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Neurobiology of Disease 22 (2006) 274 – 283

Sustained astrocytic clusterin expression improves remodeling after

brain ischemia

Anouk Imhof,a,b,e Yves Charnay,a Philippe G. Vallet,a Bruce Aronow,c Eniko Kovari,a

Lars E. French,d Constantin Bouras,a and Panteleimon Giannakopoulosa,b,*

aDepartment of Psychiatry, HUG, Belle-Idee, 2, ch. du Petit-Bel-Air, 1225 Chene-Bourg Geneva, SwitzerlandbDivision of Old Age Psychiatry, University of Lausanne School of Medicine, CH-1008 Prilly, SwitzerlandcDivision of Molecular Developmental Biology Children’s Hospital Research Foundation, Cincinnati, OH 45229-3039, USAdDivision of Dermatology, University Hospitals of Geneva, CH-1211 Geneva, SwitzerlandeCenter of Psychiatric Neurosciences, University of Lausanne School of Medicine, 1008 Prilly, Switzerland

Received 23 July 2005; revised 15 November 2005; accepted 17 November 2005

Available online 10 February 2006

Clusterin is a glycoprotein highly expressed in response to tissue injury.

Using clusterin-deficient (Clu�/�) mice, we investigated the role of

clusterin after permanent middle cerebral artery occlusion (MCAO).

In wild-type (WT) mice, clusterin mRNA displayed a sustained

increase in the peri-infarct area from 14 to 30 days post-MCAO.

Clusterin transcript was still present up to 90 days post-ischemia in

astrocytes surrounding the core infarct. Western blot analysis also

revealed an increase of clusterin in the ischemic hemisphere of WT

mice, which culminates up to 30 days post-MCAO. Concomitantly, a

worse structural restoration and higher number of GFAP-reactive

astrocytes in the vicinity of the infarct scar were observed in Clu�/� as

compared to WT mice. These findings go beyond previous data

supporting a neuroprotective role of clusterin in early ischemic events

in that they demonstrate that this glycoprotein plays a central role in

the remodeling of ischemic damage.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Astrocytes; Apolipoprotein J; Clusterin-deficient mice; Middle

cerebral artery occlusion; Neuroprotective effect

Ischemic cerebral stroke is the third cause of death and a major

cause of handicap in western countries (Dirnagl et al., 1999). In

order to improve therapeutic management and reduce health care’s

burden, it is crucial to explore the complex molecular phenomena

which take place in the course of this disorder. Soon after ischemic

brain stroke, cell death and tissue devastation caused by free

radicals, excitotoxicity and inflammation involve a number of

signaling pathways each one representing potential targets for early

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.11.009

* Corresponding author. Department of Psychiatry, HUG, Belle-Idee, 2,

ch. du Petit-Bel-Air, 1225 Chene-Bourg Geneva, Switzerland.

E-mail address: Panteleimon.Giannakopoulos@medecine.unige.ch

(P. Giannakopoulos).

Available online on ScienceDirect (www.sciencedirect.com).

therapeutic intervention (Dirnagl et al., 1999; Slevin et al., 2005).

Brain tissue directly affected by a stroke can be divided into a

necrotic core region and a surrounding peri-infarct area (also

referred to as ischemic penumbra) which initially has flow rates

adequate for survival but subsequently may become recruited in

the infarction process (Barinaga, 1998; Weinstein et al., 2004). The

long-term survival of neurons in the peri-infarct area in which the

destiny of nerve cells is not fated mainly depends on the

environment’s ability to restore local brain homeostasis (Neder-

gaard and Dirnagl, 2005). From this point of view, better

knowledge of late molecular and cellular adaptive responses to

ischemia may offer new therapeutic perspectives. Although an

impressive number of recent studies attempted to elucidate the

biological background of the early post-ischemic period (see

Dirnagl et al., 1999; Slevin et al., 2005), the molecular

determinants of the long-term structural restoration in cerebral

ischemia are surprisingly still unknown.

Among glial cell populations activated in the peri-infarct area

after stroke (Nedergaard and Dirnagl, 2005; Panickar and Noren-

berg, 2005), astrocytes have been traditionally associated with

certain essential functions such as maintaining a favorable

chemical environment and fueling of neurons (Dienel and Hertz,

2005; Pellerin and Magistretti, 2004). Various types of brain

injuries induce reactive astrogliosis characterized by cellular

morphological changes and a marked upregulation of many genes

including those encoding glial fibrillary astrocyte protein (GFAP),

protein S100 beta (Yasuda et al., 2004), trophic factors (Swanson et

al., 2004; Trendelenburg and Dirnagl, 2005), and clusterin (Cheng

et al., 1994; Jones and Jomary, 2002; Pasinetti et al., 1994;

Rosenberg and Silkensen, 1995; Walton et al., 1996; Wiggins et al.,

2003; Wilson and Easterbrook-Smith, 2000; Zoli et al., 1993). This

latter multifunctional heterodimeric glycoprotein (also called

apolipoprotein J) is constitutively synthesized by a variety of

tissues and found in most biological fluids (Jenne and Tschopp,

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283 275

1992; Jones and Jomary, 2002; Rosenberg and Silkensen, 1995;

