Chronic brain ischemia induces the expression of glial ...

Yatomi Y et al,

Chronic brain ischemia induces the expression of glial glutamate transporter

EAAT2 in subcortical white matter

Yuko Yatomi*, Ryota Tanaka*, Hideki Shimura, Nobukazu Miyamoto, Kazuo

Yamashiro*, Masashi Takanashi*, Takao Urabe, Nobutaka Hattori* 5

*Department of Neurology, Juntendo University School of Medicine, Tokyo, JAPAN

Department of Neurology, Juntendo University Urayasu Hospital, Tokyo, JAPAN

Running title: EAAT2 expression in chronic brain ischemia 10

Corresponding author:

Ryota Tanaka, MD, PhD

Department of Neurology,

Juntendo University School of Medicine 15

2-1-1 Hongo, Bunkyo ku,

Tokyo 113-0033, Japan

Tel: +81-3-3813-3111

Fax: +81-3-304-8615

E-mail: [email protected] 20

mailto:[email protected]

Yatomi Y et al,

Abstract

Glutamate plays a central role in brain physiology and pathology. The involvement of

excitatory amino acid transporters (EAATs) in neurodegenerative disorders including

acute stroke has been widely studied, but little is known about the role of glial 25

glutamate transporters in white matter injury after chronic cerebral hypoperfusion.

The present study evaluated the expression of glial (EAAT1 and EAAT2) and neuronal

(EAAT3) glutamate transporters in subcortical white matter and cortex, before and

3-28 days after the ligation of bilateral common carotid arteries (LBCCA) in rat brain.

K-B staining showed a gradual increase of demyelination in white matter after 30

ischemia, while there was no cortical involvement. Between 3 and 7 days after

LBCCA, a significant increase in EAAT2 protein levels was observed in the ischemic

brain and the number of EAAT2-positive cells also significantly increased both in the

cortical and white matter lesions. EAAT2 was detected in glial-fibrillary acidic protein

(GFAP)-positive astrocytes in both the cortex and white matter, but not in neuronal 35

and oligodendroglial cells. EAAT1 was slightly elevated after ischemia only in white

matter, but EAAT3 was at almost similar levels both in the cortex and white matter

after ischemia. A significant increase in EAAT2 expression level was also noted in the

deep white matter of chronic human ischemic brain tissue compared to the control

group. Our findings suggest important roles for up-regulated EAAT2 in chronic brain 40

ischemia especially in the regulation of high-affinity of extracellular glutamate and

minimization of white matter damage.

Yatomi Y et al,

Introduction 45

Glutamate is an essential neurotransmitter but its excess in the extracellular space is

excitotoxic to neuronal cells (Choi, 1992). Extracellular glutamate concentration is

regulated mainly through the reuptake of glutamate from the synaptic cleft via glial

excitatory amino acid transporters (EAATs). EAATs are Na+-dependent transporters;

three Na+ and one H+ are co-transported with each negatively charged glutamate 50

molecule and one K+ is counter-transported. In the normal brain, the high-affinity

plasma membrane glutamate transporters actively clear glutamate released in the

synaptic cleft to maintain glutamatergic homeostasis and prevent neurotoxicity

(Anderson and Swanson, 2000, Camacho and Massieu, 2006). Glutamatergic synaptic

dysfunction is the major mediator of excitotoxic neuronal death in neurodegenerative 55

disorders such as amyotrophic lateral sclerosis, Alzheimer’s disease (Francis, 2005),

and cerebral infarction (Kato and Kogure, 1999).

Five subtypes of glutamate transporters (EAAT1, 2, 3, 4 and 5) have so far

been identified in the central nervous system (Kanai and Hediger, 1992, Pines et al.,

1992). EAAT1 is predominantly found in the glial cells, EAAT2 is predominantly 60

present in astrocytes, EAAT3 and EAAT4 are predominantly found in neurons, and

EAAT4 and EAAT5 are, respectively, present in the retina and cerebellum (Danbolt et

al., 1998). EAAT2 is the most active glutamate transporter, and it regulates more than

90% of extracellular glutamate reuptake (Yamada et al., 2006).

