Brain slices, particularly the hippocampal slice and primary neuronal/glial cultures from cortex, hippocampus, and cerebellum of embryonic or perinatal rats and mice, have become widely used models for studying ischemia-like damage. In brain slices, ischemia-like conditions are induced by replacing the normal O2/CO2 equilibrated medium with N2/CO2 equilibrated medium; typically the cultures are maintained in an incubator containing a N2/CO2 atmosphere. When glucose is maintained in the anoxic buffer, the insult is termed hypoxia, and when glucose is omitted, the insult is termed in vitro ischemia or oxygen/glucose deprivation. Glucose deprivation alone can also induce neuronal death with some features similar to those observed in in vivo ischemia models. Hypoxia also can be induced by treatment with cyanide (NaCN or KCN) or by incubating in an anoxic atmosphere. Chemical hypoxia results in more free radical generation than does anoxia. In vitro models differ from in vivo stroke models in several aspects. Typically, a long duration of the anoxic or hypoxic insult is required to kill neurons in vitro. ATP depletion is less severe and the release of glutamate is delayed compared to ischemia in vivo. The absence of blood vessels and flow in vitro eliminates important components of the damage process present in vivo, including infiltration of inflammatory cells. In addition, the composition and responsiveness of glial cells in vitro differs from that in the intact brain.
Measurement of Stroke-Induced Damage
The measurement of dynamic changes in ischemic brain has attracted growing attention. Ischemic brain injury in both focal and global ischemia models evolves as a sequence of cellular and molecular events (Dirnagl et al., 1999). In this section we describe methods for analyzing brain injury and dynamic changes in the brain during ischemia and reperfusion.
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