Trougakos and Gonos, 2002; Wilson and Easterbrook-Smith,

2000). Clusterin mRNA expression is markedly increased during

tissue involution in response to hormonal changes or injury and

under circumstances leading to cell death by apoptosis (for a

review, see French et al., 1992). In the central nervous system,

most neurons and scarce populations of astrocytes express rather

low levels of clusterin mRNA (Pasinetti et al., 1994; Van Beek et

al., 2000; Wiggins et al., 2003). Lesions that induce neuronal loss

or deafferentation induce a substantial increase in clusterin mRNA

levels, mainly within astrocytes (Cheng et al., 1994; May and

Finch, 1992; Walton et al., 1996; Zoli et al., 1993). Clusterin

expression is also increased in various neurological conditions

such as epilepsy, dementia (Alzheimer’s disease, Pick’s disease),

multiple sclerosis, retinitis pigmentosa, glioma, scrapie, and AIDS

encephalopathy (Brown et al., 2004; Dragunow et al., 1995;

Giannakopoulos et al., 1998; Holtzman, 2004; Michel et al., 1992;

Rosenberg and Silkensen, 1995; Sasaki et al., 2002; Scolding et al.,

1998; Torres-Munoz et al., 2001; Wong, 1994). The biological

significance of this phenomenon is still unclear. Clusterin

activation could simply be an epiphenomenon of inflammation-

free removal of dying neurons or actively participate in a repair

mechanism in which glial cells attempt to terminate the active cell

death process. In this context, a neuroprotective role of clusterin

has been recently reported in axotomy-induced cell death model in

clusterin-deficient (Clu�/�) mice (Wicher and Aldskogius, 2005).

In contrast, other studies demonstrated that clusterin may

contribute to neurotoxicity (Han et al., 2001; Xie et al., 2005).

To date, only few studies explored the role of clusterin in

cerebral ischemia, leading to controversial data. Early contributions

showed that transient forebrain ischemia in rats is associated with

clusterin mRNA increase in astrocytes within the ischemic peri-

infarct area (Zoli et al., 1993). An inverse correlation between the

decrease in DNA fragmentation and the increase in clusterin-

immunoreactivity from 2 to 7 days after hypoxic–ischemic injury

in the same area was also described (Walton et al., 1996). Using a

model of permanent small middle cerebral artery occlusion

(MCAO), we demonstrated that the thickness of the peri-infarct

area, 7 days post-ischemia, is inversely related to the level of

clusterin expression in transgenic mice overexpressing human

clusterin, wild-type mice (WT) and clusterin-deficient mice

(Wehrli et al., 2001). Moreover, Wiggins et al. reported a marked

astrocytic clusterin mRNA increase after acute cortical spreading

depression and proposed a protective role of clusterin activation in

the context of acute ischemic damage (Wiggins et al., 2003).

Importantly, all previous studies considered clusterin activation as

an acute phenomenon confined to early time points after ischemia.

Using WT and Clu�/� mice, we report, for the first time to our

knowledge, a sustained glial clusterin expression over 90 days after

ischemia, which decisively influences the long-term structural

remodeling process after a permanent small MCAO.

Materials and methods

Animals

Procedures for the anesthesia and treatment of mice were

approved by the Cantonal Veterinary Office of Geneva. The

minimum number of animals required for statistical analyses was

used in experiments, and all efforts were made to minimize

suffering. Male 9- to 12-week-old Clu�/� and WT mice were used

in all experiments. Both WT and Clu�/� mice derived from a

C57BL/6J strain (Clu�/� backcross with C57BL/6J strains for at

least 10 generations). The production of Clu�/�mice was reported

in details elsewhere (McLaughlin et al., 2000). They were bred as

heterozygotes, and the absence of clusterin in Clu�/� specimens

was routinely checked by Western blot analysis in addition to poly-

merase chain amplification genotyping (McLaughlin et al., 2000).

Surgical procedures and preparation of tissues

Permanent small MCAO was performed by electrocoagulation

as described previously (De Bilbao et al., 2000). This procedure

provokes a lesion area through the frontoparietal cortex. Sham-

operated animals not subjected to MCAO were included in control

experiments. Mice were killed at various post-ischemic times (from

1 h to 90 days), and their brains were rapidly frozen by immersion

in isopentane cooled at �25-C. Brains were then stored at �80-Cuntil used. Some brains were fixed by immersion in 4%

paraformaldehyde in 0.1 M phosphate buffer saline, pH 7.4

(PBS) for 24 h, placed in 0.1 M PBS containing 7% sucrose for

24 h, frozen, and stored as above. For morphological analyses, 15-

Am cryostat coronal sections were collected on slides coated with

3-aminopropyltriethoxy-silane. For biochemical analyses including

Western blots and real-time polymerase chain amplification (RT-

PCR), the ipsilateral and contralateral sides of the dorsal half of the

coronal frozen sections encompassing the injured zone were

carefully dissected prior to their storage at �80-C.To evaluate whether local alterations in cerebral vascular

anatomy may contribute to higher susceptibility to injury in

Clu�/� mice, an additional series (n = 5) of WT and Clu�/� mice

was sacrificed on day 3 post-ischemia. They were perfused

intracardially by 0.9% NaCl solution followed by a perfusion of

a mixture (2 ml) of equal proportions of 5% gelatinous water and

black china ink warmed at 40-C. Brains were removed and

immersed for 24 h in 4% paraformaldehyde at 4-C. A careful

inspection of cerebral vasculature anatomy revealed no marked

difference in position, diameters of main cerebral arteries, and

organization of the circle of Willis between Clu�/� and WT mice.