Studies using cell cultures and experimental models described increases in 65

EAAT1 and EAAT2 after acute ischemia or hypoxia-ischemia (Arranz et al., 2010),

with selective overexpression of EAAT2 after moderate hypoxia-ischemia (Weller et

Yatomi Y et al,

al., 2008). Another study reported that administration of antisense oligonucleotides of

EAAT2 increased infarct volume and mortality after cerebral ischemia (Rao et al.,

2001b). These studies and others (Rao et al., 2001a, Kim et al., 2011) stress the 70

importance of EAAT2 changes in cerebral ischemia.

While changes in glial glutamate transporters have been reported in acute

ischemic models, there is little or no information on the regulation of excess glutamate

and changes in glial glutamate transporters in models of vascular dementia. The main

hypothesis of the present study was that chronic brain ischemia induces the expression 75

of glial glutamate transporter EAAT2 in subcortical white matter. To test this

hypothesis, we determined first the expression of glial glutamate transporters EAAT1,

2 and 3 by immunohistochemistry and immunoblotting both in the cortex and white

matter at different times after chronic cerebral hypoperfusion, ligation of bilateral

common carotid arteries (LBCCA), in rat brain. We also analyzed EAAT2 expression 80

in the human brain with damaged white matter in patients with Binswanger’s disease

and multiple lacunar infarction.

Experimental procedures

Animals and experimental design 85

Adult male Wistar rats (11-week-old) weighing 350-450 g were purchased from the

Charles River Institute (Kanagawa, Japan) and maintained on a 12-h light/dark cycle

with free access to food and water. To occlude both common carotid arteries, the rats

were anesthetized with 1–2% isoflurane in 30% oxygen and then anesthesia was

maintained with 70% nitrous oxide. During surgery, a temperature probe was inserted 90

Yatomi Y et al,

into the rectum, and a heat lamp was applied to maintain the body temperature at

37.0–37.5°C. Through a midline incision, each common carotid artery was carefully

separated from the cervical sympathetic and vagal nerves and ligated permanently.

Rats of the control group were sham-operated, which involved bilateral exposure of

the common carotid arteries. 95

Twenty-five rats were examined for histological changes and kept in cages

with food and water ad libitum. At days 3, 7, 14 and 28 after LBCCA, the rats were

re-anesthetized with 1% isoflurane and 70% N2O:30% O2, and then transcardially

perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde. The brain

was dissected out immediately, postfixed in 4% paraformaldehyde for 48 h, and stored 100

in 30% sucrose in 0.1 M PBS. For immunohistochemistry, 20-μm-thick free-floating

coronal sections of the corpus callosum were prepared for staining.

All animals were acquired and cared for according to the guidelines published

by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

All experiments described in this study were conducted after approval of the Animal 105

Care Committee of the Juntendo University.

Measurement of cerebral blood flow (CBF)

After LBCCA, CBF was measured in a left temporal window using laser Doppler

flowmetry (Laser tissue Blood Flow Meter FLO-C1; Omega Wave, Inc., Portland, 110

OR). The probe in the shape of straight rectangular sheet (7.5 mm in length and 1.0

mm in-depth) was positioned between the temporal muscle and the lateral aspect of

the skull, as described previously (Harada et al., 2005). In these experiments, there

Yatomi Y et al,

was no need for craniotomy. CBF was monitored continuously for 3-5 min at each

time, before, immediately and after, and at days 3, 7, 14 and 28 after LBCCA. 115

Reproducible recorded CBF velocity was obtained.

