Does Isoflurane Protect against Delayed Cell Death after Simulated Cerebral Ischemia? Sullivan et al. (page 189)

To address the shortcomings of in vitro  models of ischemia, Sullivan et al.  developed an organotypic slice culture model of cerebral ischemia. Because the model retains cellular integrity and sensitivity to anoxia and allows serial measurement of cell survival over many days, the team was able to test whether isoflurane prevents delayed cell death of ischemia-sensitive hippocampal neurons after simulated ischemia.

Hippocampi of 6- to 28-day-old rats were harvested and kept in culture for 7–14 days before the study began. Presence of field potentials after 7 days in culture indicated the health of the cells and persistence of synaptic connections in the slices. Propidium iodide exclusion was used to establish that a high percentage of neurons in the CA1, CA3, and dentate cell fields were living. In vitro  ischemia was simulated by anoxia combined with glucose-free media (oxygen–glucose deprivation). In the experiments involving isoflurane, the anesthetic was delivered to the chamber using a calibrated isoflurane vaporizer and remained throughout the ischemic injury period. In another experiment of 46 slices, the effect of 1% isoflurane on glutamate-induced delayed cell death was evaluated at 2, 3, and 7 days after injury. Delayed cell death was serially measured in each slide by quantifying the binding of propidium iodide to DNA with fluorescence microscopy.

Maximum cell death occurred 3–5 days after oxygen–glucose deprivation. CA1 neuronal cell death was 80 ± 18% after 3 days and 80–100% after 1 week. By the third day after oxygen–glucose deprivation, death of 70 ± 16% of CA3 neurons and 48 ± 28% of dentate gyrus neurons had occurred. Both 1% isoflurane and the N -methyl-d-aspartate antagonist MK-801 reduced cell death to levels similar to controls for 14 days after the ischemic injury. Isoflurane also reduced cell death caused by application of 100 but not 500 mm glutamate in CA1 and CA3 neurons. The researchers speculate that modulation of glutamate excitotoxicity may contribute to the protective mechanism.