Protective role of reactive astrocytes in brain ischemia role of reactive astrocytes in brain ischemia LLiet al 2 Journal of Cerebral Blood Flow & Metabolism (2007), 1–14 image analysis

Protective role of reactive astrocytes in brainischemia

Lizhen Li1, Andrea Lundkvist1, Daniel Andersson1, Ulrika Wilhelmsson1, Nobuo Nagai2,Andrea C Pardo3, Christina Nodin1, Anders Stahlberg4, Karina Aprico1, Kerstin Larsson5,Takeshi Yabe6, Lieve Moons2, Andrew Fotheringham7, Ioan Davies7, Peter Carmeliet2,Joan P Schwartz6, Marcela Pekna5, Mikael Kubista4, Fredrik Blomstrand1,Nicholas Maragakis3, Michael Nilsson1 and Milos Pekny1

1Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute ofNeuroscience and Physiology at Sahlgrenska Academy, Goteborg University, Goteborg, Sweden; 2FlandersInteruniversity Institute for Biotechnology, Center for Transgene Technology & Gene Therapy, KU Leuven, CampusGasthuisberg, Leuven, Belgium; 3Department of Neurology, Johns Hopkins School of Medicine, Baltimore,Maryland, USA; 4Department of Chemistry and Bioscience, Chalmers University of Technology and TATAABiocenter, Goteborg, Sweden; 5Department of Medical Chemistry and Cell Biology, Institute of Biomedicine,Sahlgrenska Academy, Goteborg University, Goteborg, Sweden; 6Neurotrophic Factors Section, National Institute ofNeurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA; 7School of Medicineand School of Biological Sciences, University of Manchester, Manchester, UK

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence hasbeen lacking due to the absence of suitable experimental models. Previously, we generated mice deficientin two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whoseupregulation is the hallmark of reactive astrocytes. GFAP–/–Vim–/– mice exhibit attenuated posttraumaticreactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days aftermiddle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP–/–Vim–/– than inwild-type (WT) mice; GFAP–/–, Vim–/– and WT mice had the same infarct volume. Endothelin B receptor(ETBR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct inWT mice but undetectable in GFAP–/–Vim–/– astrocytes. In WT astrocytes, ETBR colocalized extensivelywith bundles of IFs. GFAP–/–Vim–/– astrocytes showed attenuated endothelin-3-induced blockage of gapjunctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower inGFAP–/–Vim–/– than in WT mice. DNA array analysis and quantitative real-time PCR showed down-regulation 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 tochanges in glutamate transport, ETBR-mediated control of gap junctions, and PAI-1 expression.Journal of Cerebral Blood Flow & Metabolism advance online publication, 29 August 2007; doi:10.1038/sj.jcbfm.9600546

Keywords: astrocytes; GFAP; intermediate filaments; reactive gliosis (astrogliosis); vimentin

Introduction

Astroglial cells are the most abundant cells in thecentral nervous system (CNS) and are believed toplay a major role in the brain and spinal cordpathologies. Although never proved directly, astro-cytes are thought to exert a neuroprotective effect instroke by shielding neurons from oxidative stress(Kraig et al, 1995). This hypothesis is based on theability of astrocytes to engage in spatial buffering, totransport and metabolize amino acids, glucose, andother key molecules, and to upregulate antioxidantsand free radical scavengers in the ischemic region.

Reactive gliosis is a response of astrocytes toCNS injury, including brain ischemia, mechanical

Received 11 May 2007; revised 27 June 2007; accepted 16 July2007

Correspondence: Dr M Pekny, Department of Clinical Neuro-science and Rehabilitation, Institute of Neuroscience and Phy-siology, Sahlgrenska Academy at Goteborg University, GoteborgSE-405 30, Sweden. E-mail: [email protected]

This study was supported by grants from the Swedish Research

Council (11548 to Mpy, 5174 to UW, 13470 to MPa and 20114 to

MN), The Region of Vastra Gotaland (RUN), the Swedish Cancer

Foundation (3622), ALF Goteborg, Hjarnfonden, the Swedish

Stroke Association, Torsten and Ragnar Soderberg’s Foundations,

Frimurare Foundation, Rune and Ulla Amlov’s Foundation,

Trygghansa, the Swedish Society for Medicine, the Swedish

Society for Medical Research, the King Gustaf V Foundation,

Volvo Assar Gabrielsson Fond, and the Novo Nordisk Foundation

Program on Diabetic Microangiopathy, STINT Sweden, Edit

Jacobsson’s Foundation, Axel Linder’s Foundation, and Carl

Trigger’s Foundation.

Journal of Cerebral Blood Flow & Metabolism (2007), 1–14& 2007 ISCBFM All rights reserved 0271-678X/07 $30.00

www.jcbfm.com

http://dx.doi.org/10.1038/sj.jcbfm.9600546

mailto:[email protected]

http://www.jcbfm.com

trauma, neurodegenerative diseases, and tumors.Reactive astrocytes undergo changes in morphologyand in their expression of a wide range of molecules(Eddleston and Mucke, 1993; Ridet et al, 1997). Thehallmark of reactive gliosis, regardless of its origin,is increased expression of glial fibrillary acidicprotein (GFAP) and vimentin in reactive astrocytes.Glial fibrillary acidic protein and vimentin arebuilding blocks of intermediate filaments (IFs),which, together with microtubules and actin fila-ments, constitute the cytoskeleton.

We and others have generated GFAP–/– mice (Gomiet al, 1995; Liedtke et al, 1996; McCall et al, 1996;Pekny et al, 1995) and found that their astrocytesare free of IFs in the absence of any challenge, sincevimentin requires GFAP to polymerize into fila-ments (Pekny et al, 1995). However, reactive astro-cytes in GFAP–/– mice contain IFs because they alsoproduce nestin, an IF protein that copolymerizeswith vimentin in both GFAP–/– and wild-type (WT)reactive astrocytes (Eliasson et al, 1999; Pekny et al,1998a). GFAP–/– mice exhibit normal healing afterbrain and spinal cord injury (Pekny et al, 1999b;Pekny et al, 1995). In mice lacking both GFAP andvimentin (GFAP–/–Vim–/–), however, reactive astro-cytes are devoid of IFs, reflecting the inability ofnestin to self-polymerize (Eliasson et al, 1999). As aresult, reactive gliosis and glial scar formation afterneurotrauma in the brain or spinal cord areattenuated (Pekny et al, 1999b; Pekny and Pekna,2004). After neurotrauma, IF-free astrocytes ofGFAP–/–Vim–/– mice reach comparable volume oftissue as WT astrocytes; however, GFAP–/–Vim–/–

astrocytes show less prominent thickening of themain cellular processes (Wilhelmsson et al, 2004).Posttraumatic regeneration of neuronal synapses(Wilhelmsson et al, 2004) and integration of neuralgrafts (Kinouchi et al, 2003) were improved inGFAP–/–Vim–/– mice, despite more prominent synap-tic loss at the initial stage after neurotrauma(Wilhelmsson et al, 2004).