Histology

For each animal, quantification of the infarct and peri-infarct

areas was performed on the cresyl-violet and hematoxylin/eosin-

stained sections at five representative levels throughout the

rostrocaudal extent of the lesion (A 0.26, �0.22, �0.40, �0.70,and �1.2 mm relative to Bregma) (Paxinos and Franklin, 2003).

The rostrocaudal extent of the infarct was the same in both groups

of mice. Time-dependent morphological alterations up to 30 days

after MCAO have been described in details elsewhere (Mennel et

al., 2000). Morphometric parameters including total infarct volume

and maximal thickness of the peri-infarct area were estimated using

a computer-assisted image analyzing system (Software Morphom-

etry, SAMBA 2005, TITN, Alcatel, France), as previously

described (De Bilbao et al., 2000; Wehrli et al., 2001). Briefly,

the microscopic field for measurement was the infarct border

localized at the medial part of the necrotic core. Peri-infarct area

measurements were performed at �10. This area was clearly

delineated in all mice by the presence of either dark shrunken or

pale swollen neurons often surrounded by vacuoles in cresyl-violet

sections as well as a layer of cells of microglial origin (De Bilbao et

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283276

al., 2000). For each mouse, the maximal thickness of the peri-

infarct area was calculated every 140 Am throughout the

rostrocaudal extent of the infarct (n = 10 sections for each animal).

Thicknesses were summed for each animal, and a mean thickness

was calculated for each group of mice.

Clusterin and GFAP expression patterns during the post-ischemic

healing

Both clusterin and GFAP mRNA expressions were investigated

by in situ hybridization in a series of coronal sections represen-

tative of the ischemic area. For a direct comparison of the labeling

in the same sections, additional double labeling with in situ

hybridization to clusterin mRNA/GFAP immunocytochemistry

was used. Comparison between GFAP mRNA expression and

GFAP immunoreactivity (see below) in adjacent sections served as

control. The labeled riboprobes were obtained by in vitro

transcription in the presence of digoxigenin (DIG)-UTP as reported

elsewhere (Lucas et al., 2005). Briefly, total RNA from WT mouse

brain was used as template for reverse transcription-PCR using

Ready-To-Go (Amersham Biosciences, Switzerland) and then PCR

Master Mix (Promega, Switzerland). The oligonucleotides primers

were upstream 5V-gaattcgagcaggaggtctctgac-3V/downstream 5V-gcggccgcttccgcacggcttttcctgcg-3V for the amplification of clusterin

(gb access number NM_013492) and upstream 5V-gaattcatgcctcc-gagacggtgg-3V/downstream 5V-gcggccgccacgtccttgtgctcctg-3V for

the amplification of GFAP (gb access number NM_010277) mouse

genes respectively. The PCR products were cloned in TOPO pCR4,

introduced in Top10 E. coli, and positive recombinants were

selected by ampicillin and Lac-Z ccdB disruption according to the

manufacturer’s instructions (InVitrogen, Switzerland). The plas-

mids were purified from bacteria by HiSpeed Plasmid Kit (Qiagen,

Switzerland) and linearized by either Pme1 or Not1 enzymatic

restriction in order to produce antisense and sense riboprobes by

their in vitro transcription using respectively T7 or T3 polymerase

(Maxiscipt, Ambion).

The in situ hybridization procedures were performed according

to Lucas et al. (2005). Briefly, brain sections were thawed, fixed 15

min in 4% paraformaldehyde, acetylated in a mixture containing

0.5% acetic anhydride, 0.9% NaCl and 0.1 M triethanolamine, pH

8. They were then delipidated in chloroform and incubated with 5–

10 ng of DIG-labeled riboprobe in hybridizing buffer containing

1� Denhardt’s solution, 50% formamide, 20 mM Tris–HCl (pH

7.4), 300 mM NaCl, 1 mM EDTA, 10% dextran sulfate, 50%

formamide, 100 Ag/ml salmon testes DNA, and 250 Ag/ml of yeast

tRNA. After hybridization overnight at 55-C, the sections were

washed 4 � 5 min in 4� standard saline citrate buffer (SSC) at

20-C, incubated with RNase A (50 Ag/ml) for 30 min at 37-C, andwashed again 2 � 5 min in 2 � SSC, 10 min in 1 � SSC, 10 min in

0.5 � SSC at 20-C, and 30 min in 0.1 � SSC at 50-C. Thehybridizing signal was detected by immunocytochemistry using

sheep anti-DIG IgG conjugated to phosphatase alkaline (1:100;

Roche Diagnostics) and nitro blue tetrazolium/5-bromo-4-chloro-

3-indolyl phosphate as chromogenic substrate (Dakocytomation).