Immunohistochemistry

Immunohistochemistry was performed on 20-μm-thick free- floating coronal sections,

which were prepared as described previously (Miyamoto et al., 2010). After 120

incubation in 3% H2O2 followed by 10% block ace in 0.1% PBS(-), the brain sections

were immunostained overnight at 4°C using rabbit antibody against excitatory amino

acid transporter 1 (EAAT1; dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz,

CA), rabbit antibody against excitatory amino acid transporter 2 (EAAT2; dilution,

1:300; (Sasaki et al., 2009)), rabbit antibody against excitatory amino acid transporter 125

3 (EAAT3; dilution, 1:500; Abcam, Cambridge, MA), anti-glutathione-S-transferase

pi antibody (GSTπ, dilution, 1:500, Medical and Biological Laboratories, Nagoya,

Japan), anti-NG2 chondroitin sulfate proteoglycan (NG2, dilution, 1:100, Millipore,

Bedford, MA), anti-glial-fibrillary acidic protein (GFAP, dilution, 1:500; Dako Corp.,

Carpinteria, CA), anti-ionized calcium-binding adapter molecule-1 (Iba-1, dilution, 130

1:500, Wako Pure Chemical Industries, Osaka, Japan) and anti neuronal nuclear

antigen (NeuN, dilution, 1: 100, Millipore), and finally treated with secondary

antibodies (dilution, 1:500, Vectastain; Vector Laboratories, Burlingame, CA).

Immunoreactivity was visualized using the avidin–biotin complex method

(Vectastatin). Negative control sections were stained using the above-mentioned 135

immunohistochemical technique with the omission of the primary antibodies. The

Yatomi Y et al,

images were captured using a digital camera (DXM1200, Nikon, Tokyo, Japan)

attached to an Olympus CX40 microscope (Olympus, Tokyo) and analyzed using the

ACT-1 image system (version 2.20, Nikon).

140

Double immunofluorescence histochemistry

Double immunofluorescence staining was performed by simultaneous incubation of

the sections overnight at 4°C with mouse anti-GFAP (dilution, 1:500; Medical and

Biological Laboratories), mouse anti-adenomatous polyposis coli (APC, dilution,

1:100; Calbiochem, Darmstadt, Germany), mouse anti-NG2 (dilution, 1:50; Santa 145

Cruz Biotechnology), anti-platelet derived growth factor receptor-α (PDGFRα,

dilution, 1:100; Santa Cruz Biotechnology), anti-NeuN (dilution, 1:500; Millipore),

and goat anti-Iba-1 (dilution, 1:500; Abcam) antibodies. After overnight incubation

with the primary antibody, the sections were immunostained with

fluorochrome-conjugated secondary antibody (Cy3- or fluorescein isothiocyanate, 150

dilution 1:500, Jackson Immunoresearch Laboratories, West Grove, PA), and then

covered with Vectashield mounting medium (Vector Laboratories).

Klüver-Barrera staining

The corpus callosum was evaluated for white matter lesion by Klüver-Barrera staining. 155

The myelin areas in three sections per animal and both sides of the selected areas were

stained with Luxol Fast Blue. The severity of white matter lesion was graded as 0

(normal), 1 (disarrangement of nerve fibers), 2 (formation of marked vacuoles), and 3

(disappearance of myelinated fibers), according to the previously described grading

Yatomi Y et al,

system (Wakita et al., 2002). 160

SDS-PAGE and immunoblotting

Rats of each group were decapitated at 3, 7, 14 and 28 days after LBCCA (n=5 per

time point for each group). Proteins were extracted from the white matter area and

analyzed as described previously (Kobayashi et al., 2002). Briefly, aliquots containing 165

30 μg of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). The protein bands were electrotransferred onto

polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA).The

membranes were subsequently incubated with the following primary antibodies:

anti-EAAT1 (dilution, 1: 500 ), anti-EAAT2 (dilution, 1 :1000), anti-EAAT3 (dilution, 170

1 :1000) or anti-α-tubulin antibody (dilution, 5000:1, Santa Cruz Biotechnology).

After incubation with the appropriate horseradish peroxidase- conjugated secondary

antibody (dilution, 20,000:1, Amersham Life Science, Buckinghamshire, UK) for 1 h

at room temperature, immunoreactive bands were visualized in the linear range by

using an enhanced chemiluminescence Western blotting system (Amersham). Equal 175

protein loading was confirmed by measuring α-tubulin.