In this study, we subjected GFAP–/–Vim–/–, GFAP–/–,and Vim–/– mice to brain ischemia induced bymiddle cerebral artery (MCA) transection by twoindependent paradigms. After 7 days of ischemia,infarct volumes were 2- to 3.5-fold larger inGFAP–/–Vim–/– mice than in WT, GFAP–/–, or Vim–/–

mice. This increase correlated with reduced glutamateuptake in cortical slices from GFAP–/–Vim–/– mice,altered endothelin B receptor (ETBR)-mediated controlof gap junctions, and downregulation of plasminogenactivator inhibitor-1 (PAI-1) in GFAP–/–Vim–/– cultures.These findings suggest that reactive astrocytes have aprotective role in brain ischemia.

Materials and methods

Mice

Single-mutant (GFAP–/– or Vim–/–), double-mutant(GFAP–/–Vim–/–), and WT control mice were maintained

on a mixed C57Bl/129 genetic background as described(Eliasson et al, 1999; Pekny et al, 1999b). All mice wereage-matched adults except for the preparation of astrocyte-enriched cultures for which postnatal day 1 to 2 mice wereused. The mice were kept in a barrier animal facility andfed ad libitum.

Induction of Ischemia: Proximal Middle CerebralArtery Transection

Focal cerebral ischemia was induced in six WT and sixGFAP–/–Vim–/– mice by proximal MCA transection asdescribed (Fotheringham et al, 2000) with slight modifica-tions. Mice were anesthetized with isoflurane in oxygen,and body temperature was maintained at 371C with aheating pad. Under the operating microscope, the leftMCA was exposed, occluded at two points by bipolarcoagulation, and transected to ensure permanent disrup-tion (Figure 1E). The proximal end of the MCA (A) wascoagulated approximately 2.5 mm from the MCA/anteriorcerebral artery branch (as determined after brain dissec-tion). After surgery, mice were housed individually incages that were placed on a heating pad. After 1 h, themice were returned to their normal environment.

Induction of Ischemia: Distal Middle Cerebral ArteryTransection

Distal MCA transection was performed as described(Nagai et al, 1999) in two independent series of experi-ments. One set of experiments was performed in 10 WTand 11 GFAP–/–Vim–/– mice. To determine if partialdeficiency of astrocyte IFs or a deficiency of IFs inendothelial cells also affected the infarct volume, a secondset of experiments was performed in nine WT, nineGFAP–/–, eight Vim–/–, and nine GFAP–/–Vim–/– mice.Briefly, mice were anesthetized by intraperitoneal injec-tion of ketamine (75 mg/mL; Apharmo, Arnhem, TheNetherlands) and xylazine (5 mg/mL; Bayer, Leverkusen,Germany), and body temperature was maintained at 371Cwith a heating pad. Under the operating microscope, theMCA was ligated with a 10-0 suture Ethylon nylon thread(Ethylon, Neuilly, France) and transected distally (Figure1E). After surgery, the mice were returned to their cages,which were placed on a heating pad (371C) for 1 h.

Measurement of Infarct Volume 7 Days after ProximalMiddle Cerebral Artery Transection

Deeply anesthetized mice were killed by decapitation.Fresh frontal brain slices (0.5 mm thick) were cut on aVibratome, incubated for 30 mins in 0.125% triphenyl-tetrazolium chloride solution (T4375; Sigma-Aldrich, StLouis, MO, USA) in buffer containing 1.35% dimethyl-sulfoxide, 2 mmol/L MgCl2, 0.1 mol/L Na2HPO4, and0.1 mol/L NaH2PO4 (pH 7.4) at 371C, and fixed in 4%buffered formaldehyde (pH 7.4) (Fotheringham et al,2000). In each slice, the infarct area in white matter wasmeasured with a Nikon SMZ-U stereomicroscope and

Protective role of reactive astrocytes in brain ischemiaL Li et al

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Journal of Cerebral Blood Flow & Metabolism (2007), 1–14

image analysis software (Easy Image, Bergstrom Instru-ment, Goteborg, Sweden). The infarct volume was calcu-lated by integrating infarct areas on all adjacent brainslices with detectable infarction. The extent of brainedema was judged by comparing the areas of the ischemicand contralateral hemispheres.

Measurement of Infarct Volume 7 Days after DistalMiddle Cerebral Artery Transection

Deeply anesthetized mice were perfused through the leftventricle with 4% phosphate-buffered formaldehyde (pH

7.4). The brains were postfixed overnight and embeddedin paraffin. Frontal sections (8 mm thick) were stained withhematoxylin and erythrosine. Infarct areas were assessedby delineating the ischemic region, which could be clearlydiscriminated by its lighter appearance and high propor-tion of cells with pycnotic nuclei. The infarct area wasmeasured on all sections (320 mm apart) on which it wasdetectable (4 to 10 sections per mouse) as described above,and the total infarct volume was calculated.

Blood Pressure and Heart Rate Monitoring

Blood pressure and heart rate were monitored in fiveWT and four GFAP–/–Vim–/– mice during the distal MCAtransection. Under isoflurane anesthesia, the 10-0 sutureEthylon nylon thread (Ethylon, France) was passed underthe MCA. The mouse was turned on its back, an incisionwas made in the neck, the left common carotid artery wasexposed, and a 2.5F catheter was inserted and connectedto a clinical fluid-filled discardable transducer (HLT0698;Fysicon, Oss, The Netherlands). Signal from the transducerwas collected with an AD converter (PowerLab, ADInstru-ments, Chalgrove, UK). After measurement of baselineblood pressure and heart rate, the MCA was occluded bytightening the suture around the vessel. Blood pressureand heart rate were recorded 10 s, 15 mins, 30 mins, and1 h after the MCA occlusion. After the operation, the micewere kept on a heating pad (371C) for 1 h and were givenfree access to water and food. After 7 days, the mice wereanesthetized with ketamine (75 mg/mL; Apharmo) andxylazine (5 mg/mL; Bayer) and perfused first with phos-phate-buffered saline (PBS) for 7 mins and then with 4%phosphate-buffered formaldehyde (pH 7.4). The brainswere postfixed overnight and embedded in paraffin.

