Molecular Brain Research 57 Ž1998.

1–9

Research report

Microglia-specific localisation of a novel calcium binding protein, Iba1

a,b
Daisuke Ito , Yoshinori Imai a , Keiko Ohsawa a , Kazuyuki Nakajima a , Yasuo Fukuuchi b,
Shinichi Kohsaka a,)
a
Department of Neurochemistry, National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan
b
Department of Neurology, School of Medicine, Keio UniÕersity, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

Accepted 3 February 1998

Abstract

Recently it has been shown that mRNA of Iba1 Žionized calcium binding adaptor molecule 1., which was a novel calcium binding
protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells wImai et al. Biophys. Biochem. Res.
Commun., 224 Ž1996. 855–862x. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that
Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of
embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or
oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified
microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial
nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of
Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia,
especially in the activated microglia. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Iba1; Microglia; Calcium binding protein; Facial nerve axotomy; Immunocytochemistry; Immunohistochemistry

1. Introduction mapped to within the major histocompatibility complex

Calcium ions are known to be one of the most impor-
tant signal mediators in all cells including central nervous
system ŽCNS. cells. Calcium ions exert their signaling
activity through association with various calcium binding
proteins, many of which are classified into a large protein
family, the EF hand protein family w25x. We have previ-
ously reported a cDNA cloning of a novel EF hand
protein, Iba1 Žionized calcium binding adaptor molecule 1.
w12x.
Iba1 protein is a small 17-kDa protein consisting of 147
amino acids. It contains two EF hand motifs in the central
third of the molecule and this portion shows homology
with other EF hand proteins, but none of the other amino-
or carboxy-terminal regions have any homology with any
known proteins. The genomic copy of the Iba1 gene was
)
Corresponding author. Fax: q 81-423-46-1751;
kohsaka@ncnp.go.jp
ŽMHC. class III region between the BAT2 and TNF alpha
genes. Using a specific antibody, Iba1 protein was clearly
shown to be expressed in monocytic cell lines. Iba1 mRNA
has been found to be highly and specifically expressed in
microglia among the cultured brain cells. This suggested
that Iba1 protein functions as an adapter molecule that
mediates calcium signals in the monocytic lineage, includ-
ing microglia. In the present study, patterns of expression
of Iba1 protein in CNS cells were investigated in vitro and
vivo, mainly by immunocytochemical and immunohisto-
chemical techniques using mixed brain cell cultures and
adult rat brain sections.
Facial nerve axotomy is a well-established experimental
system by which microglia are locally activated to show
morphological change, proliferation and migration to at-
tach and surround the axotomized motoneurons w32x. We
have furthermore examined Iba1 expression of the acti-
vated microglia detected in the axotomized facial nucleus,
E-mail: and also discussed the role of Iba1 protein in the activation
of microglia.