The specificity of the hybridizing signal was assessed by

incubation of serial sections with sense probes. Furthermore, the

specificity of the clusterin hybridizing signal was demonstrated by

the absence of any labeling in brain sections from Clu�/� mice.

To explore whether clusterin mRNA overexpression is an

epiphenomenon of reactive astrogliosis and define the cellular

localization of the clusterin transcript in glial cell subpopulations,

we assessed the percentage of GFAP-positive astrocytes and lectin-

positive cells which expressed clusterin mRNA in the peri-infarct

area (Deng et al., 2003). Thus, in addition to the hybridization

procedures described above, adjacent sections were incubated

either with a rabbit anti-GFAP antibody (1:2000; Dakocytomation)

followed by an incubation with a goat anti-rabbit IgG conjugated to

Alexa 488 fluorescent (1:500; Molecular Probes, InVitrogen) or

biotinylated isolectin B4 (Vector Laboratories, Switzerland).

Biotinylated isolectin B4 was revealed by fluorescein–streptavidin

or HRP–streptavidin according to the manufacturer instructions

(Vector Laboratories).

Clusterin and GFAP messenger RNA quantifications

The quantification of clusterin and GFAP mRNA expressions

ipsilateral and contralateral to the lesion 0, 1, 14, 30, and 60 days

post-MCAO was performed by real-time PCR. Total RNA was

isolated and treated with DNase (Absolutely RNA Kit; Stratagene,

Switzerland). 1 Ag of RNA for each time point was converted to

cDNA using a kit (cDNA Archiv Kit, Applied Biosystems)

according to the manufacturer. For real-time transcript quantifica-

tion, the cDNA was used in a fluorogenic 5V nuclease assay

utilizing the chemistry of the TaqMan\ system on the ABI Prism

7900 Sequence Detector (Applied Biosystems). Gene-specific

TaqMan\ primers and probe sets were designed using the Primer

Express Software (Applied Biosystems). The sequences designed

were respectively (clusterin forward: 5V-gaagggccagtgtgaaagtg-3V;reverse: 5V-gggcaggattgttggttgaa-3V; probe: FAM-cacagacaagatctcc-

TAMRA) and (GFAP forward: 5V-aactttgcacaggacctcg-3V; reverse:5V-ctgtctatacgcagccaggttg-3V; FAM-ttggtttcatcttggagcttctgcctca-

TAMRA). 18S rRNA primers and probe were developed and used

according to Applied Biosystems.

PCRs included 300 nM of each primer, 100 nM probe, 1�TaqMan\ buffer A (10�: 500 mM KCl, 100 mM Tris–HCl, 0.1

mM EDTA), 5 mM MgCl2, dATP (200 AM), dCTP (200 AM),

dGTP (200 AM), dUTP (400 AM), 0.5 U AmpErase UNG, and 2.5

U AmpliTaq Gold DNA Polymerase for a final volume of 10 Al. Thereal-time PCR consisted of one cycle of each 50-C for 2 min and

95-C for 10 min, followed by 40 cycles of 95-C for 15 s and 60-Cfor 1 min. The results were quantified by DDCt method using 18S

rRNA as internal standard (Vallet et al., 2005). Controls included

the use of RNA samples extracted from Clu�/� mice, replacement

of samples by water, or omission of the reverse transcriptase step.

Western blot analyses

Estimation of clusterin and GFAP protein levels 0, 1, 7, 14, 30,

and 90 days post-MCAO was performed by densitometric analyses

of their respective immunoreactivity detected in Western blots.

Ipsilateral and contralateral brain samples collected from 12 coronal

sections (see Surgical procedures and preparation of tissues) were

dissolved in Laemmli buffer containing 2% sodium dodecyl sulfate

(SDS). For each time point, 10 Ag of proteins (BCA assay, Pierce,

Switzerland) was subjected to SDS-PAGE electrophoresis and then

electrotransferred to nitrocellulose membranes (BioRad, Switzer-

land). Membranes were blocked with non-fat dry milk (BioRad)

and incubated overnight simultaneously with goat IgG against

clusterin h-chain (1: 5000; Santa-Cruz, Biotechnology, CA), rabbit

IgG anti-GFAP (1:5000; Dakocytomation), and rabbit IgG anti-h-tubulin (1: 500; Abcam, United Kingdom). Anti-rabbit and anti-

goat IgG conjugated to horseradish peroxidase (1:500; Dakocyto-

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283 277

mation) were used as secondary antibodies. The immunocomplexes

were revealed by a chemiluminescence method (Litablot, Euro-

clone, Switzerland). Molecular weights were indicated by Magic-

Mark standard proteins (InVitrogen). Band intensities were

quantified under a GS-700 imaging scanning densitometer in the

reflectance mode (Biorad). The integrated density of h-tubulinimmunolabeled band served as loading control.

Statistical analysis

Results are mean T SE. Multiple comparisons were analyzed

with ANOVA followed by Bonferroni post hoc test. P values <0.05

were considered statistically significant.