Comparison of Cerebrovascular Architecture

The MCA territory was determined as described (Maedaet al, 1998). Briefly, the cerebrovasculature of four WTand four GFAP–/–Vim–/– mice, 8-month-old, was visualizedby detection of endothelial cells with a rat anti-CD-31antibody (1:200; BD Biosciences, San Jose, CA, USA) and ahorseradish peroxidase-conjugated secondary antibody(P0162, 1:100; Dako, Glostrup, Denmark) according tostandard immunohistochemical procedures; endogenousperoxidase activity was blocked by incubating the brainswith PBS containing 0.6% H2O2 for 30 mins, and antibodyincubations were performed at 41C overnight. The dis-tance between the dorsal midline and the line ofanastomoses connecting the peripheral branches of theMCA and the anterior cerebral artery was measured atthree points (2, 4, and 6 mm from the frontal pole) with theNikon SMZ-U stereomicroscope and image analysis soft-ware (Easy Image).

Data Analysis

Data are expressed as mean7s.e.m. The two-tailed t-testwas used for statistical analysis. Differences were con-sidered significant at P < 0.05.

Figure 1 Infarct area is 2.5- to 3.5-fold larger in GFAP–/–Vim–/–

(GV) than in WT mice 7 days after MCA transection. The infarctarea was visualized by triphenyltetrazolium chloride andhematoxylin/erythrosin staining 7 days after proximal (A) anddistal (C) transection of the MCA. (B and D) Infarct volume wasmore than two-fold larger in GFAP–/–Vim–/– than in WT mice (B,*P < 0.05; D, ***P < 0.001 and **P < 0.01). (D) In GFAP–/–

(G) and Vim–/– (V) mice, the infarct volume was not significantlydifferent from that in WT mice. (E) Proximal MCA transectionwas performed at point X after bipolar coagulation at points Aand B. Distal MCA transection was performed at point C(approximately 1.5 mm distal to point A). Values are mean7s.e.m.


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Immunohistochemistry

Coronal cryosections (35mm), made from perfused andpostfixed brains of four WT and three GFAP–/–Vim–/– mice,were incubated in 0.05% glycine in PBS for 1 h at roomtemperature and permeabilized overnight in PBS contain-ing 0.5% Tween 20 and 1% bovine serum albumin at roomtemperature. S100b and ETBR were detected with rabbitantibodies against S100b (Z0311, 1:200; Dako) and ETBR(1:100; Alomone Labs, Jerusalem, Israel) and Alexa 488-conjugated anti-rabbit antibodies (1:500; MolecularProbes, Eugene, OR, USA). Glial fibrillary acidic proteinand glutamine synthase (GS) were detected with mouseantibodies against GFAP (clone GA5, 1:100; Sigma-Aldrich) and GS (1:100; Chemicon Europe Ltd, Hampshire,UK), and Alexa 568-conjugated anti-mouse antibodies(1:500; Molecular Probes). Glutamate transporter-1 (GLT-1) was detected with guinea-pig antibodies against GLT-1(1:100; Chemicon Europe Ltd.) and Alexa 488-conjugatedanti-guinea pig antibodies (1:100; Molecular Probes).Microglia were detected with biotinylated tomato lectin(30mg/mL; Sigma-Aldrich) and Cy3-conjugated streptavi-din (1:100; Sigma-Aldrich). Antibodies were diluted in1% bovine serum albumin and 0.01% Tween 20 in PBS.Cell nuclei were stained with ToPro-3 (1:1,000; MolecularProbes).

Astrocyte-Enriched Cultures

Primary astrocyte-enriched cultures were prepared frompostnatal day 1 GFAP–/–Vim–/– and WT mice as described(Pekny et al, 1998a). The cultures for immunocyto-chemistry were grown in 16-chamber slides (Nalge NuncInternational, Naperville, IL, USA) in Dulbecco’s modifiedEagle’s medium (D5671; Sigma-Aldrich) containing 10%fetal calf serum, 2 mmol/L L-glutamine, and penicillin–streptomycin (Invitrogen, Paisley, UK). Cultures for quan-titative real-time PCR analysis of mRNA were maintainedin 10% fetal calf serum (ETBR and connexin 43 (Cx43)) or1 or 10% fetal calf serum (PAI-1).

Immunocytochemistry

Cells were fixed in methanol at �201C for 5 mins andwashed with PBS. After nonspecific binding was blockedwith 5% normal goat serum (Dako) for 15 mins at roomtemperature, cells were incubated with rabbit antibodiesagainst ETBRs (1:100; Alomone Labs) and mouse anti-bodies against GFAP (clone GA5, 1:100; Sigma-Aldrich)and then with Alexa 488-conjugated anti-rabbit and Alexa568-conjugated anti-mouse antibodies (1:500; MolecularProbes). All antibodies were diluted in PBS.

Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis and Immunoblotting for Endothelin BReceptors

Medium was removed from confluent cultures of astrocytes(10 to 14 days after plating). After two washes with PBS,cells were harvested by scraping into sodium dodecyl

sulfate-extraction buffer consisting of protease inhibitorcocktail (Roche, Mannheim, Germany), 1 mmol/L Na+

orthovanadate, and 1 mmol/L phenylmethanesulfonylin 2% sodium dodecyl sulfate, and the cell lysatewas frozen at �801C. Thawed lysates were sonicated for5 secs, and the protein content was determined withthe Bicinchoninic Acid Protein Assay (Sigma-Aldrich).Protein (50mg) was separated on 4 to 12% bis-Tris gels(Invitrogen, Carlsbad, CA, USA) and transferred to anitrocellulose membrane. The membrane was stainedwith Ponceau S solution (Sigma-Aldrich) to control forequal protein loading. ETBRs were detected with poly-clonal rabbit antibodies (1:500; Alomone Labs), andb-actin (internal loading control) was detected with amouse monoclonal antibody (1:1,000; Abcam, Cambridge,UK). To enhance the signal for ETBR, the immunoblot wasincubated with biotin-conjugated monoclonal anti-rabbitimmunoglobulins (Sigma-Aldrich) and probed with per-oxidase-conjugated streptavidin (Dako) or with peroxi-dase-conjugated donkey anti-mouse immunoglobulins forb-actin detection (Jackson Immunoresearch, Suffolk, UK).Immunoreactivity was visualized by chemiluminescenceand quantified with Multi Gauge v2.3 software (FujiPhoto Film Co., Ltd, Tokyo, Japan). Band densities werecompared by one-way analysis of variance followed byBonferroni’s multiple comparison test performed with theSPSS statistical package v11.5.1 (SPSS, Chicago, IL, USA).