0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 4 0 - 0
2 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9
2. Materials and methods
2.1. Immunoblot analysis of primary cultured cells
Primary cultures of neurons, microglia, astroglia, and
meningeal fibroblasts were prepared from rat brain accord-
ing to the method described previously w12,19,28,33x. After
seeding, neurons were cultured in poly-L-lysine-coated cul-
ture dishes for 1 day in minimum essential medium ŽMEM.
containing 10% fetal calf serum ŽFCS.. Microglia were
cultured for 1 day in Dulbecco’s modified Eagle medium
ŽDMEM. containing 10% FCS and astroglia and meningeal
fibroblasts were cultured for 7 days in same type medium.
Individual types of cells were then extracted in 50 mM
Hepes–NaOH, pH 7.0 containing 0.1% Nonidet-P40, 250
mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM dithio-
threitol, 1 mM phenylmethylsulfonyl fluoride, and 50
m grml aprotinin, as described previously w12x. Protein
concentrations were measured by the method of Bradford
w1x with a kit from Bio-Rad. Cell extracts Ž10 m g. were
loaded on a 12% SDS-PAGE gel w13x, and separated
proteins were transferred onto an Immobilon P membrane
ŽMillipore.. The membrane was blocked, and incubated
with an anti-Iba1 antibody, as described previously w12x.
After washing, Iba1 protein was then visualized by using a
goat anti-rabbit IgG antibody conjugated with horseradish
peroxidase ŽHRP. ŽBio-Rad. and the ECL system
ŽAmersham..
2.2. Immunocytochemical staining of mixed culture brain
cells
Neocortical tissue was removed from 17-day rat em-
bryos, and dissociated cells were seeded into the wells of a
PLL-coated dish, cultured in DMEM containing 10% FCS,
and maintained for the number of days indicated. After
fixation with 4% paraformaldehyde, the cells were washed
in phosphate-buffered saline ŽPBS. and blocked overnight
in PBS containing 3% normal goat serum and 3% bovine
serum albumin ŽBSA. ŽSigma. at 48C. The cells were the
incubated with rabbit polyclonal anti-Iba1 antibody and
either mouse anti-microtubule-associated protein 2 ŽMAP2.
monoclonal antibody ŽmAb. ŽSigma., mouse anti-glial fib-
rillary acidic protein ŽGFAP. mAb ŽLipshaw., mouse anti-
myelin basic protein mAb ŽMBP. ŽBoehringer., or mouse
anti-ED-1 mAb ŽSertec. overnight at 48C. They were
subsequently washed in PBS and incubated for 1 h with a
fluorescein isothiocyanate ŽFITC.-conjugated anti-mouse
IgG antibody ŽTAGO. and a Texas Red ŽTX.-conjugated
anti-rabbit IgG antibody ŽAmersham..
2.3. Immunohistochemistry
Adult Wistar rats were perfused through the left ventri-
cle of the heart with PBS and then with 4% paraformal-
dehyde in PBS. The fixed brains were immersed in 20%
sucrose in PBS overnight, quickly frozen with dry ice
powder, sliced into 12–14 m m sections with a cryostat,
and stored at y808C.
For the Iba1 staining, sections were washed in PBS,
incubated in 0.3% H 2 O 2 in methanol for 30 min to inhibit
endogenous peroxidase activity, washed in PBS, and
blocked in PBS containing 1.5% normal goat serum and
1% BSA for 2 h at room temperature. The sections were
then incubated with a rabbit anti-Iba1 polyclonal antibody
overnight at 48C, washed in PBS, and incubated with a
HRP-conjugated goat anti-rabbit IgG antibody ŽBio-Rad.
for 2 h at room temperature. They were then incubated
with 50 mM Tris-HCl ŽpH 7.2. containing 0.05% di-
aminobenzidine tetrahydrochloride ŽDAB. and 0.01%
H 2 O 2 . For control staining, normal rabbit IgG was used as
the primary antibody.
For OX-42 staining, the sections were incubated with
MRC OX-42 ŽSerotect. overnight at 48C. Antigen was
subsequently detected with a Vectastain Elite ABC-kit
ŽVector..
After the reaction, the sections were counterstained with
Mayer haematoxylin, dehydrated in ethanol steps, and
mounted.
2.4. Immunohistochemical staining of the facial nucleus
after axotomy
Under ether anesthesia, the right facial nerve of a
Wistar rat Ž7–8 weeks old. was transected near its exit
from the cranium via the stylomastoid foramen by the
method described previously w20x. Rats were sacrificed at
the times indicated, and the brain sections were prepared
as described in Ref. w20x.
The coronal section corresponding to the level of the
facial nucleus was incubated with the anti-Iba1 antibody
and then incubated with TX-conjugated anti-rabbit IgG
ŽAmersham.. The same sections were also incubated with
FITC-labeled B 4-isolectin from Griffonia simplicifolia
ŽGSA I-B 4 . ŽSigma. according to the procedure of Persen-
chini et al. w25x and Streit and Kreutzberg w31x for double-
staining. Iba1 staining and GSA I-B 4 binding were anal-
ysed by fluorescence microscopy.
2.5. Immunoblot analysis of the facial nucleus after facial
nerÕe axotomy
At the end of the desired period following axotomy, the
