Imaging Anoxic Depolarization During Ischemia-Like Conditions in the Mouse Hemi-Brain Slice

Abstract

Focal ischemia evokes a sudden loss of membrane potential in neurons and glia of the ischemic core termed the anoxic depolarization (AD). In metabolically compromised regions with partial blood flow, peri-infarct depolarizations (PIDs) further drain energy reserves, promoting acute and delayed neuronal damage. Visualizing and quantifying the AD and PIDs and their acute deleterious effects are difficult in the intact animal. In the present study, we imaged intrinsic optical signals to measure changes in light transmittance in the mouse coronal hemi-brain slice during AD generation. The AD was induced by oxygen/glucose deprivation (OGD) or by ouabain exposure. Potential neuroprotective strategies using glutamate receptor antagonists or reduced temperature were tested. Eight minutes of OGD (n = 18 slices) or 4 min of 100 microM ouabain (n = 14) induced a focal increase of increased light transmittance (LT) in neocortical layers II/III that expanded concentrically to form a wave front coursing through neocortex and independently through striatum. The front was coincident with a negative voltage shift in extracellular potential. Wherever the LT front (denoting cell swelling) propagated, a decrease in LT (denoting dendritic beading) followed in its wake. In addition the evoked field potential was permanently lost, indicating neuronal damage. Glutamate receptor antagonists did not block the onset and propagation of AD or the extent of irreversible damage post-AD. Lowering temperature to 25-30 degrees C protected the tissue from OGD damage by inhibiting AD onset. This study shows that anoxic depolarization evoked by global ischemia-like conditions is a spreading process that is focally initiated at multiple sites in cortical and subcortical gray. The combined energy demands of O(2)/glucose deprivation and the AD greatly exacerbate neuronal damage. Glutamate receptor antagonists neither block the AD in the ischemic core nor, we propose, block recurrent PID arising close to the core.

Sequential Release of GABA by Exocytosis and Reversed Uptake Leads to Neuronal Swelling in Simulated Ischemia of Hippocampal Slices

“…To analyze the effect of blocking GABA receptors on the swelling that occurred at the time of the AD, we used the fact that in submerged slices swelling leads to less scattering of light passing through the slice and so increases the intensity of transmitted light (Kreisman et al, 1995;Joshi and Andrew, 2001). This is because intracellular organelles scatter light and reduce the transmitted light, but when cells swell, the organelles are diluted, reducing scattering and increasing transmittance.…”

“…To investigate this, we used the fact that cell swelling leads to a decrease of light scattering and an increase of light transmittance through the slice (Kreisman et al, 1995;Joshi and Andrew, 2001), as schematized in Figure 7A. Figure 7B shows a specimen record of light transmittance during application of normal ischemic solution.…”

Spreading Depression: Imaging and Blockade in the Rat Neocortical Brain Slice

“…In addition, we have recently reported that certain sigma-one receptor ( 1 R) ligands are effective in preventing the propagating wave of anoxic depolarization (AD) (Anderson et al 2000). This wave arises under simulated ischemia and, if unchallenged, leaves cells damaged in its wake (Obeidat and Andrew 1998;Jarvis et al 2001;Joshi and Andrew 2001). Similarities between normoxic and hypoxic SD have been noted (Aitken et al 1998).…”

Live, optical experimental neuroimaging reveals variations of the cerebral blood flow response to ischemic spreading depolarization

“…The rate of SD propagation is predicted to increase when (1) extracellular K + removal is hampered, (2) the extracellular space is smaller (cell swelling), and (3) extracellular K + and/or glutamate concentrations are increased [73]. In line with the predictions, hypoxic SDs tended to propagate approximately 1 mm/min more rapidly than normoxic SDs as observed in brain slices [74,75]. These data in combination with our findings suggest that the tissue at the SD focus may be more hypoxic than in the surrounding area.…”