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The Journal of Neuroscience, July 15, 2002, 22(14):5865-5878
A Dual Role for the SDF-1/CXCR4 Chemokine Receptor System in
Adult Brain: Isoform-Selective Regulation of SDF-1 Expression Modulates
CXCR4-Dependent Neuronal Plasticity and Cerebral Leukocyte Recruitment
after Focal Ischemia
Ralf K.
Stumm1,
Jutta
Rummel1,
Vera
Junker2,
Carsten
Culmsee2,
Manuela
Pfeiffer1,
Josef
Krieglstein2,
Volker
Höllt1, and
Stefan
Schulz1
1 Institute of Pharmacology and Toxicology,
Otto-von-Guericke University Magdeburg, 39120 Magdeburg, Germany, and
2 Institute of Pharmacology and Toxicology, Philipps
University Marburg, 35037 Marburg, Germany
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ABSTRACT |
The chemoattractant stromal cell-derived factor-1 (SDF-1) and its
receptor CXC chemokine receptor 4 (CXCR4) are key modulators of immune
function. In the developing brain, SDF-1 is crucial for neuronal
guidance; however, cerebral functions of SDF-1/CXCR4 in adulthood are
unclear. Here, we examine the cellular expression of SDF-1 isoforms and
CXCR4 in the brain of mice receiving systemic lipopolysaccharide (LPS)
or permanent focal cerebral ischemia. CXCR4 mRNA was constitutively
expressed in cortical and hippocampal neurons and ependymal cells.
Hippocampal neurons targeted the CXCR4 receptor to their
somatodendritic and axonal compartments. In cortex and hippocampus,
CXCR4-expressing neurons exhibited an overlapping distribution with
neurons expressing SDF-1 transcripts. Although neurons synthesized
SDF-1 mRNA, the SDF-1 isoform was selectively expressed by
endothelial cells of cerebral microvessels. LPS stimulation
dramatically decreased endothelial SDF-1 mRNA expression throughout
the forebrain but did not affect neuronal SDF-1. After focal
cerebral ischemia, SDF-1 expression was selectively increased in
endothelial cells of penumbral blood vessels and decreased in
endothelial cells of nonlesioned brain areas. In the penumbra, SDF-1
upregulation was associated with a concomitant infiltration of
CXCR4-expressing peripheral blood cells, including macrophages.
Neuronal SDF-1 was transiently downregulated and neuronal CXCR4 was
transiently upregulated in the nonlesioned cerebral cortex in response
to ischemia. Although endothelial SDF-1 may control cerebral
infiltration of CXCR4-carrying leukocytes during cerebral ischemia, the
neuronal SDF-1/CXCR4 system may contribute to ischemia-induced
neuronal plasticity. Thus, the isoform-specific regulation of SDF-1
expression modulates neurotransmission and cerebral infiltration via
distinct CXCR4-dependent pathways.
Key words:
CXC chemokine; stromal cell-derived factor-1; CXCR4; focal cerebral ischemia; lipopolysaccharide; in situ
hybridization; immunocytochemistry; antibody
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INTRODUCTION |
Chemokines are small proteins with
chemotactic effects on leukocytes. Among chemokines expressed in brain,
stromal cell-derived factor-1 (SDF-1) attracted much attention because
it was shown to be crucial for cerebellar development (Ma et al., 1998 ;
Zou et al., 1998; Klein et al., 2001; Lu et al., 2001). The isoforms SDF-1 and SDF-1 arise by alternative splicing from the SDF-1 gene. They share an identical 5'-untranslated and coding region but
differ at the 3'-end (Tashiro et al., 1993; Nagasawa et al., 1994;
Shirozu et al., 1995). A rat SDF-1 mRNA has been described, which is
similar to SDF-1 but contains a 2.5 kb insertion after codon 89 (Gleichmann et al., 2000). The SDF-1 and SDF-1 proteins are
identical except four C-terminal amino acids and represent endogenous
ligands of the CXC chemokine receptor 4 (CXCR4), which also functions
as coreceptor for human immunodeficiency virus (HIV-1) in lymphocytes
(Bleul et al., 1996b; Feng et al., 1996; Oberlin et al., 1996).