Fig. 1. In situ hybridization of clusterin mRNA in the brains of (A)

control and (B) sham-operated wild-type mice. Note the laminar patterns

of distribution of the specific hybridizing signal through the cerebral

cortex, mostly within neurons. Note also in panel B the occasional

presence of strongly labeled astrocytes in the corpus callosum (CC).

Clusterin mRNA expression through the ipsilateral brain side of wild-type

mice at 1 h, 24 h, 7 and 14 days post-MCAO was illustrated in panels

(C–F) respectively. In addition to a weak expression of clusterin mRNA

in most neurons, a strong hybridizing signal was visualized within

astrocytes occurring through the peri-infarct zone, adjacent areas and

whole corpus callosum. The clusterin-expressing astrocytes were partic-

ularly abundant between 7 and 14 days post-MCAO (compare C–F). CP:

caudoputamen. Scale bars (C–F) 2 mm; (B) 0.5 mm; (insert B) 60 Am;

(insert E) 40 Am.

Results

The present study revealed three main findings. First, in WT

mice brain, the vast majority of neurons expressed a low level of

clusterin independently of MCAO. Second, MCAO induced a

long-lasting (up to 90 days post-MCAO) astrocytic expression of

clusterin, ipsilateral to the lesion. Third, a comparison between WT

and Clu�/� mice revealed that the absence of clusterin is

associated with a significant slower tissue remodeling during the

healing process.

MCAO stimulates clusterin expression in astrocytes

In situ hybridization experiments showed that clusterin mRNA

was widely expressed in cortical neurons as well as in subpopulation

of ependymal cells and choroid plexuses, in both non-operated and

sham-operated WT mice (Figs. 1A, B). The hybridizing signal

localized in the cytoplasm of neurons was weak but specific since no

signal was detected in brain sections fromClu�/�mice (see Fig. 1B

insert). Occasionally, few astrocytes displaying a strong signal of

hybridization were visualized in the corpus callosum (Fig. 1B).

From 1 to 14 days post-MCAO, there was a progressive increase of

the number of astrocytes expressing clusterin mRNA (Figs. 1C–F).

Ipsilateral to the lesion, through the peri-infarct areas and to a lesser

extent in the corpus callosum, the maximal number of clusterin-

expressing astrocytes was reached between 14 and 30 days post-

MCAO (see Figs. 1C–F and 2A). Few labeled astrocytes were

additionally visualized through the corpus callosum, contralateral

homotopic cortical area of the unlesioned hemisphere, and caudate

putamen (not shown). At 90 days post-MCAO, only few clusterin

mRNA-positive astrocytes restricted to a narrow band along the scar

remained observable (see Fig. 2E).

As illustrated in Fig. 3, real-time PCR confirmed the presence

of high levels of clusterin mRNA ispsilateral to the lesion, mainly

at 30 days post-MCAO. In parallel, a slight but significant increase

in clusterin mRNA levels was also observed in the contralateral

side. Beyond 30 days post-MCAO, clusterin mRNA expression

markedly decreased. Importantly, there was a substantial increase

of GFAP mRNA in Clu�/� compared to WT mice up to 30 days

post-MCAO.

As expected, Western blot analysis in brain extracts and serum

of WT mice revealed a single band of clusterin h-chainimmunoreactivity between 40 and 45 kDa (see Figs. 4A, B). The

absence of corresponding labeling in tissue extracts and serum from

Clu�/� mice demonstrated the specificity of the immunoreaction.

Two bands corresponding respectively to GFAP (50 kDa) and h-

tubulin (55 kDa) immunoreactivity were clearly visualized in brain

extracts of WT and Clu�/� mice (Figs. 4A, B). Western blot

analyses indicated that maximal level of clusterin was expressed

between 14 and 30 days post-MCAO, ipsilateral to the lesion (Fig.

4B). From 14 to 30 days post-MCAO, the levels of GFAP measured

in Clu�/� brains were significantly higher than those observed in

WT mice, ipsilateral to the lesion (Fig 4C). At 30 days post-

ischemia, there was a 19.8% increase of GFAP immunoreactivity in

Clu�/� compared to WT mice. The antibody specific to clusterin

h-chain successfully used in our Western blot analyses and two

other antibodies against the whole recombinant clusterin were not

suitable for immunohistochemistry in mouse brain sections.

Slow-down of the restoration process in Clu�/� mice

No neurological deficits (forelimb, circling) were observed after

surgery. The infarction was confined primarily to the neocortex

(frontal associative, motor and somatosensory cortices) and was

limited medially by a small subcortical infarct in the dorsolateral

caudate putamen. Generally, cortical infarction extended rostro-

Fig. 3. Quantitative estimation of clusterin (A) and GFAP mRNAs (B) by

real-time PCR ipsilateral (i) and contralateral (c) to the ischemic damage at

0, 1, 14, 30, and 60 days post-ischemia. The RNA 18S was used for the

standardization (see Materials and methods for further information). N = 10

(A), 8 (B) for each time post-MCAO. In panel A, the asterisks indicate

significant differences in clusterin mRNA levels compared to controls. In

panel B, the asterisks indicate significant differences in GFAP mRNA levels

between WT and Clu�/� mice ( P < 0.05).

