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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.025031

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CCAAT/enhancer binding protein β deficiency provides cerebral protection following excitotoxic injury

¹ Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28029-Madrid, Spain
² Centro Nacional de Investigaciones Cardiovasculares, Instituto de Salud Carlos III, Melchor Fernandez Almagro, 3, 28029-Madrid, Spain
³ Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense de Madrid, 28040-Madrid, Spain

View larger version (67K): [in this window] [in a new window]   Fig. 1. Increased expression of C/EBPβ in mouse glial primary cultures after treatment with different stimuli. (A) Western blot showing an increase in C/EBPβ expression in microglial cells treated with LPS and KA and in astrocytes treated with LPS, staurosporine, KA or glutamate for 24 hours. Only a small induction was observed in astrocyte cultures after treatment with KA or glutamate. (B,C) Primary astroglial (B) and microglial (C) cultures prepared from C/EBPβ+/+ were treated as in A, and C/EBPβ expression was evaluated by immunofluorescence analysis and confocal microscopy using a specific anti-C/EBPβ antibody (clone A16), as described in the Materials and Methods. Cultures were also stained with anti-CD11b (clone M1/70.15) or anti-GFAP antibodies to detect microglia or astrocytes, respectively. (D,E) To confirm the specificity of the anti-C/EBPβ antibody, primary microglial (D) and astroglial (E) cultures from C/EBPβ–/– mice were treated with LPS for 24 hours, and C/EBPβ expression was evaluated by immunofluorescence analysis and confocal microscopy. Representative results from three experiments are shown. Scale bar, 10 µm. Nuclei were counterstained by DAPI (blue). B, basal; LPS, lipopolysaccharide; St, staurosporine; KA, kainic acid; Glu, glutamate.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Induction of pro-inflammatory mediators is attenuated in microglial primary cultures from C/EBPβ-deficient mice. (A) Western blots showing a reduction in the expression of IL1β and COX2 in C/EBPβ–/– microglial cultures after treatment with LPS or KA for 24 hours, compared with cultures established from C/EBPβ+/+ mice. (B,C) Primary microglial cultures prepared from C/EBPβ+/+ or C/EBPβ–/– mice were treated as in A, and the expression of C/EBPβ (green), IL1β (blue in B) and COX2 (blue in C) was evaluated using immunofluorescence techniques, and confocal microscopy using specific antibodies as described in the Materials and Methods. The last column of panels in each set shows colocalization of C/EBPβ with IL1β (B) or COX2 (C). The microglial marker CD11b is shown in red. Representative results from three experiments are shown. Scale bar, 10 µm. B, basal; KA, kainic acid; LPS, lipopolysaccharide.