CXCR4-mediated apoptosis in neuronal cultures induced by HIV-1
glycoprotein gp120 and SDF-1 indicates a potential role of this
receptor in neurodegeneration (Hesselgesser et al., 1998; Kaul and
Lipton, 1999). Microglia-enhanced SDF-1-stimulated release of TNF
and glutamate from astrocytes in vitro was proposed as a
novel excitotoxic pathomechanism in neurodegenerative diseases (Bezzi
et al., 2001). Because SDF-1 modulates synaptic transmission via
neuronal CXCR4 receptors in the developing cerebellum (Limatola et al.,
2000), SDF-1 may exert physiological functions also in other brain regions.
Our understanding of the role of the SDF-1/CXCR4 receptor system in the
adult brain has been hampered, however, by the lack of information
about the cellular expression of CXCR4 and SDF-1 isoforms. Previous
contradictory studies described SDF-1 expression either as widespread
in neurons and oligodendrocytes (Gleichmann et al., 2000) or as
restricted to distinct neuronal populations and endothelial cells (Tham
et al., 2001). Moreover, the differential cellular expression,
regulation, and function of SDF-1 isoforms has not been characterized
in the brain.
SDF-1 is involved in inflammation and wound healing (Nanki et al.,
2000; Fedyk et al., 2001). SDF-1 triggers adhesion of lymphocytes to
activated endothelial cells in vitro (Kantele et al., 2000). Most likely, endothelial SDF-1 plays a crucial role in
trans-endothelial migration of leukocytes (Campbell et al.,
1998; Pablos et al., 1999; Grabovsky et al., 2000). This raises the
question to what extent SDF-1 in the brain may exert chemotactic
effects on monocytes and lymphocytes after endothelial activation by
systemic LPS-induced immune challenge (Bleul et al., 1996a; Quan et
al., 1997; Lebel et al., 2000; Rivest et al., 2000). The potential
excitotoxic and chemotactic activities of SDF-1 may also be relevant in
focal cerebral ischemia, a condition involving disruption of the
blood-brain barrier (BBB), excitotoxic neurodegeneration, and glial
activation (Stoll et al., 1998).
Here, we established the cellular expression patterns of SDF-1 isoforms
and CXCR4 in the adult mouse brain by in situ hybridization. The subcellular distribution of CXCR4 was characterized by confocal microscopy using a novel antiserum. We provide evidence for
differential expression of SDF-1 isoforms in endothelial and neuronal
cells. Furthermore, our results suggest that isoform-specific
regulation of SDF-1 and SDF-1 selectively modulates
CXCR4-dependent neurotransmission and cerebral leukocyte infiltration.
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MATERIALS AND METHODS |
Generation of anti-peptide antisera
Rabbit polyclonal antisera were generated against the C-terminal
portion of the mouse CXCR4 receptor (residues 338-359,
KGKRGGHSSVSTESESSSFHSS), which is identical in mouse, rat, and human.
The peptide was coupled to keyhole limpet hemocyanin, and the conjugate
was emulsified with Freund's adjuvant and injected into three rabbits
(2144-2146) for immunization as described (Schreff et al., 2000). Two
of the three antisera (2144 and 2145) developed a titer against their immunizing peptide and yielded essentially identical results during antibody characterization. Antiserum 2144 was subjected to
immunoaffinity purification and used throughout this study.
Western blot analysis
The mouse CXCR4 cDNA (kind gift of Dr. Martin Lipp and Dr.
Reinhold Förster, Max Delbrück Center for Molecular
Medicine, Berlin, Germany) was tagged at its N terminus with the T7
epitope tag sequence MASMTGGQQMG and subcloned into pcDNA3.1 expression vector (Invitrogen, Groningen, The Netherlands) as described (Koch et
al., 2001). Cell membranes were prepared from human embryonic kidney
(HEK) 293 cells stably transfected with the CXCR4 receptor, as well as
from mouse brain, and solubilized in lysis buffer (150 mM
NaCl, 5 mM EDTA, 3 mM EGTA, 20 mM
HEPES, pH 7.4, containing 4 mg/ml dodecyl--maltoside, and proteinase
inhibitors: 1 mM phenylmethylsulfonylfluoride, 1 µM pepstatin, 10 µg/ml leupeptin, and 2 µg/ml
aprotinin). The lysate was cleared and subjected to immunoprecipitation
with anti-CXCR4 antibodies. Alternatively, glycoproteins were partially
purified using wheat germ lectin agarose (WGA; Vector Laboratories,
Burlingame, CA). Beads were washed and eluted with SDS-sample buffer
for 20 min at 60°C. Either crude membrane proteins (100 µg per
lane) or WGA extracts purified from 500 µg membrane proteins were
subjected to 10% SDS-PAGE and immunoblotted onto nitrocellulose. Blots
were incubated with 1 µg/ml affinity-purified anti-CXCR4 (2144) or rat monoclonal anti-CXCR4 antibody clone 2B11, which is directed against the N terminus (Forster et al., 1998), overnight at 4°C. Bound primary antibodies were then detected using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. For absorption controls, anti-CXCR4 antibody was preincubated with 10 µg/ml of its
cognate peptide for 2 hr at room temperature.