Fig. 2. In situ hybridization of clusterin mRNA (A, B, E, F) and GFAP

mRNA (C, D, G, H) expression in consecutive sections through represen-

tative brain regions of WT (A, C, E, G) and Clu�/� mice (B, D, F, H),

ipsilateral to the lesion, at 30 and 90 days post-MCAO. Note the absence of

specific hybridizing signal for clusterin in Clu�/� mice (B, F). Note also in

parallel sections the larger population of GFAP-expressing cells in the brain

of Clu�/�mice (compare C and D). The asterisk in panels B and D indicates

a disorganized zone suggesting a worse restoration of the tissue in Clu�/�mice (compare C and D). At 90 days post-MCAO, only a narrow band of

astrocytes expressing GFAP remained visible along the scar (arrows) in WT

and Clu�/�mice (G and H). At this time point, the patterns of distribution of

clusterin expression overlap that of GFAP expression (compare E and G).

Scale bars: (A–H) 2 mm; (insert A) 60 Am; (insert E) 40 Am.

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283278

caudally from the coronal level of olfactory nuclei to the first

coronal levels of the substantia nigra. By 7 days post-occlusion, the

margin between infarcted and non-infarcted area became obvious

as the extent of the peri-infarct area was clearly delineated. In all

animals, the topography of the lesions was similar (De Bilbao et

al., 2000). At 7 days post-MCAO, WT and Clu�/� mice displayed

similar necrotic core infarct volumes (5.3 T 0.3 mm3 vs. 5.7 T 0.5

mm3) confirming the reproducibility of the MCAO paradigm.

Morphometric analyses indicated that Clu�/� mice displayed a

significantly larger peri-infarct area than WT mice (Table 1).

Moreover, from 14 to 90 days post-MCAO, the healing process

was significantly slower in Clu�/� than in WT mice. Both in situ

hybridization (Fig. 2) and immunohistochemistry (Fig. 5) showed

that at 30 days post-MCAO, Clu�/� mice still displayed

significantly higher numbers of GFAP-reactive astrocytes in the

peri-infarct area compared to WT mice (Fig. 5, compare B and D).

At this time point, a good structural restoration was present in WT

mice documented in hematoxylin/eosin-stained sections by the

poor astrocytic reaction, rarity of macrophages, and neovasculari-

zation (Fig. 5). In contrast, a more active cellular reaction was

observed in Clu�/� mice characterized by the presence of

numerous GFAP-expressing astrocytes and several foamy macro-

phages (Figs. 5C, D, and insert). At 90 days post-MCAO, a larger

scar area was observed in Clu�/� mice compared to WT mice

(Fig. 6). Moreover, a weak cellular reaction was still observed in

Clu�/� mice but not in WT mice (Fig. 6, compare C and F).

Dissociation between the activation of glial clusterin mRNA and

reactive astrogliosis

Double labeling study in WT mice brains at 7, 14, 30, and 90

days post-MCAO revealed that GFAP-immunoreactive astrocytes

frequently coexpressed clusterin mRNA (Fig. 7). At 30 days post-

MCAO, 55 T 8% of GFAP-positive astrocytes coexpressed clusterin.

At this time point, a substantially higher number of astrocytes were

GFAP-negative but expressed clusterin mRNA (Figs. 7E, F).

At 3 and 7 days post-MCAO, isolectin B4-positive cell

populations (macrophages and resident microglial cells) were

Fig. 4. Time-course of clusterin and GFAP immunoreactivity using Western blot analyses in WT and Clu�/� mice from 0 to 90 days post-MCAO. For each

immunolabeled band visualized in panel A, the integrated density in arbitrary units (AU) was normalized with h-tubulin (i: ipsilateral c: contralateral side to

MCAO). Note that the 40–45 kDa band detected in brain extracts and serum of WT mice was never observed in Clu�/� mice. The clusterin level estimates in

WT mice were illustrated in panel B. GFAP level estimates in WT and Clu�/� were illustrated in panel C. N = 6 mice for each time post-MCAO. The asterisks

indicate significant differences compared to controls (P value < 0.05).

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283 279

mainly observed in the core infarct. These microglial cells were

consistently clusterin mRNA-negative (Fig. 8).

Discussion

Strengths of this report include the concomitant analysis of

clusterin mRNA and protein on both early and late time points after

Table 1

Maximal thickness of the peri-infarct area 7 and 14 days post-MCAO

respectively in WT (n = 11) and Clu�/� mice (n = 10)

Days post-MCAO 7 14

WT 547.7 T 100.9 682.1 T 153.0*

Clu�/� 606.1 T 134.8 805.1 T 125.3**

Values were means T SE in micrometer.

* P < 0.05.

** P < 0.01.

ischemia in WT mice as well as the detailed morphological

analysis of cerebral scars up to 90 days post-ischemia in WT and

Clu�/� mice. Our data reveal a sustained clusterin production in

astrocytes localized through the scaring area for at least 90 days

after MCAO. This unusually long-lasting expression of clusterin

was associated with better structural recovery and decreased

activation of GFAP-positive astrocytes in WT mice compared to

that observed in Clu�/� mice. In the initial post-ischemic period,

both in situ hybridization and real-time PCR analysis revealed a

steady increase of clusterin transcript in the peri-infarct area.

