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To explore the role of neurotrophin-3 (NT-3) during cerebral ischemia, NT-3-deficient brains were subjected to transient focal ischemia. Conditional mutant brains produced undetectable amounts of NT-3 mRNA, whereas the expression of the neurotrophin, BDNF, the NT-3 receptor, TrkC, and the nonselective, low-affinity neurotrophin receptor p75NTR, were comparable to wild-type. Baseline absolute blood flow, vascular and neuroanatomical features, as well as physiological measurements were also indistinguishable from wild-type. Interestingly, the absence of NT-3 led to a significantly decreased infarct volume 23 h after middle cerebral artery occlusion. Consistent with this, the addition of NT-3 to primary cortical cell cultures exacerbated neuronal death caused by oxygen-glucose deprivation. Coincubation with the oxygen free radical chelator, trolox, diminished potentiation of neuronal death. NT-3 also enhanced neuronal cell death and the production of reactive oxygen species caused by oxidative damage inducing agents. We conclude that endogenous NT-3 enhanced neuronal injury during acute stroke, possible by increasing oxygen-radical mediated cell death.

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD50 for exposure to glutamate (10(-3)-10(-5) M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.

Controlled release of drugs to specific locales in the brain has engendered considerable interest. Here we evaluate the safety and biocompatibility of 6-microm diameter particles composed of dipalmitoylphosphatidylcholine and chondroitin sulfate A, when delivered into the cerebral parenchyma and ventricles, and in the case of intravascular injection. Some particles were loaded with fluorescein-labeled albumin to facilitate detection. Particles placed in medium with cultured murine primary cortical neurons did not increase cell death at concentrations as high as 4 mg/ml. When particles (100 microg in 2 microl) were placed stereotactically in the striatum and lateral ventricles, there was no histological evidence on hematoxylin-eosin stained sections of tissue injury outside of the needle track in any animal 3, 7, and 14 days after injection (n=6 each), and no inflammation. Ventricular size was not significantly different between animals given intraventricular injections of particles and albumin solution at those time points (n=4 each). Intracarotid injection of particles at concentrations of 0.2 and 1 mg/ml (n=4 each) did not affect relative cerebral blood flow, and there were no embolic events on histology. In one animal in the group injected with 5 mg/ml (n=3), there was a profound decrease in rCBF, with patchy emboli on histology. These novel biodegradable particles are biocompatible in and around the brain, and may be safe for intracranial sustained drug delivery either in the parenchyma or into the CSF.