Immunocytochemistry
Wild-type HEK 293 cells or HEK 293 cells stably transfected with
T7-tagged CXCR4 receptor were grown on coverslips overnight. For
internalization studies, cells were treated with SDF-1 in concentrations ranging from 1 to 1000 ng/ml for 30 min and fixed with
4% paraformaldehyde and 0.2% picric acid in 0.1 M
phosphate buffer, pH 7.4, for 1 hr at room temperature. Cells were
washed and stained with affinity-purified anti-CXCR4 or anti-T7
antibodies (Gramsch Laboratories) as described (Pfeiffer et al., 2001).
Bound primary antibodies were detected with biotinylated anti-rabbit IgG antibodies (Vector) followed by cyanine 3.18 (Cy3)-conjugated streptavidin (Amersham).
Immunohistochemistry
Male NMRI mice or male Wistar rats were deeply anesthetized with
chloral hydrate and transcardially perfused with Tyrode's solution
followed by Zamboni's fixative containing 4% paraformaldehyde and
0.2% picric acid in 0.1 M phosphate buffer, pH 7.4. Brains were rapidly dissected and postfixed in the same fixative for 2 hr at
room temperature. Tissue was cryoprotected by immersion in 30% sucrose
for 48 hr at 4°C before sectioning using a freezing microtome.
Free-floating sections (30-40 µm) were washed and incubated with 1 µg/ml affinity-purified anti-CXCR4 antibodies. Staining of primary
antibody was detected using Cy3-conjugated anti-rabbit antibody or the
tyramine amplification procedure as described (Schulz et al., 1998).
For dual immunofluorescence of CXCR4 and GFAP, sections were first
stained for CXCR4 as described above and subsequently labeled with
guinea pig anti-GFAP antibody (1:2000) (Progen, Heidelberg, Germany),
which was visualized with a Cy5-conjugated secondary antibody. For
immunocytochemical controls, the primary antibody was omitted, replaced
by preimmune sera, or absorbed with several concentrations ranging from
1 to 10 µg/ml of homologous or heterologous peptides for 2 hr at room
temperature. Specimens were examined using a Leica TCS-NT laser
scanning confocal microscope (Heidelberg, Germany) equipped with a
krypton/argon laser. Cy3 was imaged with 568 nm excitation and 570-630
nm bandpass emission filters, and Cy5 was imaged with 647 nm excitation
and 665 nm longpass emission filters.
Probes used in in situ hybridization
The expression of the SDF-1 isoforms was characterized using
riboprobes derived from three partial mouse cDNAs. The first probe
(called SDF-1) was complementary to nucleotides (nt) 11-273 of
SDF-1 and SDF-1 (GI:12025675 and GI:12025674). This corresponds to parts of the 5'-untranslated as well as the coding region (nt 82-351 of GI:12025675). The nt 19-262 of this probe are also 96% identical to rat SDF-1, SDF-1, and SDF-1. The second probe (SDF-1) was complementary to nt 1005-1780 of GI:12025675, which are
specific for mouse SDF-1. The mouse SDF-1 cDNA (Tashiro et al.,
1993) was kindly provided by Dr. T. Honjo and Dr. K. Tashiro (Kyoto
University, Kyoto, Japan). The third probe (SDF-1) was complementary
to nt 366-1186 of SDF-1 (GI:12025674) and exhibited no similarity
with SDF-1. In the rat, a SDF-1 mRNA has recently been described,
which is similar to SDF-1 but contains a 2.5 kb insertion after
codon 89 (Gleichmann et al., 2000). Although a similar isoform has not
been described so far in mice, our SDF-1 probe would be expected to
hybridize also to such a putative mouse SDF-1 mRNA. For simplicity,
we refer to the detected mRNA as SDF-1 mRNA. The SDF-1 and SDF-1
cDNAs as well as the cDNAs of the rat CXCR4 (U90610, nt 1-1050), the
polypeptide of the mouse complement component 1q (C1q; nt 35-1041)
(Wood et al., 1988), mouse GFAP (nt 189-1054) (Lewis et al., 1984),
rat pre-prosomatostatin (nt 32-307) (Goodman et al., 1985), and mouse
interleukin-1 (IL-1; nt 205-724) (Telford et al., 1986) were
amplified by RT-PCR. C1q was used as marker for both brain microglial
cells and macrophages (Schwaeble et al., 1995; Schafer et al., 2000).