Reverse Transcription and Quantitative Real-TimePCR

cDNA was generated using the iScript cDNA Synthesis Kit(Bio-Rad Laboratories, Hercules, CA, USA) with a mixtureof random hexamers and oligo(dT) primers, according tothe manufacturer’s instructions, except that for ETBR andCx43 expression analyses, the incubation time at 421C wasincreased from 30 to 60 mins. The reverse transcriptionwas run in duplicate in 10-mL reactions (Stahlberg et al,2004) using 1.5 mg of total RNA extracted from primaryastrocyte cultures as described below. The cultures wereprepared from four WT and four GFAP–/–Vim–/– mice (forETBR and Cx43), and five WT and three GFAP–/–Vim–/–

mice (for PAI-1). Gene-specific SYBR-Green I-based PCRassays were designed for ETBR (GenBank accessionnumber NM_007904), PAI-1 (GenBank accession numberM33960), and Cx43 (GenBank accession number M63801).Formation of expected PCR products was confirmed withagarose gel electrophoresis and melting curve analysis.The primer sequences were as follows: 50-AAGAATGCCCAAGAGAAAAC-30 (forward) and 50-AAAAAGGAAGGAAGGAAAATC-30 (reverse) for ETBR; 50-CAGACAATG GAAGGGCAACA-30 (forward) and 50-GCGGGCTGAGATGACAAA-30 (reverse) for PAI-1; and 50-ACCCAACAGCAGCAGACTT-30 (forward) and 50-ACCGACAGCCACACCTTC-30

(reverse) for Cx43. Real-time PCR experiments wererun on a Rotor-Gene 3000 (Corbett Research, Sydney,Australia) and were analyzed as described (Stahlberg et al,2004). All gene expression data were normalized to totalRNA concentration. The statistical significance of differ-ences between GFAP–/–Vim–/– and WT mice was tested


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with two-tailed t-tests. Polymerase chain reaction runconditions and thermocycler programs are available onrequest.

Scrape Loading/Dye Transfer

Astrocyte gap-junctional communication (AGJC) wasassessed by using the scrape loading/dye transfer techni-que as described (Blomstrand et al, 1999). In brief,confluent astrocyte cultures from five WT and fourGFAP–/–Vim–/– mice were incubated for 7 mins in HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buf-fered salt solution (137 mmol/L NaCl, 5.4 mmol/L KCl,0.41 mmol/L MgSO4, 0.49 mmol/L MgCl2, 1.26 mmol/LCaCl2, 0.64 mmol/L KH2PO4, 3 mmol/L NaHCO3,5.5 mmol/L glucose, and 20 mmol/L HEPES (pH 7.4)),with or without a nonsaturating dose of 10 nmol/Lof endothelin-3 in this and subsequent steps, and followedby four quick washes in Ca2 +-free buffer. Luciferyellow (0.1% w/v) in Ca2 +-free buffer was added, andtwo parallel scrapes were performed with a scalpel.The Lucifer yellow was removed after 1 min, andCa2 +-containing buffer was added. 8 mins after scraping,four images from each culture were captured with a NikonOptiphot 2 equipped with a Hamamatsu C5810 chilledthree-chip color charge-coupled device camera. The dyespread was quantitated by using Easy Image Analysis 2000(Tekno Optik AB, Stockholm, Sweden) to measure thefraction of a predefined area occupied by Lucifer yellow-positive cells.

Glutamate Uptake

Fresh brain slices microdissected from five WT and nineGFAP–/–Vim–/– mice were homogenized in 5 mmol/L Tris/320 mmol/L sucrose with a protease inhibitor cocktail(Complete; Roche, Indianapolis, IN, USA), using a hand-held pellet pistol. Samples were then washed three timesin ice-cold PBS (pH 7.4) and resuspended in Na+-containing Krebs buffer (120 mmol/L NaCl, 25 mmol/LTris–HCl, pH 7.4, 5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L KH2PO4, 1 mmol/L MgSO4, and 10% glucose) or inNa+-free Krebs (120 mmol/L choline-Cl substituted forNaCl). The samples were then incubated with 10 mmol/L3H-glutamate for 4 mins at 371C, harvested with a tissueharvester (Brandel, Gaithersburg, MD, USA) on a What-man GF/B paper (FPD-100; Brandel), and washed threetimes in Tris buffer (pH 7.4). Radioactivity was deter-mined by standard scintillation counting. MeanNa+-dependent uptake was determined in duplicate bysubtracting the mean uptake in a Na+-free duplicate.To determine the contribution of GLT-1-mediated trans-port, a separate set of samples was also incubated with500mmol/L dihydrokainate and 10 mmol/L 3H-glutamatefor 4 mins at 371C, harvested, and washed three times inTris buffer (pH 7.4) on the Whatman GF/B paper using atissue harvester. Radioactivity and mean Na+-dependentuptake were determined as described above.

Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis and Immunoblotting for GlutamateTransporter-1

Brain slices (approximately 2� 2 mm) taken fromthe cortex of the left hemisphere of six WT andnine GFAP–/–Vim–/– mice were homogenized by sonicationin Tris-EDTA (ethylenediaminetetraacetic acid) buffercontaining protease inhibitors (aprotinin, pepstatin,and leupeptin). Samples were stored at �701C untiluse. Aliquots of homogenized samples (1 to 20 mg protein)were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (8% polyacrylamide gels) andtransferred to nitrocellulose membranes (Hybond ECL,Amersham, Buckinghamshire, UK) by electroblotting(100 V, 60 mins). Western blot analysis was performedas described (Rothstein et al, 1994). In brief, blotswere blocked and washed in 50 mmol/L Tris-bufferedsaline containing 5% nonfat milk and 0.5% Tween 20,and probed with affinity-purified polyclonal antibodiesspecific for GLT-1 (1:10,000; C terminus-directed anti-body) using horseradish peroxidase-conjugated donkeyanti-rabbit IgG (1:5,000; Amersham) as secondary anti-body. The blots were also probed for actin (1:2,000) as aprotein-loading standard. In all cases, protein levelsvaried by < 10% between lanes. Immunoreactivity wasvisualized by enhanced chemiluminescence and quanti-fied densitometrically with Quantity One v4.0 software(Bio-Rad Laboratories).