View larger version (115K): [in this window] [in a new window]   Fig. 3. Induction of C/EBPβ expression after KA injection in the adult mouse brain. (A) Immunohistochemical analyses of coronal brain sections prepared from C/EBPβ+/+ mice 24 and 72 hours after KA injection. C/EBPβ protein levels were substantially enhanced in the stratum radiatum (SR) and in the granular layer (GL) of the dentate gyrus of the hippocampus of C/EBPβ+/+ mice 24 hours and 72 hours post injection, compared with vehicle-injected controls. Images are representative for experiments from five animals. Scale bar, 25 µm. (B) Double immunofluorescent labeling and confocal microscopic analyses of C/EBPβ after KA injection in C/EBPβ+/+ mice. Double-labeling studies were performed with an antibody recognizing C/EBPβ (green) and different reagents to detect astrocytes (anti-GFAP), microglia (tomato lectin), and neurons (Neurotrace) (red labeling). Complete colocalization (yellow) was found in neurons, although 24 hours and 72 hours after KA injection some astrocytes and microglia were found that express C/EBPβ (arrows). Scale bar, 10 µm. (C) Immunohistochemical analyses of coronal brain sections prepared from C/EBPβ–/– mice 24 hours and 72 hours after KA injection. As expected, C/EBPβ–/– mice did not present any C/EBPβ staining. Images are representative for experiments from five animals. Scale bar, 25 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Deficiency of C/EBPβ ameliorates KA-induced gliosis. (A,B) C/EBPβ+/+ and C/EBPβ–/– adult mice were injected with vehicle (V) or KA and sacrificed 24 hours and 72 hours post injection. Coronal sections (30 µm) were stained with antibodies against (A) GFAP or (B) CD11b to detect astrocytes or microglia, respectively. A significant decrease in both astrogliosis and microgliosis is observed in C/EBPβ–/– mice. Substantial gliosis is observed in C/EBPβ+/+ mice after KA-injection, which is prevented in C/EBPβ–/– mice (A). Immunohistochemical detection using anti-CD11b antibodies reveals highly ramified microglia in the hippocampus of KA-treated C/EBPβ–/– mice (B). By contrast, in C/EBPβ+/+ mice the activated microglial cells exhibit shorter and thicker processes, and larger cell bodies. Insets show higher magnification of the images corresponding to C/EBPβ+/+ mice. (C,D) Quantification of the number of reactive astrocytes (C) and microglial cells (D) analyzed in the hilus, stratum radiatum or molecular layer of the hippocampus. Values are the mean ± s.e. from five different animals and two independent sections per animal. #P0.05, ##P0.01, ###P0.001; vehicle vs KA-injected animals. *P0.05, ***P0.001; knockout vs wild-type KA-injected animals; SR, stratum radiatum; Hil, hilus; ML, molecular layer. Images representative for each group are shown. Scale bars, 25 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 5. C/EBPβ deficiency impairs KA induction of pro-inflammatory mediators. (A-D) C/EBPβ+/+ and C/EBPβ–/– adult mice were injected with KA and sacrificed 24 and 72 hours post-injection. Coronal sections (30 µm) were stained with antibodies against (A,B) IL1β and (C,D) COX2. Neurotrace was used to identify neurons, and anti-GFAP and tomato lectin to detect astrocytes and microglia, respectively. IL1β expression was mainly localized in the hilus (Hil) and the molecular layers (ML) surrounding the dentate gyrus, and was completely absent in the C/EBPβ–/– mice after KA injection, compared with their C/EBPβ+/+ littermates. COX2 immunoreactivity was mainly detected in the stratum radiatum (SR) and the granule cell layer of the dentate gyrus (GL). In contrast to controls, COX2 immunoreactivity was noticeably reduced in C/EBPβ–/– mice. Confocal analysis of (B) IL1β and (D) COX2 subcellular localization indicates that IL1β is expressed primarily in glial cells, whereas COX2 immunostaining is restricted to neurons. Images are representative for experiments from five animals. Scale bars, 25 µm (A,C) and 10 µm (B,D).

View larger version (88K): [in this window] [in a new window]   Fig. 6. Deficiency of C/EBPβ decreases KA-induced expression of LCN2 and histidine decarboxylase. (A,B) C/EBPβ+/+ and C/EBPβ–/– adult mice were injected with vehicle (V) or KA and sacrificed 24 hours post-injection. Coronal sections (30 µm) were stained with antibodies against (A) LCN2 or (B) histidine decarboxylase. C/EBPβ–/– mice exhibited lower levels of LCN2 and histidine decarboxylase after KA injection, as compared with littermate controls. Representative results from three independent experiments are shown. Scale bars, 25 µm. SR, stratum radiatum; GL, granular layer; Hil, hilus.

View larger version (82K): [in this window] [in a new window]   Fig. 7. Deficiency of C/EBPβ protects neurons from excitotoxic brain damage. (A,B) C/EBPβ+/+ and C/EBPβ–/– adult mice were injected with KA and sacrificed 24 hours and 72 hours post-injection. Coronal sections (30 µm) were stained with (A) Fluoro-Jade B and (B) anti-NeuN antibody. C/EBPβ–/– mice exhibited low levels of neuronal degeneration (as detected by Fluoro-Jade B staining) and a diminished loss of neurons in the CA1 and CA3 regions (as shown by NeuN staining) when compared with littermate controls. Scale bars, 10 µm (A) and 25 µm (B). (C) Quantification of degenerating neurons analyzed in the CA1 and CA3 areas of the hippocampus. Values represent the mean ± s.e. from five different animals and two independent sections per animal. (D) The extent of neuronal damage in the CA1 and CA3 areas of the hippocampus was quantified as described in Materials and Methods. Data were normalized against the mean values obtained from vehicle-injected mice. Values represent the mean ± s.e. from five different animals and two independent sections per animal. *P0.05, **P0.01, ***P0.001, C/EBPβ–/– mice versus C/EBPβ+/+ mice at each time point.

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