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.
Intimate partner violence (IPV) is a leading cause of morbidity and mortality among women of childbearing age. This study aimed to describe delivery of IPV education in Australian pre-vocational medical degrees, and barriers and facilitators influencing this delivery. Eighteen Australian medical schools offering pre-vocational medical degrees were identified. Two were excluded as they had not finalized new curricula. One declined to participate. At least one staff member from each of the remaining 15 schools completed a telephone survey. Main outcome measures included whether IPV education was delivered within the degree, at what stage, and whether it was compulsory; mode and number of hours of delivery; and barriers and facilitators to delivery. Twelve of the medical schools delivered IPV education (median time spent per course = 2 hr). IPV content was typically included as part of Obstetrics and Gynecology or General Practice curriculum. Barriers included time constraints and lack of faculty commitment, resources, and funding. The two schools that successfully implemented a comprehensive IPV curriculum used an integrated, advocacy-based approach, with careful forward planning. Most Australian pre-vocational medical students receive little or no IPV education. The need remains for a more consistent, comprehensive approach to IPV education in medical degrees.
Transporters for L-glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial EAATs, we employed [(3)H]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [(3)H]4MG in astrocytic membranes at 4 degrees C represented 90% of total binding. Binding was rapid (apparent t(1/2) approximately 7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (n(H) = 0.8) with a K(d) of 6.0 +/- 1.5 microM and B(max) = 9.7 +/- 2.9 pmol/mg protein. Binding of [(3)H]4MG to astrocytic homogenates was Na(+)-dependent and inhibited by K(+). Compounds acting at EAATs, such as L-glutamate (Glu), D-aspartate (D-Asp), L-(2S,3S,4R)-2-(carboxycyclopropyl)glycine and L-trans-pyrrolidine-2,4-dicarboxylate displaced binding to nonspecific levels. L-Serine-O-sulphate, an EAAT1-preferring ligand, fully displaced binding of [(3)H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L-threo-3-methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [(3)H]4MG binding. Studies with [(3)H]D-Asp reinforced evidence that [(3)H]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [(3)H]4MG was also accumulated rapidly (apparent t(1/2) approximately 4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K(m) of 146 +/- 24 microM, V(max) = 336 +/- 27 nmol/mg protein/min), which possessed an EAAT1-like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [(3)H]4MG can be utilized in various preparations including cultured astrocytes.
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