DNA Array Analysis

RNA extraction, DNase treatment, poly (A + ) RNAenrichment, probe synthesis, column chromatography,and hybridization of the probes to the array wereperformed as described in the Atlas Pure Total RNALabeling System User Manual and the Atlas cDNAExpression Arrays User Manual (Clontech Laboratories,Palo Alto, CA, USA). Confluent astrocyte cultures wereharvested by scraping into RNase-free PBS and centri-fuged at 500g for 5 mins at 41C. The arrays were exposed tophosphor screens (Molecular Dynamics, Buckingham-shire, UK) overnight. The screens were scanned in aStorm instrument (Storm 820, Molecular Dynamics), andthe images were analyzed with AtlasImage 1.5 (ClontechLaboratories).

Results

Larger Infarct Volume in GFAP–/–Vim–/– than WT Miceafter Proximal Middle Cerebral Artery Transection

Seven days after proximal MCA transection, infarctvolume was 257% higher in GFAP–/–Vim–/– micethan in WT controls (14.073.20 vs 5.4371.81 mm3,P < 0.05; Figures 1A and 1B). No difference wasfound in the extent of brain edema (1.0070.08 vs1.0470.08, respectively).


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Larger Infarct Volume in GFAP–/–Vim–/– than WT Miceafter Distal Middle Cerebral Artery Transection

To determine if the transection point affectsthe difference in the infarct volume between WTand GFAP–/–Vim–/– mice, we transected theMCA more distally (Figure 1E). In the first set ofexperiments, the infarct volume was 351% larger inGFAP–/–Vim–/– than in WT mice 7 days after distalMCA transection (4.6070.62 vs 1.3170.40 mm3,P < 0.001; Figures 1C and 1D). To determineif partial deficiency of IFs in astrocytes or deficiencyof IFs in endothelial cells also affected theinfarct volume, single GFAP–/– and Vim–/– micewere included in a second experimental series.The infarct volume was 214% larger in GFAP–/–-

Vim–/– than in WT mice (4.7170.62 vs 2.2070.40 mm3, P < 0.01; Figure 1D), and there wereno significant differences in the infarctvolume between GFAP–/– and Vim–/– mice (1.3470.62 and 1.3270.46 mm3, respectively) and WTmice (Figure 1D).

Median Blood Pressure and Heart Rate andCerebrovascular Architecture are not Altered inGFAP–/–Vim–/– Mice

To address a possible primary effect of the absenceof GFAP and Vim on blood pressure and cerebro-vascular architecture, we recorded blood pressureand heart rate during and after the MCA transectionand compared the anastomoses between branches ofthe MCA and the anterior cerebral artery. During theMCA transection and for 60 mins thereafter, meanblood pressure and heart rate did not differ in WTand GFAP–/–Vim–/– mice (data not shown). The linesof anastomoses between branches of the MCA andthe anterior cerebral artery were comparable in WTand GFAP–/–Vim–/– mice (Figure 2).

Normal Number of Astrocytes and Microglia in thePenumbra of GFAP–/–Vim–/– Mice

Next, we examined the penumbra and the infarctarea histologically. Except for the larger infarct areain GFAP–/–Vim–/– mice, hematoxylin/erythrosinestaining showed no difference in the appearanceof the penumbra and the infarct region betweenWT and GFAP–/–Vim–/– mice. Immunostaining forS100b revealed comparable numbers of astrocytes inthe two groups (62.1373.29 vs 57.3373.44 per0.25 mm2 for WT and GFAP–/–Vim–/– mice, respec-tively; P = 0.37), although GFAP–/–Vim–/– astrocyteshad less prominent hypertrophy of the main cellularprocesses (Figures 3A and 3B). Comparable numbersof astrocytes in the two groups were also confirmedby GS immunostaining (data not shown). Tomatolectin staining showed no differences in the appear-ance, distribution, and number of microglial cells in

the penumbra and the infarct region between WTand GFAP–/–Vim–/– mice (54.572.84 vs 61.3375.24per 0.0625 mm2, respectively; P = 0.27 and data notshown).

Absence of Endothelin B Receptor-Immunoreactivityin Astrocytes in the Ischemic Penumbra ofGFAP–/–Vim–/– Mice

Endothelin B receptor expression by astrocytes inthe injured CNS was proposed as one of thesteps leading to astrocyte activation and reactivegliosis (Koyama et al, 1999). In the ischemicpenumbra and in the corpus callosum 7 daysafter MCA transection, ETBRs were highly expressedon reactive astrocytes in WT mice (Figures 3Eand 3K) but were essentially undetectable onastrocytes of GFAP–/–Vim–/– mice (Figures 3F and3L). Similarly, in WT astrocytes in cultures preparedfrom postnatal day 1 mouse brains, ETBRs werereadily detectable and the ETBR immunoreactivityexhibited a characteristic filamentous appearance(Figure 4). In contrast, ETBR immunoreactivity wasundetectable in the cytoplasm of GFAP–/–Vim–/–

astrocytes (Figure 4). Interestingly, numerousGFAP–/–Vim–/– astrocytes exhibited a granular ETBRimmunoreactivity in the cell nucleus (Figure 4).This finding suggests that IFs are required for the

Figure 2 The territory supplied by the MCA is comparable inWT and GFAP–/–Vim–/– (GV) mice. Comparison of the line ofanastomoses (A) at 2, 4, and 6 mm from the frontal pole (B).Values are mean7s.e.m.


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production, stability, or distribution of ETBRs inreactive astrocytes.

Endothelin B Receptor Immunoreactivity Colocalizeswith Intermediate Filament Bundles in ReactiveAstrocytes

To investigate further the relationship between ETBRand astrocyte IFs, we analyzed their distributionsin cultured astrocytes by laser-scanning confocalmicroscopy. This analysis revealed remarkablecolocalization between bundles of IFs and ETBRimmunoreactivity in WT astrocytes, with theETBR immunoreactivity decorating bundles of IFs(Figure 5).

Intermediate Filaments Determine Endothelin BReceptor Distribution in Reactive Astrocytes

To determine if IFs are required for the production,stability, or distribution of ETBR in reactiveastrocytes, we performed quantitative real-timePCR and Western blot analyses on cultured astro-cytes from GFAP–/–Vim–/– and WT mice. EndothelinB receptor mRNA levels were 43% higher inGFAP–/–Vim–/– than in WT astrocytes (P < 0.05), butthe amounts of ETBR protein were comparable(Figure 6). Thus, in the absence of IFs, astrocytescontain a normal amount of ETBRs, which do notassociate with IFs but seem to be distributedthroughout the cell.

