Research reportExcitotoxic swelling occurs in oxygen and glucose deprived human cortical slices
Introduction
Glutamate is the major fast excitatory neurotransmitter in the mammalian central nervous system. It produces neuronal depolarization by gating the opening of membrane receptor/ionophores which are selectively permeable to sodium and calcium ions [24]. These receptor/ionophores are named for the synthetic amino acids that most selectively activate them: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), N-methyl-d-aspartate (NMDA), and kainate (KA). Of these three subclasses of glutamate-gated receptor/ionophores, the NMDA receptor is most permeable to calcium. During normal synaptic transmission, glutamate receptors are exposed to transmitter for only a few milliseconds. However, exposure of these receptors to high glutamate concentrations (>50 μM) for seconds to minutes leads to permanent injury or death. The proximal trigger for glutamate neurotoxicity is the entry of calcium, and likely sodium as well, since both ions flow down their electrochemical gradient into the neuron. The sodium influx is associated with obligate chloride and water entry, leading to neuronal swelling and shrinkage of the extracellular space [23].
There is a considerable body of experimental data indicating that ischemia leads to an elevation of extracellular glutamate in the brains of experimental animals, probably by limiting uptake and increasing efflux from neurons [6]. The glutamate concentration can reach toxic levels 3, 9. Consistent with this, glutamate antagonists limit neuronal loss in a variety of in vitro and in vivo models of neuronal ischemia [14]. Despite the extensive experience with glutamate antagonists in animal stroke models, there is still no clinical data demonstrating a role for excessive extracellular glutamate in human stroke.
Therefore, we developed a technique to investigate whether excessive glutamate release participates in the early events in human neocortical slices deprived of oxygen and glucose (OGD). We found that electrical resistance, an index of cellular swelling, increases in neocortical slices following OGD, and that this resistance increase is blunted by concurrent treatment of the slice with a glutamate receptor antagonist. This is the first direct evidence that glutamate is involved in early events that ultimately lead to ischemic neuronal death in man.
Section snippets
Rat cortical slice preparation
Rats of both genders (10- to 21-day-old) were anesthetized in a CO2 chamber (70% CO2, 30% O2) and decapitated. The brains were removed to ice cold saline composed of, in mM, 200 sucrose, 30 NaCl, 5 KCl, 9 MgCl2, 1 MgSO4, 10 glucose, 20 HEPES, 3 NaHCO3, and 1 NaHPO4. The pH was 7.4. Recent reports indicate that this solution reduces swelling and improves slice viability [1]. The anterior portions of both hemispheres were transected, glued to a vibratome chuck and supported by an agar block.
Results
We first determined that the amplifier gave a linear response when the current amplitude was increased (Fig. 1C,D). Second, we verified that perturbations known to produce neuronal swelling would increase tissue resistance. Current flow is predominantly through the extracellular space 21, 26, and when cells swell, the extracellular space is decreased, thereby elevating electrical resistance 11, 26. Slices were exposed to NMDA by including 30 μM NMDA in the superfusate. The relative voltage drop
Discussion
In previous brain slice experiments on hypoxia, we and others have used intracellular and extracellular recording to assess the integrity of the tissue 8, 14, 12. It is possible to directly monitor the resting membrane potential of individual neurons during an experiment to show that hypoxia leads to membrane depolarization (sometimes preceded by hyperpolarization) and that neuroprotective therapies prevent or delay this depolarization. Another approach has been to monitor the evoked,
Acknowledgements
We would like to thank Nancy Lancaster for excellent help with all the slice experiments, Camille Caupp RN and Shelly Weible RN, for making the transfer of slices from the operating room to the laboratory go smoothly and Mark P. Goldberg MD, for critical discussion of the manuscript. This research was supported by NIH grants R01 NS19988 (SMR), P20 NS 32568 (TSP and SMR), and T32 NS07027 (JLW).
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