brainstem was sectioned in a cryostat from caudal to
rostral until the facial nuclei were reached. The facial
nucleus was excised on both the operated and the unoper-
ated side and immediately homogenized in 10 mM N-2-
hydroxyethylpiperazine-N X-2-ethanesulfonic acid–NaOH,
pH 7.4 containing 5 mM EDTA, 50% glycerol, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
Lysates were centrifuged at 14,000 rpm for 10 min to
remove insoluble debris. The protein concentration of the
 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9
resulting supernatant was measured as described above and
stored at y808C. A portion of the supernatant Ž25 m g
protein. was examined by immunobloting to detect Iba1
protein, as described above.
3. Results
3.1. Microglia-specific expression of Iba1 protein in cul-
tured brain cells
In our previous reports w12,21x, we have reported cDNA
cloning of iba1. RNA blot analysis demonstrated that Iba1
mRNA is expressed in cultured microglia but not in neu-
rons or astroglia. To confirm the cell-type localisation of
Iba1 protein in the brain, microglia, neurons, astroglia, and
meningeal fibroblasts were prepared and immunoblotted
with the anti-Iba1 antibody. A 17-kDa band corresponding
to Iba1 protein was clearly detected in cultured microglia,
but not in neurons, astroglia or meningeal fibroblasts ŽFig.
1.. Furthermore, mixed primary-cultured brain cells were
double-stained with the anti-Iba1 antibody and either anti-
MAP2, anti-GFAP, anti-MBP or anti-ED-1 mAbs, antigens
of which are specific markers for neurons, astroglia, oligo-
dendroglia and microglia, respectively. As shown in Fig.
Fig. 1. Immunoblot analysis of Iba1 protein in cultured brain cells.
Neurons and microglia were cultured for 1 day and astroglia and
meningeal fibroblasts were cultured for 7 days, and cell extracts were
immunoblotted with anti-Iba1 antibody. The arrowhead indicates the
position of Iba1 protein.
3
2G and H, the Iba1-positive cells and the ED-1-positive
cells were identical. By contrast, none of the Iba1-positive
cells were positive for MAP2 ŽFig. 2A,B., GFAP ŽFig.
2C,D., or MBP ŽFig. 2E,F.. These results clearly indicated
that the Iba1 protein was specifically expressed in the
microglia among brain cells under primary culture condi-
tions.
3.2. Immunohistochemical analysis of Iba1 expression in
rat brain
In order to determine whether Iba1 protein is expressed
in microglia in the brain itself, sections were prepared
from adult rat brain for immunohistochemical staining
with anti-Iba1 antibody. In the parenchyma of the cere-
brum and hippocampus, Iba1-positive cells had small nu-
clei, scant cytoplasm, and thin, branched processes ŽFig.
3A,D.. This kind of cell was found throughout the
parenchyma of the white and grey matter. The distinctive
morphology and widespread distribution of these cells
were highly consistent with the classical descriptions of
ramified microglia w2,23x. In addition, we compared these
immunostaining patterns with staining for OX-42, a classi-
cal marker for microglia w27x. The population and mor-
phology of Iba1-positive cells were quite similar to OX-
42-positive cells in the cerebrum ŽFig. 3A,C.. These find-
ings indicated that the type of cells in normal adult rat
brain that are Iba1-positive are ramified microglia.
In addition to the ramified microglia, perivascular cells
were also stained positive for Iba1 ŽFig. 3E.. These cells
had elongated cell bodies and were closely adherent to the
blood vessels. Based on their morphology and location,
they appeared to be the perivascular microglia described in
earlier reports w4,11x. Cells with macrophage-like morphol-
ogy, including a round nucleus, abundant cytoplasm, and a
few short processes in the leptomeninges and choroid
plexus were also positive for Iba1 ŽFig. 3F..
3.3. Iba1 expression in actiÕated microglia in facial nu-
cleus after axotomy of the facial nerÕe
In response to various stimuli, microglia are known to
transform into activated forms that can be distinguished by
their morphology and antigenicity w30x. Facial nerve axo-
tomy is a well-established experimental system for detect-
ing activated microglia in the facial nucleus w32x. In addi-
tion, macrophages can not invade the facial nucleus from
the blood stream under these conditions w9x. We therefore
examined the changes in expression of Iba1 in the acti-
vated microglia in the facial nucleus following axotomy.
The facial nuclei were first examined by immunohisto-
chemistry with anti-Iba1 antibody 7 days after the axotomy
ŽFig. 4.. Low magnification of the brainstem sections
clearly showed an increase in Iba1-immunoreactivity on
the operated side compared with the unoperated side ŽFig.
4A.. At higher magnification, heavily stained cells sur-
4 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9