Cell count and statistical analysis

In each of the EAAT1-, 2-, 3-, GFAP-, GSTπ-, and Iba-1-stained sections, the

numbers of stained cells in white matter lesions (0.25 mm2) in predefined areas (SVZ, 180

RMS, granular cell layer of olfactory bulb (Grl)), were counted (n=5 rats for each

group) by an investigator blinded to the experimental group, using Axio-Vision

Yatomi Y et al,

software (Carl Zeiss, Jena, Germany). One-way analysis of variance (ANOVA)

followed by post hoc Fisher’s protected least significant difference test was used to

examine the differences in various parameters among the different groups. A P value 185

<0.05 denoted the presence of a statistically significant difference.

Autopsy Specimens

Postmortem human brains of six patients (three with chronic ischemia and three

control) were obtained from Juntendo University School of Medicine. The clinical 190

data of these six subjects are summarized in Table 1. The Human ethics review

committee of Juntendo University approved this part of the study. Formalin-fixed

paraffin-embedded samples from the white matter frontal lobe were immunostained.

Cerebral hemispheres were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate

buffer, pH 7.4, and embedded in paraffin. The cortex and white matter from the 195

frontal lobe were cut into 30-μm-thick sections. After removal of paraffin, antigens

were retrieved with autoclaving and endogenous peroxidase activity was quenched for

30 min with 3% H2O2 in methanol. After incubation in 3% H2O2 followed by 10%

block ace in 0.1% PBS(-) for 1 h, the brain sections were immunostained overnight

with rabbit antibody against EAAT2 (dilution, 1:300; (Sasaki et al., 2009)) at 4°C 200

followed by treatment with secondary antibodies (dilution, 1:500, Vectastain; Vector

Laboratories). Immunoreactivity was visualized using the avidin–biotin complex

method (Vectastatin). The images were captured using a digital camera (DXM1200,

Nikon) attached to an Olympus CX40 microscope (Tokyo) and analyzed using the

ACT-1 image system (version 2.20, Nikon). The numbers of stained cells found in the 205

white matter were counted in EAAT2 immunohistochemistry section. One way t-test

Yatomi Y et al,

followed by post hoc Fisher’s protected least significant difference test was used to

examine differences in various parameters among the different groups.

Results 210

Histological findings

Hypoperfusion was confirmed by reduction in CBF after LBCCA relative to the

baseline (before LBCCA), whereas no significant change was noted in control rats

(post-LBCCA: 35% immediately after, 55% at day 3, 66% at day 7, 78% at day 14 215

and 83% at day 28, relative to the baseline) (Fig. 1). Extensive changes in the

Klüver-Barrera staining pattern appeared in the corpus callosum at 7 days after

LBCCA. After the initial changes of disarrangement of the nerve fibers in the corpus

callosum, the myelinated fibers were reduced in number while the remaining fibers

showed further and gradual disorganization. The numbers of intracellular vacuoles 220

increased in the corpus callosum at day 28 after LBCCA. There was no significant

hemorrhage or infarction in any brain region. The overall grade of white matter injury

was significantly higher from day 14 after ischemia compared with the control (Fig. 2).

In comparison, no changes were noted in the cerebral cortex after ischemia (data not

shown). 225

The density of astrocytes positive for GFAP in the white matter at 3 and 7 days

in the LBCCA group was significantly higher than in the sham group, although the

density gradually returned to the baseline level. In the cerebral cortex, the number of

GFAP-positive cells was significantly higher at day 3 after ischemia, and the increase

Yatomi Y et al,

continued until day 28 post-LBCCA. The number of GSTπ-positive oligodendrocytes 230

was transiently higher at days 7 and 14 days after ischemia but was significantly lower

at day 28 post-LBCCA than the control (Fig. 3C). On the other hand, the number of

NG2-positive oligodendrocyte progenitor cells in the white matter increased

gradually and significantly from 7 days after ischemia relative to the control (Fig. 3D).

Although the number of Iba-1 positive microglial cells was significantly higher at day 235

3 post-LBCCA, it decreased gradually after day 7 post-LBCCA (Fig. 3E).

