).…”

A novel method for inducing focal ischemia in vitro

“…Using this novel technique, we were successful in producing a focal infarct within the brain slice and a subsequent region of neuronal death that increased as a function of time. The major disadvantage of current in vitro ischemic models is that OGD solution is applied to the entire tissue (slice or cultured neurons) thus mimicking a global, rather than a focal ischemic event (Garcia de Arriba et al, 1999;Jarvis et al, 2001;Lipski et al, 2007). It can be argued that these models mimic the events that occur within the core region of a stroke but not the events that occur in the penumbra that still has a supply of both oxygen and glucose as compared to the core region.…”

“…However, in order to efficiently characterize such compounds, it is desirable to have a model that allows researchers to rapidly determine efficacy as well as to determine mechanism of action of the compounds. The most common models currently used in stroke research include: in vivo focal ischemia models (Weng and Kriz, 2007;Saleh et al, 2009), in vitro dissociated cell models (Larsen et al, 2005;Ye et al, 2009) and in vitro brain slice models (Garcia de Arriba et al, 1999;Jarvis et al, 2001). In vivo focal ischemia models (whole animal models) are used extensively to study stroke and involve invasive surgery to expose and occlude a cerebral artery (Saleh et al, 2009).…”

“…As well, using this type of model, it is difficult to determine the mechanism of action of therapeutic interventions. The use of in vitro models where oxygen-glucose deprivation (OGD) is used to mimic stroke (Jarvis et al, 2001;Wise-Faberowski et al, 2009;Ye et al, 2009) overcomes many of these problems. Of the in vitro models, the dissociated cell models are popular for screening due to their high throughput, low cost and ease of use.…”

“…One limitation of current in vitro slice models of stroke is that OGD media is applied to the entire brain slice and thus all of the cells in the slice are subject to the same ischemic condition (Jarvis et al, 2001;Wise-Faberowski et al, 2009). This is referred to as global ischemia and thus the entire slice is representative of the "core".…”