Fig. 2. Double immunocytochemical staining of cultured mixed brain cells. Cells were cultured for 3 days ŽA–D, G and H. or 10 days ŽE, F., and
double-stained with the anti-Iba1 and anti-cell-type-specific antibodies. Neurons, astroglia, oligodendroglia, and microglia were recognised by anti-MAP2
ŽA., anti-GFAP ŽC., anti-MBP ŽE., and anti-ED-1 ŽG. mAbs, respectively, and visualized with a TX-conjugated secondary antibody. Iba1 staining was
visualized with FITC-conjugated anti-rabbit IgG in ŽB., ŽD., ŽF., and ŽH., which are the same fields as in ŽA., ŽC., ŽE., and ŽG., respectively. Scale bar, 50
m m.

rounding the motoneurons were observed on the operated rather weak. To confirm that the heavily stained cells on
side ŽFig. 4B., suggesting that the immunopositive cells the operated side were activated microglia, the sections
were activated microglia. Ramified microglia stained on were double-stained with anti-Iba1 antibody and GSA
the control side ŽFig. 4C., but their immunoreactivity was I-B 4 , which is known to bind to both ramified and acti-
 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9 5

Fig. 3. Immunohistochemical staining of adult rat brain. Sections of cerebral cortex were stained with the anti-Iba1 antibody ŽA., normal rabbit IgG ŽB.,
and OX-42 ŽC.. Sections of the hippocampus were immunostained with the anti-Iba1 antibody ŽD.. Sections containing blood vessels in the parenchyma of
cerebrum ŽE. and leptomeninges ŽF. were also immunostained with the anti-Iba1 antibody. Immunopositive cells in ŽE. and ŽF. are indicated by
arrowheads. All of the sections were counterstained with haematoxylin. Scale bars, 50 m m.

vated microglia w29,31x. The anti-Iba1 antibody and GSA
I-B 4 recognised the same cells surrounding motoneurons
on day 7 after the axotomy ŽFig. 4D,E.. These findings
indicate that Iba1 expression is upregulated in activated
microglia following axotomy.
The changes in level of Iba1 expression after axotomy
were examined by immunohistochemistry and immunoblot
analysis. Three days after axotomy, heavily stained mi-
croglia with altered shapes appeared on the operated side
ŽFig. 5A.. The stained cells on the operated side had
extensive cytoplasm and short processes, and some were
located in perineuronal positions. On post-operative day 7,
Iba1 immunoreactivity was dramatically increased in the
activated microglia surrounding the motoneurons ŽFig. 5C..
On day 28, the proportion of perineuronal cells had de-
creased, and most of the cells had long processes, but
6 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9

Fig. 4. Immunohistochemical staining of the facial nucleus with the anti-Iba1 antibody 7 days after axotomy of the facial nerve. ŽA. A general view of a
brainstem section corresponding to the level of the facial nucleus is shown. The section was immunostained with the anti-Iba1 antibody. Scale bar, 10 m m.
High magnifications of the facial nucleus on the side of the facial nerve axotomy ŽB. and on the unoperated side ŽC.. Scale bar, 50 m m. A section of the
facial nucleus on the operated side was double-stained with the anti-Iba1 antibody and a TX-conjugated secondary antibody ŽD. and with FITC-conjugated
GSA I-B 4 ŽE.. Scale bar, 50 m m.

some were still apposed to neuronal soma ŽFig. 5E.. The
immunoreactivity of these cells at this time appeared to be
weaker than on day 7. On the control side, Iba1 immuno-
reactivity did not change in the ramified microglia at any
of the stages examined ŽFig. 5B,D,F.. The changes in
expression of Iba1 in the microglia following axotomy
were further investigated by immunoblot analysis using the
facial nuclei excised on the operated side. Immunoblot
analysis revealed a slightly increased level of Iba1 even on
1 day after the axotomy and a continued increase until day
7 followed by a gradual decline until day 28 ŽFig. 6.. The
above findings indicated that expression of Iba1 protein is
 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9 7

Fig. 5. Immunohistochemical staining of the facial nuclei after axotomy of the facial nerve. Sections were prepared 3 days ŽA, B., 7 days ŽC, D. and 28
days ŽE, F. following the axotomy. ŽA., ŽC., and ŽE.: operated side; ŽB., ŽD., and ŽF.: control side. The sections were immunostained with the anti-Iba1
antibody and counterstained with haematoxylin. Scale bar, 50 m m.