Figure 3 Astrocytes around the ischemic lesion show ETBR immunoreactivity in WT but not GFAP–/–Vim–/– (GV) mice. (A and B) Nodifference was detected in the distribution of astrocytes in the penumbra in the two groups, although astrocytes of GFAP–/–Vim–/– miceshowed less prominent hypertrophy of cellular processes. Green, S100b; blue, cell nuclei visualized by ToPro-3. Reactive astrocytesadjacent to the ischemic lesion in WT mice (visualized by antibodies against GFAP, C and I) were ETBR-positive (E and K) 7 days afterMCA transection. (F and L) In contrast, no ETBR immunoreactivity was detected in astrocytes in GFAP–/–Vim–/– mice. (G and M)Merged images of (C and E) and of (I and K), respectively. (C to N) Red, GFAP; green, ETBR; blue, nuclei visualized by ToPro-3. Scalebar = 50 mm.


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Endothelin-3-Induced Blockage of Gap Junctions isAttenuated in GFAP–/–Vim–/– Astrocytes

Endothelins are well-known blockers of astrocytegap-junctional communication (AGJC) (Blomstrand

et al, 1999). To determine if the altered cytoplasmicdistribution of ETBRs affects AGJC and thus thefunction of the astrocyte syncytium, we stimulatedETBRs with the selective ligand endothelin-3. Nosignificant difference in basal AGJC was observedbetween WT and GFAP–/–Vim–/– astrocytes, butendothelin-3-mediated inhibition of AGJC was lessprominent in GFAP–/–Vim–/– than in WT astrocytes(72.378.9 vs 39.872.6% of control levels; P < 0.01;Figure 7A). Gap junctions are made of connexins,with Cx43 being the most predominant gap junctionprotein in astrocytes (Giaume and McCarthy, 1996).Using quantitative real-time PCR, we found thatCx43 mRNA expression was 44% higher inGFAP–/–Vim–/– in than WT astrocytes (P < 0.05; Figure7B), further linking astrocyte IFs and AGJC.

Reduction in Glutamate Transport in GFAP–/–Vim–/–

Mice

The ability of astrocytes to remove glutamatereleased by neurons was proposed to reduce infarctsize by limiting the excitotoxic cell death (reviewedin Nedergaard and Dirnagl, 2005). To determine ifglutamate transport is altered in GFAP–/–Vim–/– mice,we examined total glutamate uptake and glutamateuptake mediated by GLT-1, the primary glutamatetransporter in astrocytes, in freshly dissected hemi-cortices of GFAP–/–Vim–/– and WT mice. Mean totalglutamate uptake in GFAP–/–Vim–/– mice was 44%lower than in WT controls (1.9970.79 vs 3.537

Figure 4 ETBR immunoreactivity had a filamentous appearancein the cytoplasm of cultured WT astrocytes but was absent inthe cytoplasm of GFAP–/–Vim–/– astrocytes, where it was oftenconfined to the cell nuclei. Green, ETBR. Scale bar = 50 mm.

Figure 5 ETBR and bundles of IFs colocalize in cultured WT astrocytes. Laser-scanning confocal microscopy revealed a filamentousappearance of ETBR immunostaining, which colocalized with GFAP-positive bundles of IFs. Red, GFAP; green, ETBR. Scalebar = 20mm.


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0.17 nmol/mg per min; P < 0.05). Similarly,GLT-1-dependent transport was 50% lower thanin WT mice (1.3870.78 vs 2.8570.29 nmol/mgper min, P < 0.05; Figure 8A). Western blot analysisshowed comparable amounts of GLT-1 in WT andGFAP–/–Vim–/– cerebral cortex (2273 vs 2074 U;Figures 8B and 8C) and immunohistochemicalanalyses of the ischemic penumbra showed lessprominent GLT-1 immunostaining in astrocytes ofGFAP–/–Vim–/– compared with WT mice (Figure 8D).Thus, astrocytes of GFAP–/–Vim–/– mice are lesscapable of removing glutamate from the ischemicbrain tissue.

Plasminogen Activator Inhibitor-1 is Downregulatedin GFAP–/–Vim–/– Astrocytes

To address further, at a molecular level, the effect ofattenuated reactive gliosis, we performed microarrayanalysis with the Atlas cDNA Expression Arrays(Clontech Laboratories). The expression of 1,200genes was analyzed in primary astrocyte culturesderived from WT and GFAP–/–Vim–/– mice andmaintained in the presence of 10% serum. Only asingle gene, PAI-1, an inhibitor of tissue plasmino-gen activator (tPA), fulfilled the criteria of a three-fold or higher downregulation in GFAP–/–Vim–/– than

Figure 6 ETBR protein levels are comparable in WT and GFAP–/–Vim–/– (GV) cultured astrocytes. (A) Coomassie blue stainingconfirmed equal loading of protein. (B) Western blot analysis showed comparable levels of ETBR protein in WT and GFAP–/–Vim–/–

astrocytes. b-Actin was used as a control. (C) Quantification of ETBR protein levels from (B). (D) Quantitative real-time PCR showedhigher levels of ETBR mRNA in GFAP–/–Vim–/– than in WT astrocytes (*P < 0.05). Values are mean7s.e.m. AU, arbitrary units.


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in WT astrocytes (the ratio was 0.3 for GFAP–/–-

Vim–/–/WT astrocyte cultures). Next, we performedquantitative real-time PCR analysis of PAI-1 mRNAin primary GFAP–/–Vim–/– and WT astrocytes main-tained in the presence of either 1 or 10% serum, thelatter mimicking some aspects of reactive gliosis.Compared with WT, GFAP–/–Vim–/– astrocytesshowed a 69% reduction in PAI-1 mRNA in the

presence of 1% serum (P < 0.05), and an 86%reduction in 10% serum (P < 0.01; Figure 9).

Discussion

In the absence of a suitable experimental model,direct proof that astrocytes have a protective effect

Figure 7 (A) Endothelin-3 (ET3)-induced blockage of gapjunction is attenuated in GFAP–/–Vim–/– (GV) astrocytes(*P < 0.05 and ***P < 0.001). (B) Quantitative real-timePCR shows higher levels of connexin 43 (Cx43) mRNA inGFAP–/–Vim–/– than in WT astrocytes (*P < 0.05). Values aremean7s.e.m.

