We examined the effects of GABA receptor stimulation on the neuronal death induced by exogenously added excitatory amino acids or combined oxygen-glucose deprivation in mouse cortical cell cultures. Death induced by exposure to NMDA, AMPA, or kainate was attenuated by addition of GABA or the GABAA receptor agonist, muscimol, but not by the GABAB receptor agonist, baclofen. The antiexcitotoxic effect of GABAA receptor agonists was blocked by bicuculline or picrotoxin. In contrast, GABA or muscimol, but not baclofen, markedly increased the neuronal death induced by oxygen-glucose deprivation. Muscimol potentiation of neuronal death was associated with increased glutamate efflux to the bathing medium, and increased cellular 45Ca2+ accumulation; it was blocked by MK-801, but not NBQX, suggesting mediation by NMDA receptors. Bicuculline only weakly attenuated muscimol potentiation of oxygen-glucose deprivation-induced neuronal death, probably because it itself increased this death. Present results raise a note of caution in the proposed use of GABAA receptor stimulation to limit ischemic brain damage in vivo.
These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor-mediated neuronal death consequent to hypoxic-ischemic insults.
Long-term survival and integration of NT2N cells in the periinjured cortex of immunocompetent rats provides the researcher with an important cellular system that can be used to study maturation, regulation, and neurite outgrowth of transplanted neurons following TBI.
S100B protein in brain is produced primarily by astrocytes, has been used as a marker for brain injury and has also been shown to be neurotrophic and neuroprotective. Using a well characterized in vitro model of brain cell trauma, we examined the potential role of exogenous S100B in preventing delayed neuronal injury. Neuronal plus glial cultures were grown on a deformable Silastic membrane and then subjected to strain (stretch) injury produced by a 50 ms displacement of the membrane. We have previously shown that this injury causes an immediate, but transient, nuclear uptake of the fluorescent dye propidium iodide by astrocytes and a 24-48 h delayed uptake by neurons. Strain injury caused immediate release of S100-beta with further release by 24 and 48 h. Adding 10 or 100 nM S100B to injured cultures at 15 s, 6 h or 24 h after injury reduced delayed neuronal injury measured at 48 h. Exogenous S100B was present in the cultures through 48 h. These studies directly demonstrate the release and neuroprotective role of S100B after traumatic injury and that, unlike most receptor antagonists used for the treatment of trauma, S100B is neuroprotective when given at later, more therapeutically relevant time points.
Traumatic brain injury causes alterations in cerebral blood flow that are thought to influence secondary pathophysiology and neurologic outcome in humans. Since it is difficult to study early changes in blood flow in head-injured patients, animal models of brain injury must be employed. However, techniques to monitor brain blood flow in animals are labor intensive and generally provide discontinuous flow measurements. The present study examines the application of laser-Doppler flowmetry for measurement of cerebral blood flow following experimental brain injury. This method allows continuous monitoring of local cerebral blood flow before, during, and after injury. Rats (n = 9) were prepared for lateral fluid percussion injury under barbiturate anesthesia. Injury (2.10 +/- 0.02 atm) was induced over the right parietal cortex, and blood flow was monitored in the contralateral cortex. Seconds after the peak hypertension after injury, blood flow in the left parietal cortex increased 226% +/- 18% (means +/- SEM). This increase was transient, with blood flow falling below control values within minutes. Five minutes after injury, blood flow was 83% +/- 8% of control, and at 1 h, this value had fallen to 56% +/- 6%. Blood flow at 60 min was 93% +/- 5% of control in the sham-injured group (n = 10). The reduction in cerebral blood flow in our laser-Doppler study was of similar magnitude as previously reported in rats injured at a similar intensity when blood flow was examined with radiolabeled microspheres. Given these results, we believe laser-Doppler flowmetry can be used to continuously monitor posttraumatic blood flow following experimental brain injury.
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