upregulated in activated microglia after the facial nerve
axotomy and peaks on day 7.
4. Discussion
In the present study we have shown that Iba1 is specifi-
cally expressed in microglia on the protein level, but not in
neurons, astroglia, or oligodendroglia, both in vitro and in
vivo. Immunocytochemical staining of cultured mixed brain
cells showed that the cells that stained with the anti-Iba1
antibody also stained with the anti-ED-1 antibody ŽFig.
2G,H., which is one of the well-established antibody reacts
with microglia w3x. In addition, immunohistochemical
staining of normal rat brain sections showed that Iba1
protein was expressed in ramified microglia ŽFig. 3A,D.,
8 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9
Fig. 6. Immunoblot analysis of Iba1 protein expression in the facial nuclei
after axotomy. Protein was extracted from the facial nuclei on the
operated side on the days indicated and immunoblotted with the anti-Iba1
antibody. The arrowhead indicates the position of Iba1 protein.
which also stained for another microglia-specific marker,
OX-42 ŽFig. 3C. w7,27x.
Microglia have been reported to exhibit antigen hetero-
geneity in the brain w6–8,14,24,34x. For example, an anti-
body against rat complement type 3 receptor was reported
to react with ramified microglia but not with perivascular
microglia w7x. By contrast, an anti-ED2 antibody reacts
with perivascular microglia, but not with ramified mi-
croglia w8x. Gehrmann and Kreutzberg w6x reported that
both MUC 101 and 102 mAb, which were raised against
cultured rat microglia, specifically recognise microglia, but
that the distribution patterns of the cells that stained with
these antibodies differ. MUC 101 frequently stained rami-
fied microglia in white matter but not in grey matter, while
MUC 102 strongly stained ramified microglia throughout
both white and gray matter. These findings suggest that
antigen heterogeneity exists among ramified microglia and
that this heterogeneity reflects possible subpopulations of
microglia cells. In the present study, the anti-Iba1 antibody
prepared by our laboratory could recognised ramified mi-
croglia throughout the white and gray matter and perivas-
cular microglia in the brain. This pattern of expression
suggested that Iba1 is expressed in common by different
subpopulations of microglia.
We have reported that Iba1 protein is expressed in
monocytic cell lines w12,21x. In our preliminary study, the
anti-Iba1 antibody immunohistochemically stained other
types of cells in the monocyte–macrophage lineage, such
as splenic macrophages and Kupffer cells Ždata not shown..
These findings suggested that Iba1 should be applicable to
the identification of cells in the monocyte–macrophage
lineage, including microglia.
Microglia are known to transform into activated mi-
croglia in response to various environmental stimuli. Al-
though their precise functions remain uncertain, activated
microglia are thought to be involved in either the injury
repair processes or the pathogenesis of certain neurological
diseases w5,10,15–18,22,26,35,36x. To determine whether
Iba1 is involved in regulating the function of activated
microglia, we investigated the expression of Iba1 protein
in microglia following facial nerve axotomy. In this study,
we showed that Iba1 protein was upregulated in microglia
surrounding the axotomised motoneurons in the facial
nucleus. The time course of morphological changes in the
activated microglia after axotomy corresponded well to
that of Iba1 protein levels in the facial nucleus ŽFigs. 4–6..
This suggested that Iba1 protein plays some role in regulat-
ing the function of activated microglia. In view of the
location of the iba1 gene within the major histocompatibil-
ity complex class III region w12x, the Iba1 upregulation
shown in the activated microglia is of great interest, be-
cause microglia are known to act as antigen-presenting
cells in the brain w34x. The functional role of this protein in