Figure 8 Total and GLT-1-mediated glutamate uptake isdecreased in the cortex of GFAP–/–Vim–/– (GV) mice. (A) Totaland GLT-1-mediated glutamate uptake was lower in GFAP–/–-

Vim–/– than in WT mice (*P < 0.05). (B) Western blot analysisshowed comparable levels of GLT-1 protein in WT andGFAP–/–Vim–/– astrocytes. Actin was used as a control. (C)Quantification of GLT-1 protein levels from (B). Values are mean7s.e.m. (D) Glutamine synthase (GS)-positive astrocytes adja-cent to the ischemic lesion in WT mice show distinct GLT-1immunoreactivity, whereas in GV mice, GLT-1 immuno-reactivity is less prominent. Arrowheads, astrocytes. Green, GLT-1; red, GS; nuclei visualized by ToPro-3. Scale bar = 25mm.


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in the ischemic brain has largely been lacking.Attempts to generate mammalian models in whichastrocytes would be globally or regionally elimi-nated resulted in phenotypes too severe or complexfor studying the role of astrocytes in the pathogen-esis of CNS diseases. In mice expressing herpessimplex virus thymidine kinase from the GFAPpromoter, ablation of dividing astroglial cells bytreatment with ganciclovir during early postnataldevelopment led to severe developmental abnorm-alities (Delaney et al, 1996). The same approach wasapplied in adult mice to selectively eliminate thesubpopulation of dividing reactive astrocytes afterneurotrauma and resulted in a massive invasion ofleukocytes in response to dying astrocytes, in-creased neurodegeneration, and failed blood–brainbarrier (BBB) repair (Bush et al, 1999), implyingneuroprotective activities of astrocytes in thelesioned area (Faulkner et al, 2004).

Upregulation of IFs is a hallmark of reactivegliosis after trauma or stroke and is a key step inastrocyte activation (Pekny et al, 1999b). To addressthe role of reactive astrocytes in stroke, we subjectedGFAP–/–, Vim–/–, and GFAP–/–Vim–/– mice, which lackone or both of the key components of IFs in reactiveastrocytes, to proximal or distal MCA transection. Inthree independent experiments, GFAP–/–Vim–/– micehad 2- to 3.5-fold larger infarct volumes after 7 daysof ischemia than WT controls. The infarct volumesin GFAP–/– or Vim–/– mice were not significantlydifferent from those in WT mice. The increasedinfarct volumes in GFAP–/–Vim–/– mice were linkedto an altered distribution of ETBR, less efficientETBR-mediated inhibition of AGJC, reduced abilityto take up glutamate, and lower levels of tPAinhibitor PAI-1 in IF-free GFAP–/–Vim–/– astrocytes.These results provide evidence that reactive astro-cytes have a protective role in brain ischemia.

In GFAP–/– mice, reactive astrocytes containreduced amounts of IFs, which are composed of

vimentin and nestin (Eliasson et al, 1999; Peknyet al, 1998a). Reactive astrocytes in Vim–/– mice alsocontain reduced amounts of IFs, and the IF bundlesare abnormally compact, reflecting reduced spacebetween individual filaments (Eliasson et al, 1999).Despite these abnormalities, reactive astrocytes inthese single-mutant mice were not compromisedwith regard to their function in stroke. However,the complete absence of IFs in reactive astrocytesin GFAP–/–Vim–/– mice profoundly affected infarctvolume. These results are consistent with thefindings that healing after brain or spinal cordtrauma was prolonged and synaptic loss wasincreased in GFAP–/–Vim–/– mice, which have acomplete deficiency of IFs in reactive astrocytes,but there was normal healing in GFAP–/– or Vim–/–

mice, which have only a partial deficiency (Menetet al, 2003; Pekny et al, 1999b; Wilhelmsson et al,2004). However, we cannot exclude the possibilitythat partial IF deficiency had a subtle effect thatescaped detection. In another study, GFAP–/– miceand WT controls had similar infarct volumes after 2days of permanent MCA occlusion; however, whenMCA occlusion was combined with transient occlu-sion of the carotid artery, the GFAP–/– mice hadlarger infarcts than the controls (Nawashiro et al,2000).

Vimentin is also expressed in other cell types inthe CNS, particularly endothelial cells, oligoden-droglia, and microglia. Endothelial cells in Vim–/–

mice are devoid of IFs because the other IF proteinexpressed in these cells, nestin, cannot formfilaments on its own (Eliasson et al, 1999; Peknyet al, 1999b). Thus, with regard to IF production inreactive astrocytes, the only difference betweenVim–/– and GFAP–/–Vim–/– mice is the partial orcomplete absence, respectively, of IFs. Since strokevolumes were not significantly different in Vim–/–

mice and WT controls, the absence of IFs inendothelial cells and other cell types does notaffect infarct size. GFAP–/–Vim–/– and WT mice hadcomparable cerebrovascular architecture, as well ascomparable mean blood pressure and heart ratebefore and during the focal brain ischemia. Thisimplies that the increased infarct size after MCAtransection in GFAP–/–Vim–/– mice is a consequenceof the absence of IFs in astrocytes.

How might the absence of IFs in reactive astro-cytes explain the increased infarct size inGFAP–/–Vim–/– mice? The lack of IFs may compro-mise several functions of astrocytes, namely AGJC,uptake and transport of molecules such as gluta-mate, glucose, and ascorbate, protection against tPA-mediated neurotoxicity, reconstruction of damagedBBB, migration into the injured area, and regulationof cell volume.

We saw a close colocalization between bundlesof IFs and ETBR immunoreactivity in cultured WTastrocytes and altered ETBR immunoreactivity inGFAP–/–Vim–/– astrocytes both in vitro and in vivo.These findings suggest that ETBRs associate with IFs

Figure 9 Plasminogen activator inhibitor-1 mRNA levels arelower in GFAP–/–Vim–/– (GV) than in WT astrocytes cultured inthe presence of 1 or 10% fetal calf serum (**P < 0.01 and*P < 0.05). Values are mean7s.e.m.


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and are freely distributed throughout the cell inIF-free astrocytes, which also exhibit less efficientETBR-mediated inhibition of AGJC. Astrocyte gap-junctional communication was proposed to promotesecondary expansion of focal injury, since openastrocytic gap junctions can mediate the propaga-tion of cell death signals or undesirable backflow ofATP from living to dying cells (Lin et al, 1998).Thus, less efficient ETBR-mediated inhibition ofAGJC in GFAP–/–Vim–/– mice may have contributed tolarger infarct volume in these mice.