Although clusterin expression was detected in both neurons and

astrocytes, in situ hybridization revealed prominent changes in the

number of astrocytes expressing clusterin rather than variation in

the intracellular labeling intensity. Thus, it can be reasonably

assumed that increased levels in clusterin mRNA or protein

immunoreactivity respectively estimated by real-time PCR and

Western blot analyses post-MCAO mainly reflected size variations

of the astrocytic population. The present observations are

consistent with previous data indicating that following brain injury,

Fig. 5. Morphological characteristics of the scar area in brain sections from

WT (A, B) and Clu�/� (C, D) at 30 days post-MCAO. Sections were

stained with hematoxylin-eosin (A, C) and GFAP immunohistochemistry

(B, D). Note the very active cellular reaction in Clu�/� mice with

numerous astrocytes and foamy macrophages (insert of panel C) as

compared to the good structural restoration observed in WT mice (A).

Note that the population of GFAP astrocytes visualized in Clu�/� (D)

exceed that detected in wild-type (B) mice (arrows). Scale bars: (A–C) 200

Am and (insert in C) 15 Am.

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283280

astrocytes represent the major producers of clusterin (Pasinetti et

al., 1994; Van Beek et al., 2000; White et al., 2001; Wiggins et al.,

2003).

Extending our previous observations (Wehrli et al., 2001), the

size of the peri-infarct area was substantially larger in Clu�/�mice compared to WT mice both at 7 and 14 days post-ischemia.

This implies that clusterin gene activation may be a basic

mechanism of cell protection against decreased blood supply,

and its absence cannot be compensated by the activation of other

stress proteins, at least in this cerebral ischemia paradigm. In

addition to what was originally thought of this stress-induced

Fig. 6. Morphological characteristics of the scar area in brain sections from WT (A

hematoxylin/eosin. The arrows in panels A and D point to the extension of the res

restoration of the cerebral tissue in WT compared to Clu�/� mice. In panel C, arr

also in panel F the abundance of small pycnotic nuclei presumably of glial cells.

glycoprotein (Cheng et al., 1994; May et al., 1992; Walton et al.,

1996; Zoli et al., 1993), the present data reveal that clusterin

production is not confined to the acute post-stroke period but

represents a long-lasting phenomenon. Our results indicate a

maximal expression of clusterin transcript confined to astrocytes

30 days post-MCAO. Western blot analysis also confirmed the

marked increase of clusterin up to this time point. Although one

could argue that this activation of clusterin is an epiphenomenon of

reactive astrogliosis in the peri-infarct area, this is an unlikely

scenario since our double-labeling experiments identified a large

population of clusterin mRNA expressing astrocytes were GFAP-

negative. It is also unlikely that we underestimate the population of

GFAP-immunoreactive astrocytes visualized in double-labeling

experiments since their distribution patterns overlapped that

revealed by GFAP in situ hybridization. Importantly, several

evidences indicate that upregulation of GFAP is not a prerequisite

for the development of reactive gliosis in response to brain injury.

For instance, GFAP-deficient mice still display post-traumatic

reactive gliosis comparable to that observed in wild-type mice

(Pekny et al., 1995). Furthermore, subpopulations of brain

negative-GFAP astrocytes were previously reported in various

models of brain ischemia (see Giffard and Swanson, 2005). Future

studies should explore the molecular characteristics and post-

traumatic destiny of the astrocyte subpopulations coexpressing

clusterin and GFAP compared to those expressing only clusterin or

GFAP.

The chronic expression of astrocytic clusterin is closely related

to the healing process in the peri-infarct area as demonstrated by

the worse structural restoration and marked increase of GFAP

mRNA-positive astrocytes in the peri-infarct area observed in

Clu�/� compared to WT mice up to 30 days post-ischemia.

Supporting these morphological findings, biochemical quantitative

investigations also indicated that both GFAP mRNA and GFAP

protein upregulation is more intense in Clu�/� than WT mice at

this time period. Interestingly, morphological comparisons between

WT and Clu�/� mice showed that this late positive effect of

clusterin is still present even after 90 days post-MCAO, when the

levels of clusterin transcript and protein were comparable between

ischemic and non-ischemic hemispheres in WT mice. The role of

–C) and Clu�/� (D–F) at 90 days post-MCAO. Sections were stained with

idual scar at low magnification. High magnification (C, F) indicates a better

ows point to spared neurons rarely observed in Clu�/� brain sections. Note

Scale bars: (A, D) 1 mm; (B, E) 420 Am; (C, F) 80 Am.