Changes in expression of EAATs

The expression of EAAT1, EAAT2 and EAAT3 was evident in both the cerebral

cortex and white matter of the normal brain. The expression level of EAAT2 was 240

relatively intense in both areas compared to EAAT1 and EAAT3. In the normal brain,

immunostaining for EAAT2 was observed mainly in astrocytes in both areas, while

immunostaining for EAAT1 and EAAT3 was detected in oligodendrocytes present in

the white matter area and also in cortical neurons (Fig. 4a, f, k, p).

The detection of EAAT1, EAAT2 and EAAT3 signals in western blot analysis 245

prompted a time-course study of EAAT expression at days 3, 7, 14, and 28

post-ischemia. EAAT2 protein appeared as a band of ~62 kDa in the subcortical white

matter tissue, and quantitative analysis indicated a significant increase in the protein at

day 3 post-ischemia (to 123% of the baseline), which persisted up to day 14 (P <

0.001), consistent with the results of immunohistochemical analysis. Further analysis 250

showed a gradual reduction at day 14 post-ischemia, with EAAT2 protein level

reduced at ~90% of the control at day 28 post-LBCCA. Analysis of the cerebral cortex

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for serial changes in EAAT2 protein level showed a pattern of changes in this protein

similar to that observed in the white matter, with a moderate increase of EAAT2

protein at days 3 and 7 post-LBCCA. 255

Analysis of EAAT1 protein level showed a slight increase at 3 days (to 114%

of the baseline), and a further increase at day 7 post-ischemia (to 116% of the

baseline) (P < 0.05). However, EAAT1 protein level decreased after day 14

post-LBCCA. In comparison, EAAT1 protein level was observed in 84% of the

control animals at day 28 post-ischemia. Only a negligible increase in EAAT3 protein 260

level was observed in the white matter and cortex, but the change was not significant

compared to the control (Fig. 5).

Immunohistochemical staining showed more intense and coherent staining for

EAAT2 in the white matter than in other areas. Staining for EAAT2 at day 1

post-LBCCA was similar to the control brain (data not shown), but a significant 265

increase in EAAT2 level was observed in the white matter at days 3-7 post-ischemia.

Immunohistochemical analysis showed no significant changes in EAAT1 and EAAT3

expression levels after hypoperfusion (Fig. 4).

Localization of EAAT2 270

Double-labeling immunofluorescence analysis was conducted to determine the

distribution of EAAT1, EAAT2 and EAAT3. The expression of EAAT2 was localized

to astrocytes in both the white matter and the cortex throughout the study period.

Staining for EAAT2 was not observed in other glial cells such as oligodendrocytes or

microglia (Fig. 6A). 275

Yatomi Y et al,

The expression of EAAT1 was limited in the white matter to mature

oligodendrocytes stained with antibodies to APC, a marker of oligodendroglia.

However, in the cortex, staining for EAAT1 was noted in neurons. Both astrocytes

and microglias were negative for EAAT1 staining (Fig. 6B).

Staining for EAAT3 was observed in the white matter in immature 280

oligodendrocytes stained with antibodies to PDGFRα, a marker of oligodendroglial

progenitor cells. In the cortex, staining for EAAT3 was limited to neurons. Both

astrocytes and microglias were negative for EAAT3 (Fig. 6C).

Changes in expression of EAAT2 in human brain 285

Finally, we analyzed the change in EAAT2 expression pattern in human ischemic

brain tissue. EAAT2 was expressed in the white matter of the control brains. The

number of EAAT2-positive cells was significantly higher in ischemic brain than the

control (Fig. 7). Furthermore, morphological variation and hypertrophy of

EAAT2-positive cells were noted in the ischemic brain, compared to the control 290

EAAT2-positive cells with few axons.

Discussion

The results of this study showed that chronic cerebral hypoperfusion resulted in 295

transient up-regulation of glial glutamate transporters in the subcortical white matter

and cortex. Among the three glutamate transporters examined in this study, EAAT2

increased significantly in both the rat brain ischemia model used in this study and

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human brain tissue.