activated microglia is currently being investigated in our
laboratory.
Expression of Iba1 protein was detectable only in
monocytic lineage including microglia and macrophage by
RNA blot and immunoblot w12x as well as by immunocyto-
chemistry and immunohistochemistry as shown in this
article. These findings strongly demonstrates a highly mi-
croglia enriched concentration of Iba1 protein. However,
the data cannot exclude the possibility that Iba1 protein
might be present in other cell types at lower concentra-
tions.
Finally, the anti-Iba1 polyclonal antibody used in this
study was raised against a synthetic peptide corresponding
to the Iba1 carboxy-terminal sequence that is completely
conserved among human, rat, and mouse Iba1 proteins
Ždata not shown.. The anti-Iba1 antibody is therefore able
to recognise human, rat, and mouse Iba1 proteins w21x.
Furthermore, it should be noted that the anti-Iba1 antibody
is the only polyclonal antibody currently available by
which microglia can be double-stained with other mono-
clonal antibodies.
Acknowledgements
This study was supported by Grants from the Japanese
Ministry of Health and Welfare, the Science and Technol-
ogy Agency, Japan, and by a Grant-in-Aid for Scientific
Research on Priority Areas from the Japanese Ministry of
Education, Science, Sports and Culture.
References
w1x M.M. Bradford, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding, Anal. Biochem. 72 Ž1976. 248–254.
 D. Ito et al.r Molecular Brain Research 57 (1998) 1–9
w2x P. del Rio-Hortega, El tercer elemento de los centros nerviosos: I. La
microglia en estados normal; II. Intervencio de la microglia en los
processos patologicas; III. Naturaleza probable de la microglia, Bol.
Soc. Espanola Biol. 9 Ž1919. 69–120.
w3x C.D. Dijkstra, E.A. Dopp, P. Joling, G. Kraal, The heterogeneity of
mononuclear phagocytes in lymphoid organs: distinct macrophage
subpopulations in the rat recognised by monoclonal antibodies ED1,
ED2 and ED3, Immunology 54 Ž1985. 589–599.
w4x Dolman, C.L., Microglia, in: R.L. Davis, D.M. Robertson ŽEds..,
Textbook of Neuropathology, Microglia, Williams and Wilkins,
Baltimore, 1985, pp. 117–137.
w5x J. Gehrmann, P. Bonnekoh, T. Miyazawa, U. Oschlies, E. Dux, K.A.
Hossman, G.W. Kreutzberg, The microglia reaction in the rat hip-
pocampus following global ischemia: immuno-electron microscopy,
Acta Neuropathol. 84 Ž1992. 588–595.
w6x J. Gehrmann, G.W. Kreutzberg, Characterisation of two new mono-
clonal antibodies directed against rat microglia, J. Comp. Neurol.
313 Ž1991. 409–430.
w7x M.B. Graeber, W.J. Streit, G.W. Kreutzberg, Axotomy of the rat
facial nerve leads to increased CR3 complement receptor expression
by activated microglial cells, J. Neurosci. Res. 211 Ž1988. 8–24.
w8x M.B. Graeber, W.J. Streit, G.W. Kreutzberg, Identity of ED2-posi-
tive perivascular cells in rat brain, J. Neurosci. Res. 22 Ž1989.
103–106.
w9x M.B. Graeber, W. Teztlaff, W.J. Streit, G.W. Kreutzberg, Microglial
cells but not astrocytes undergo mitosis following rat facial nerve
axotomy, Neurosci. Lett. 85 Ž1988. 317–321.
w10x S. Haga, K. Akai, T. Ishii, Demonstration of microglial cells in and
around senile Žneuritic. plaques in the Alzheimer brain, Acta Neu-
ropathol. 77 Ž1989. 569–575.
w11x W.F. Hickey, H. Kimura, Perivascular microglial cells of the CNS
are bone marrow-derived and present antigen in vivo, Science 239
Ž1988. 290–292.
w12x Y. Imai, I. Ibata, D. Ito, K. Ohsawa, S. Kohsaka, A novel gene iba1
in the major histocompatibility complex class III region encoding an
EF hand protein expressed in a monocytic lineage, Biochem. Bio-
phys. Res. Commun. 224 Ž1996. 855–862.
w13x U.K. Laemmli, Cleavage of structural proteins during the assembly
of the head of bacteriophage T4, Nature 227 Ž1970. 680–685.
w14x L.J. Lawson, V.H. Perry, P. Dri, S. Gordon, Heterogeneity in the
distribution and morphology of microglia in the normal, adult mouse