Astrocytes play a major role in the transport andmetabolism of a range of molecules, in particularthose utilized in nutrition, cell–cell signaling, andneurotransmission. A failure to carry out thesefunctions may constitute a major pathogenic com-ponent in stroke and other CNS pathologies (re-viewed in Kraig et al, 1995). In primary cultures ofreactive astrocytes from WT, GFAP–/–, Vim–/–, andGFAP–/–Vim–/– mice, we found no differences in theirability to transport glucose and ascorbate (Peknyet al, 1999a). Previously, we showed that despitecomparable availability of GS (Wilhelmsson et al,2004), glutamine levels in reactive astrocytes fromboth GFAP–/– and GFAP–/–Vim–/– mice were increasedby 70 to 100% (Pekny et al, 1999a). Our present datashow that glutamate transport is reduced in braincortices of GFAP–/–Vim–/– mice. It is tempting tospeculate that the reduced ability of the GFAP–/–-

Vim–/– mice to remove glutamate from the ischemicbrain tissue, possibly due to deficient intracellulartrafficking of GLT-1, is the consequence of intra-cellular glutamine accumulation. It was shownrecently that GLT-1 immunoreactivity in astrocytescolocalized with bundles of IFs (Shobha et al, 2007)and GLT-1 trafficking and cluster formation withinastrocyte processes were proposed to be dependenton the cytoskeleton (Zhou and Sutherland, 2004).Notably, GLT-1–/– mice show more extensive ischemia-induced damage (Mitani and Tanaka, 2003), andwhen GLT-1+ /– mice are crossed to superoxidedismutase-1 (G93A) mice, a model of amyotrophiclateral sclerosis, they have more prominent loss ofmotor neurons and muscle strength than SOD-1(G93A) mice alone (Pardo et al, 2006). Thesefindings indicate that glutamate transporter dys-function influences astrocyte–neuron interactionsafter injury.

Among 1,200 genes whose expression was com-pared between WT and GFAP–/–Vim–/– astrocytes,PAI-1 was the only gene that showed at least three-fold downregulation in the mutants. Quantitativereal-time PCR confirmed the downregulation ofPAI-1 mRNA in GFAP–/–Vim–/– astrocytes. Plasmino-gen activator inhibitor-1 inhibits tPA, which has aneurotoxic effect in the ischemic penumbra, prob-ably mediated through the activation of both micro-glia and N-methyl D-aspartate receptors (Sheehanand Tsirka, 2005). Mice deficient in tPA havesmaller infarcts than WT controls in a transientfocal ischemia model, and administration of tPA to

both tPA-deficient and WT mice increases infarctvolume (Tsirka et al, 1995). Similarly, mice over-expressing PAI-1 have smaller infarcts than WTcontrols after focal brain ischemia (Nagai et al,2005). Thus, reduced levels of PAI-1 mRNA couldenhance the neurotoxic effects of tPA in theischemic penumbra of GFAP–/–Vim–/– mice.

Reactive astrocytes play a key role in the recon-struction of damaged BBB after both trauma andstroke. In a previous study with the in vitro BBBmodel, we demonstrated that IF-deficient astrocyteswere either unable to induce BBB properties inendothelial cells or did so less efficiently (Peknyet al, 1998b). Thus, less efficient reconstruction ofthe BBB in the penumbra may contribute to theincreased infarct volume in GFAP–/–Vim–/– mice.

In response to CNS injury, astroglial cells migrateover considerable distances to the injured region,where they join the local pool of reactive astrocytes(Johansson et al, 1999). We have shown that partialor complete deficiency of IFs in reactive astrocytesin vitro reduces their ability to migrate (Lepekhinet al, 2001). However, since the overall number ofastrocytes and microglia around the infarct area didnot differ in GFAP–/–Vim–/– and WT mice, reducedmigration does not seem to contribute to theincreased infarct volume.

The ability of astrocytes to regulate their volumeby releasing osmotically active molecules, such astaurine, is considered to be a mechanism forcounteracting cytotoxic brain edema in stroke(Kimelberg, 1991). When subjected to hypotonicstress, GFAP–/–Vim–/– reactive astrocytes in vitrorelease 25 to 46% less taurine than WT controls,indicating a reduced capacity to counteract cellswelling (Ding et al, 1998) that could havecontributed to the increased infarct volumes inGFAP–/–Vim–/– mice. Consistent with this possibility,we showed that mice with IF-free nonreactiveastrocytes have more prolonged changes in extra-cellular space diffusion parameters than WT con-trols during cell swelling evoked by hypotonic stressor high [K+] (Anderova et al, 2001).

Previously, we showed that attenuation of reactivegliosis in GFAP–/–Vim–/– mice increases synaptic lossat the initial stage after neurotrauma, but allowscomplete synaptic regeneration later on (Wilhelmssonet al, 2004). Thus, reactive gliosis seems to play apositive role soon after CNS injury, although itseems to pose an obstacle to regeneration at a laterstage (reviewed in Pekny and Pekna, 2004). Theresults of a recent study using conditional ablationof Stat3 and Socs3 also point to a dual role ofreactive astrocytes after spinal cord injury (Okadaet al, 2006). Here we show a similar neuroprotectiveeffect of reactive gliosis in brain ischemia. Geneticablation of astrocyte IFs led to altered intracellulardistribution of ETBR immunoreactivity in reactiveastrocytes, both in the dentate gyrus affected byneurodegeneration induced by entorhinal cortexlesion (Wilhelmsson et al, 2004) and in ischemic


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tissue as shown here. Thus, less efficient ETBR-mediated inhibition of AGJC in GFAP–/–Vim–/– astro-cytes might contribute to both increased synapticloss in the acute stage after entorhinal cortex lesionand to larger infarct volume after focal brainischemia in these mice.

In summary, our findings provide in vivo evidencethat reactive astrocytes play a protective role inischemic stroke.

Acknowledgements

We thank Dr Anders Hamberger, Dr John Eriksson,and Hanna-Mari Pallari for their input in this projectand Dr Martin Rydmark for his advice on stereo-meric evaluation of the infarct volume.

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Protective role of reactive astrocytes in brain ischemia role of reactive astrocytes in brain ischemia LLiet al 2 Journal of Cerebral Blood Flow & Metabolism (2007), 1–14 image analysis