Glutamate transporter EAAT2 expression is up-regulated in reactive astrocytes in human periventricular leukomalacia

Abstract

The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% +/- 5.6%) compared with control white matter (21.4% +/- 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation.

Fig. 1

Neuropathological elements of PVL. Diffuse PVL in cerebral white matter from a 34-week-old case showing reactive astrocytosis (A), with DAB staining of anti-GFAP, and activated microglia (B), with DAB staining of CD68 counterstained with hemotoxylin. C: CD68 immunostaining counterstained with hemotoxylin in the same case showing macrophage infiltration in the focal necrosis of PVL. Scale bars = 50 μm.

Fig. 2

Densitometric analysis of EAAT2 protein expression in developing human cerebral white matter. Cerebral white matter lysates from cases of different ages were run a on an SDS-PAGE (8–18%) with 40 μg protein per lane and immunoblotted with anti-nGLT1 antibody. Densitometric values are plotted as percentage of adult human standard control.

Fig. 3

DAB staining of EAAT2 in PVL. Cerebral white matter from a 34-week PVL case showing EAAT2 staining in reactive astrocytes (arrows). Arrowheads point to laminar processes that appear morphologically axonal. Scale bar = 50 μm.

Fig. 4

Expression of EAAT2 in reactive astrocytes in cerebral white matter from PVL brains. Confocal fluorescence microscopy (×60) of reactive astrocytes in cerebral white matter from a 40-week-old infant diagnosed with PVL labeled with GFAP monoclonal antibodies (A) counterstained with anti-nGLT1 antibodies (B). Overlay of A and B shows EAAT2 labeling in reactive astrocytes (C); bisbenzamide for nuclear staining is blue. Scale bar = 50 μm.

Fig. 5

Percentage of EAAT2-positive GFAP-expressing cells in PVL vs. control cases. The number of EAAT2-immunopositive GFAP-labeled cells was divided by the number of GFAP-positive cells alone to yield the percentage of EAAT2-positive, GFAP-positive cells. Scale bar = 50 μm.

Fig. 6

Expression of EAAT2 in macrophages in cerebral white matter from PVL brains. DAB staining using anti-nGLT1 antibodies was performed on paraffin-embedded PVL tissue. Digital microscopy (×40) of cerebral white matter from a 40-week-old infant diagnosed with PVL labeled with anti-nGLT1 antibodies. Arrows show EAAT2-stained macrophages. Scale bar = 50 μm.

Fig. 7

Expression of EAAT2 in macrophages in cerebral white matter from PVL brains. Fluorescence microscopy (×40) of cerebral white matter from a 32-week-old preterm infant diagnosed with PVL labeled with anti-nGLT1 antibodies (B) counterstained with the CD68 monoclonal antibodies (A). Overlay of A and B shows EAAT2 labeling in macrophages (C). Scale bar = 50 μm.

Fig. 8

DAB staining of EAAT2 in paraffin-embedded PVL human cerebral white matter. DAB staining of anti-nGLT1 in a 40-week PVL case. Arrows point to developing OLs staining with anti-nGLT1. Arrowheads indicate mature MBP OLs. Scale bar = 50 μm.

Fig. 9

Immunofluorescent double labeling of EAAT2⁺ and O4⁺ OLs in human developing cerebral white matter with PVL. Fluorescence microscopy (×40) of cerebral white matter in a 32-week-old preterm infant diagnosed with PVL labeled with the O4 monoclonal antibody (A) counterstained with anti-nGLT1 antibodies (B). Overlay of A and B shows EAAT2 expression in O4⁺ OLs (C). Scale bar = 50 μm.