Glutamate-mediated excitotoxicity is one of the major etiologies of 300

neurodegenerative diseases, such as ALS and AD. In fact, selective loss of EAAT2

was reported in both the motor cortex and spinal cord of patients with ALS (Rothstein

et al., 1995), whereas overexpression of EAAT2 in ALS-associated mutant SOD mice

improved L-glutamate induced cytotoxicity and delayed motor neuron loss (Guo et al.,

2003). In Alzheimer’s disease, EAAT2 expression in the brain is significantly 305

decreased (Masliah et al., 1996), even in the early stages of the disease (Jacob et al.,

2007). Interestingly, partial loss of EAAT2 was recently reported to result in

acceleration of the disease process in animal models of Alzheimer’s disease

(Mookherjee et al., 2011). These results indicate that EAAT2 plays an important role

in the pathogenesis of neurodegenerative disease. 310

Several studies have examined the effects of ischemia on EAATs, which

resulted in and variable changes in their expression patterns according to the protocol

of acute ischemia model (Gottlieb et al., 2000, Cimarosti et al., 2005).

EAAT2 levels were reported to decrease in the cortex and caudate-putamen at

day 3 of recovery from brain ischemia (Rao et al., 2001a), increase in the white matter 315

at day 1 after reperfusion following middle cerebral artery occlusion (MCAO), but

decrease at 7 days after ischemia (Arranz et al., 2010). However, overexpression of

EAAT2 induced by intraperitoneal injection of ceftriaxone (beta-lactams) or

intracerebral injection of viral vector is reported to exert significant neuroprotective

effects against focal cerebral ischemia (Chu et al., 2007, Harvey et al., 2011). Thus, 320

overexpression of EAAT2 seems to protect against neuronal death induced by

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ischemic insult. In the present study, we used ligation of the left and right common

carotid arteries in rats, which is reported to be an appropriate model for human

ischemic white matter lesions, as confirmed histologically (Kobayashi et al., 2002,

Thomas et al., 2002, Tomimoto et al., 2003, Chida et al., 2011). Our results showed 325

increased amounts of EAAT2 up to 7 days after ischemia. Glutamate is known to be

released from damaged oligodendroglia and axons following reversal of uptake

carriers in conditions of energy deprivation (Karadottir and Attwell, 2007, Tekkok et

al., 2007). In this regard, excitotoxic injury of the white matter (infiltration of

immunocytes, loss of myelin and oligodendrocytes, and axonal damage) is considered 330

an important pathophysiology in multiple sclerosis, and is enhanced by deficiency of

glutamate detoxification. EAAT2 was selectivity lost from oligodendrocytes found

around active MS lesions, especially in early active lesions, extending into adjacent

white matter (Werner et al., 2001). These results suggest that excess glutamate plays a

critical role in tissue injury not only in the cortical area but also in white matter region. 335

In periventricular white matter injury, hypoxia-ischemia induces glutamate production

from oligodendrocyte lineage cells and axons, which in turn activates receptors on

adjacent oligodendrocytes, such as NMDA receptors, with cause white matter injury

(Back et al., 2007).

Considered together, the results suggest that overexpression of EAAT2 after 340

chronic hypoperfusion was induced by high extracellular levels of glutamate. Thus,

prevention of such up-regulation could protect against white matter damage. These

results are in agreement with the previous finding of up-regulation of EAAT2

expression in reactive astrocytes in human periventricular leukomalacia (Desilva et al.,

Yatomi Y et al,

2008). Our results also showed large numbers of EAAT2-positive cells in ischemic 345

white matter of autopsy cases.

One recent study showed that elimination of EAAT2 by antisense

oligonucleotide worsened the neurological status after transient MCAO (Rao et al.,

2001b). In contrast, the present study demonstrated that pretreatment with β-lactam

antibiotics, like ceftriaxon, which is reported to induce up-regulation of EAAT2 350

(Rothstein et al., 2005, Lee et al., 2008), reduced infarct volume after transient MCAO

(Chu et al., 2007). These results suggest that long-term up-regulation of EAAT2 after

chronic hypoperfusion might result in minimization of white matter lesion by removal

of extracellular glutamate. Further studies are needed to understand the effects of

changes in EAATs expression after chronic hypoperfusion. 355

Acknowledgment

This work was supported by a grant from Research Institute for Disease of Old Age,

Juntendo University, Tokyo.

