Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP(-/-)Vim(-/-) mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP(-/-)Vim(-/-) than in wild-type (WT) mice; GFAP(-/-), Vim(-/-) and WT mice had the same infarct volume. Endothelin B receptor (ET(B)R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP(-/-)Vim(-/-) astrocytes. In WT astrocytes, ET(B)R colocalized extensively with bundles of IFs. GFAP(-/-)Vim(-/-) astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP(-/-)Vim(-/-) than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET(B)R-mediated control of gap junctions, and PAI-1 expression.

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1-3 days in vitro)against induced apoptosis and mature cells (5؉ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFB activation. PEDF induced an increased phosphorylation of IB␣, decreased levels of IB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IB, or N-acetylLeu-Leu-norleucinal, which blocks proteosome degradation of IB, demonstrating that NFB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brainderived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.

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