brain, Neuroscience 39 Ž1990. 151–170.
w15x P.J. Maddon, A.G. Dagleish, J.S. McDougal, P.R. Calpham, R.A.
Weiss, R. Axel, The T4 gene encodes the AIDS virus receptor and is
expression in the immune system and the brain, Cell 47 Ž1986.
333–348.
w16x P.L. McGeer, S. Itagaki, B.E. Boyes, E.G. McGeer, Reactive mi-
croglia are positive for HLA-DR in the substantia nigra of Parkin-
son’s and Alzheimer’s disease brains, Neurology 38 Ž1988. 1285–
1291.
w17x L. Meda, M.A. Cassatella, G.I. Szendrei, L. Otvos Jr., P. Baron, M.
Villalba, D. Ferrari, F. Rossi, Activation of microglial cells by
beta-amyloid protein and interferon-gamma, Nature 374 Ž1995.
647–650.
w18x T. Morioka, A.N. Kalehua, W.J. Streit, Progressive expression of
9
immunomolecules on microglia cells in rat dorsal hippocampus
following transient forebrain ischemia, Acta Neuropathol. 83 Ž1992.
149–157.
w19x K. Nakajima, M. Hamanoue, N. Takemoto, T. Hattori, K. Kato, S.
Kohsaka, Plasminogen binds specifically to alpha-enolase on rat
neuronal plasma membrane, J. Neurochem. 63 Ž1994. 2048–2057.
w20x K. Nakajima, M. Reddington, K. Kohsaka, G.W. Kreutzberg, Induc-
tion of urokinase-type plasminogen activator in rat facial nucleus by
axotomy of the facial nerve, J. Neurochem 66 Ž1996. 2500–2505.
w21x K. Ohsawa, Y. Imai, K. Nakajima, S. Kohsaka, Generation and
characterization of a microglial cell line, MG5, derived from p53-de-
ficient mouse, Glia 21 Ž1997. 285–298.
w22x V.H. Perry, S. Gordon, Modulation of CD4 antigen on macrophages
and microglia in rat brain, J. Exp. Med. 166 Ž1987. 1138–1143.
w23x V.H. Perry, S. Gordon, Macrophages and microglia in the nervous
system, Trends Neurosci. 11 Ž1988. 273–277.
w24x V.H. Perry, D.A. Hume, S. Gordon, Immunohistochemical localisa-
tion of macrophages and microglia in the adult and developing
mouse brain, Neuroscience 15 Ž1985. 313–326.
w25x A. Persenchini, N.D. Moncrief, R.H. Kretsinger, The EF-hand fam-
ily of calcium-modulated proteins, TINS 11 Ž1989. 462–467.
w26x A. Probst, H. Brunnschweiler, V. Lautenschlager, J. Ulrich, A
special type of senile plaque, possibly an initial state, Acta Neu-
ropathol. 74 Ž1987. 133–141.
w27x A.P. Robinson, T.M. White, D.W. Mason, Macrophage heterogene-
ity in the rat as delineated by two monoclonal antibodies MRC
OX-41 and MRS OX-42, the latter recognising complement receptor
type 3, Immunology 57 Ž1986. 239–247.
w28x S. Saitoh, N. Iijima, M. Ikeda, K. Nakajima, M. Kimura, M.
Katsuki, T. Mori, S. Kohsaka, De novo production of alpha 2-
macroglobulin in cultured astroglia from rat brain, Brain Res. Mol.
Brain Res. 12 Ž1992. 155–161.
w29x W.J. Streit, An improved staining method for rat microglial cells
using the lectin from Griffonia simplicifolia, J. Histochem. Cy-
tochem. 38 Ž1990. 1683–1686.
w30x W.J. Streit, M.B. Graeber, G.W. Kreutzberg, Functional plasticity of
microglia: a review, Glia 1 Ž1988. 301–307.
w31x W.J. Streit, G.W. Kreutzberg, Lectin binding by resting and acti-
vated microglia, J. Neurocytol. 16 Ž1987. 249–260.
w32x W.J. Streit, G.W. Kreutzberg, Response of endogenous glial cells to
motor neuron degeneration induced by toxic ricin, J. Comp. Neurol.
2682 Ž1988. 48–263.
w33x N. Takei, J. Kondo, K. Nagaike, K. Ohasawa, K. Kato, S. Kohsaka,
Neuronal survival factor from bovine brain is identical to neuron-
specific enolase, J. Neurochem. 57 Ž1991. 1178–1184.
w34x W.E. Thomas, Brain macrophages: evaluation of microglia and their
functions, Brain Res. Rev. 17 Ž1992. 61–74.
w35x B.A. Watkins, H.H. Dorn, W.B. Kelly, R.C. Armstrong, B.J. Potts,
F. Michaels, C.V. Kufta, M. Dubois-Dalcq, Specific tropism of
HIV-1 for microglia cells in primary human brain cultures, Science
249 Ž1990. 549–553.
w36x M.N. Woodroofe, A.S. Bellamy, M. Feldman, A.N. Davison, M.L.
Cuzner, Immunocytochemical characterisation of the immune reac-
tion in the central nervous system in multiple sclerosis. Possible role
for microglia in lesion growth, J. Neurol. Sci. 74 Ž1986. 135–152.