360

Yatomi Y et al,

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Figure Legends 480

Fig. 1. Temporal changes in cerebral blood flow (CBF). Pre: before ligation of

bilateral common carotid artery (LBCCA); post: immediately after LBCCA, and at 3,

7, 14 and 28 days after LBCCA. Values are mean ± SEM of five rats in each group.

*P <0.05, **P < 0.001, compared with the baseline (before occlusion, pre). 485

Fig. 2. Klüver-Barrera staining of the corpus callosum. Pre: before ligation of bilateral

common carotid artery (LBCCA); post: immediately after LBCCA, and at 3, 7, 14 and

28 days after LBCCA. Vacuolation and demyelinated fibers were prominent at 28

days after LBCCA, compared with the control group. Scale bar = 20 μm. 490

Fig. 3. (A) Photomicrographs of GFAP-positive cells (a–j), GSTπ-positive cells (k-o),

NG2-positive cells (p-t) and Iba-1-positive cells (u-y) after ligation of bilateral

common carotid artery (LBCCA). Scale bar = 50 μm. (B) Density of GFAP-positive

cells in white matter and cortex, (C) density of GSTπ-positive cells in the white matter, 495

(D) density of NG2-positive cells in white matter and (E) density of Iba-1-positive

cells in the white matter. Data are mean ± SEM of five rats in each group. *P < 0.05,

**P < 0.001, compared with the baseline (pre).

Fig. 4. Photomicrographs of EAAT2-positive cells in the white matter (a–e) and 500

Yatomi Y et al,

cortex (f-j), EAAT1-positive cells in the white matter (k-o), and EAAT3-positive cells

(p-t) after ligation of bilateral common carotid artery (LBCCA). Scale bar = 50 μm.

EAAT2 labeling increased significantly at 3 days after LBCCA.

Fig. 5. (A, B) Western blotting analysis of EAAT1, EAAT2 and EAAT3 after ligation 505

of bilateral common carotid artery (LBCCA) in the: (A) cortex and (B) white matter.

Note the 60-, 62- and 57-kDa bands corresponding to EAAT1, EAAT2, EAAT3

proteins, respectively. Equal protein loading was confirmed by measuring α-tubulin.

(A’, B’). Densitometric analysis of EAAT1, EAAT2 and EAAT3 protein after

LBCCA (A’) in the cortex and (B’) white matter. Representative results of three 510

experiments with similar results. Data in A’ and B’ are mean ± SEM of five rats in

each group, *P < 0.05, **P < 0.001, compared with the baseline (pre).

Fig. 6. (A) (a) Proportion of EAAT2/GFAP double-stained cells, (b) EAAT2/APC

double-stained cells, (c) EAAT2/NG2 double-stained cells and (d) EAAT2/Iba-1 515

double-stained cells in the white matter. (B) Proportion of EAAT1/APC

double-stained cells. (C) Proportion of EAAT3/PDGFRα double-stained cells in the

white matter after ligation of bilateral common carotid artery.

Fig. 7. (A) Photomicrographs of EAAT2-positive cells in human brain. Scale bar = 520

500 μm. (B) Density of EAAT2-positive cells in the white matter. Data are mean ±

SEM, **P < 0.001, compared with the control group.

Yatomi Y et al,

Table 1. Characteristics of autopsy cases. 525

Case Age

(yrs) Sex Group Diagnosis

1 76 M 1 Multiple cerebral infarction

2 80 F 1 Multiple cerebral infarction

3 91 M 1 Binswanger disease

4 84 M 2 Chronic heart failure

5 41 M 2 Lipid aturage myopathy, chronic renal failure

6 67 M 2 Left putaminal hemorrhage

Cases were divided into two groups based on the presence (or absence) of white

matter damage: group 1: patients with chronic hypoperfusion, group 2: patients 530

without white matter damage.

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