Fig. 7. Double-labeling clusterin mRNA/GFAP (ipsilateral side) in the

cerebral cortex ofWTmice at 30 days post-MCAO. The micrographs (A–B)

were taken from different areas as indicated in the drawing. High densities of

labeled astrocytes were detected along the scar. Numerous GFAP-immuno-

reactive astrocytes (green fluorescence) also expressed clusterin messenger

(black staining) (see arrowed cells in B–D). The reverse is not true since the

area occupied by astrocytes expressing clusterin exceeds that of GFAP-

reactive astrocytes (e.g., E and F). In panel A, note the strong expression of

clusterin in the choroid plexuses (cp) and within a subpopulation of

ependymal cells (arrows). Scale bars: (A–C, E) 35 Am; (D) 15 Am; (F) 20 Am.

Fig. 8. Double-labeling clusterin mRNA/isolectin B4 (Griffonia simplici-

folia) in the cerebral cortex of WT mice 7 days post-MCAO. The lectin

(IB4) binding on macrophages and resident microglial cells was revealed by

brown reaction in panels A and B or fluorescein in panel C, whereas the

hybridizing signal of clusterin mRNA corresponds to the dark reaction (see

Materials and methods for details). Note that the respective distribution of

lectin-positive cells in the infarct (i) and just beneath in the peri-infarct area

differs from that of clusterin-expressing cells (see B and arrows in C). Scale

bars: (A) 1.2 mm; (B) 60 Am; (C) 100 Am.

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283 281

GFAP mRNA-positive astrocytes in healing process is still matter

of debate. A recent study has shown that the sustained proliferation

of GFAP mRNA-positive astrocytes may inhibit synaptic regener-

ation and thus actively participate to the worst repair process

observed in Clu�/� mice (Pekny and Nilsson, 2005). Alterna-

tively, the accumulation of GFAP mRNA-positive astrocytes in

Clu�/� mice may be a simple epiphenomenon reflecting the

extent of ischemic damage (Nedergaard and Dirnagl, 2005).

Possibly reflecting the multifunctional role of this protein,

several endogenous clusterin-binding partners were reported

(see Bajari et al., 2003; Jones and Jomary, 2002; Wilson and

Easterbrook-Smith, 2000). Among the various molecular inter-

actions of this protein, the inhibition of complement-mediated

cytolysis was mentioned as a possible early protective mechanism

in response to brain ischemia (Jones and Jomary, 2002; Van Beek

et al., 2000). Clusterin was also considered as a putative secreted

chaperone stabilizing stressed proteins following thermal, me-

chanical, or oxidative injury (Hochgrebe et al., 2000; Jones and

Jomary, 2002; Wilson and Easterbrook-Smith, 2000). Bearing in

mind that clusterin is an apolipoprotein (Apo J), other physio-

logical functions including lipoprotein transport and cholesterol

homeostasis may also contribute to regenerative processes (Bajari

et al., 2003; Jones and Jomary, 2002; Kamada et al., 2005; White

et al., 2001). The sustained expression of clusterin, beyond the

acute phase of inflammation, strongly suggests that this protein

participates to long-term plasticity changes and brain remodeling

(White et al., 2001), a role compatible with its expression in

astrocytic population surrounding the scar (Trendelenburg and

Dirnagl, 2005).

The molecular mechanisms fostering the long-term expression

of clusterin following stress remain conjectural. It was reported that

transforming growth factor-beta (TGF-h) stimulates clusterin gene

expression in cell cultures (Jin and Howe, 1999). TGF-h is a

neuroprotective factor strongly upregulated following ischemia-

induced brain damage, especially in the penumbral area (Buck-

walter and Wyss-Coray, 2004; Buisson et al., 2003; Trendelenburg

and Dirnagl, 2005). It has been previously reported that, in the

kidney, angiotensin stimulates both TGF-h and clusterin expres-

sion, and that clusterin can bind to TGF-h receptors (Yoo et al.,

2000). Whether clusterin synergizes with TGF-h to mitigate the

progression of brain infarction remains an intriguing question.

A recent paper by Criswell et al. (2005) indicates that

transactivation of the early growth response-1 (Egr-1) transcription

factor regulates the expression of clusterin, promoting a prosur-

vival cascade after ionizing radiations. It was reported elsewhere

that brain ischemia upregulated Egr-1 (Lu et al., 2003). Further-

more, it was shown that this factor could contribute to brain

ischemic tolerance (Kawahara et al., 2004). Thus, the exploration

of the Egr-1 expression’s status and its upstream transduction

factors during healing may be also of particular interest in our

model.

The main limitation of our study is that it was not possible to

identify long-term motor or behavioral changes related to clusterin

A. Imhof et al. / Neurobiology of Disease 22 (2006) 274–283282

expression. Although the model of permanent small MCAO allows

for a reliable investigation of morphological changes at late post-

ischemia time points, it is known to be associated with only

marginal changes in these domains (Gonzalez and Kolb, 2003).

Detailed behavioral/motor assessments in transient ischemia

models and future morphological studies including administration

of recombinant cargo clusterin in Clu�/� mice are warranted to

explore the therapeutic potential of this protein in cerebral

ischemia.

Acknowledgments

This study was supported by a grant from the Swiss National

Science Foundation (FNRS no. 3100-06789; YC, PV, LF, PG), the

Jerome Tissieres Foundation (PG) and the University Hospitals of

Lausanne. We thank Mrs. M. Demeri, B. Greggio and Mr. G. Erba

for their expert technical assistance.

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