All described cDNAs were cloned into the pGEM-Teasy vector (Promega)
and subjected to double-strand DNA sequencing. A riboprobe for
glutamate acid decarboxylase was used previously (Stumm et al., 2001).
For the mouse CXCR4 receptor (Heesen et al., 1996; Nagasawa et al.,
1996), the cDNA of Dr. M. Lipp and Dr. R. Förster was used.
Riboprobes in antisense and sense orientation were generated from the
linearized vector constructs by in vitro transcription
(Melton et al., 1984) using [35S]-UTP
(1000 Ci/mmol; 15 µM concentration in the
transcription reaction) or digoxigenin-UTP (DIG) (Roche Diagnostics,
Mannheim, Germany) as label. The probes were subjected to mild alkaline hydrolysis (Angerer et al., 1987) and purified using P-30 spin columns
(Bio-Rad).
In situ hybridization histochemistry
Radioactive in situ hybridization was performed as
described previously (Stumm et al., 2001). Fixation of slide-mounted
frozen sections (14 µm thickness) was performed in phosphate-buffered 4% paraformaldehyde for 60 min. After washing in PBS, the slides were
incubated for 10 min in 0.4% Triton X-100, rinsed in distilled water,
and incubated for 10 min in 0.1 M
triethanolamine, pH 8.0 (Sigma, Deisenhofen, Germany) containing 0.25%
v/v acetic anhydrate (Sigma). After washing in PBS, the sections were
dehydrated in 50 and 70% 2-propanol, air dried, and stored at
 20°C. Riboprobes were diluted in hybridization buffer [3× sodium
chloride/sodium citrate (SSC), 50 mM
NaPO4, 20 mM
dithiothreitol, 1 × Denhardt's solution, 0.25 gm/l yeast tRNA,
10% dextran sulfate, and 50% formamide] to 50,000 dpm/µl.
Hybridization was performed for 14 hr at 60°C. The slides were washed
in 2× SSC and 1× SSC before a 30 min treatment with 1 U/ml RNase T1
and 20 µg/ml RNase A (Roche Diagnostics) at 37°C in 10 mM Tris, pH 8.0, 0.5 M
NaCl, 1 mM EDTA. Slides were extensively washed
in 1× and 0.2× SSC at room temperature and subsequently in 0.2× SSC
for 60 min at 60°C. After washing in water, the tissue was dehydrated
in 50 and 70% 2-propanol. Slides were exposed together with
[14C] standards (ARC, St. Louis, MO) to
x-ray film for 2-5 d. For high-power bright- and dark-field
microscopic analysis, autoradiographic detection of
35S was performed with NTB-2 nuclear
emulsion (Eastman Kodak, Rochester, NY). Exposure times were 10-42 d.
Cresyl violet was used as counterstain.
Double in situ
hybridization histochemistry
The visualization of two different RNA transcripts in the same
tissue section was performed by combining radioactive and
nonradioactive in situ hybridization essentially as
described (Stumm et al., 2001). Digoxigenin-labeled riboprobes (see
above) were diluted in radioactive hybridization solution to 1 µg/ml,
hybridized, and washed as above. To detect nonradioactive hybrids,
slides were equilibrated in buffer 1 (0.1 M
maleic acid, 150 mM NaCl, pH 7.5) containing
0.05% Tween 20 (Merck, Darmstadt, Germany). After blocking for 1 hr in
blocking buffer (buffer 1 containing 2% blocking reagent; Roche),
alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche) were
applied overnight at a concentration of 0.5 U/ml blocking buffer.
Slides were washed twice for 30 min in buffer 1 and equilibrated in
buffer 2 (100 mM Tris, 100 mM NaCl, 50 mM
MgCl2, pH 9.4, 0.05% Tween 20) before a 16 hr
color reaction using 0.2 mM
5-bromo-4-chloro-3-indolyl phosphate and 0.2 mM
nitroblue tetrazolium salt (Roche). The reaction was stopped by washing
the slides in distilled water. The
35S-labeled probes were detected by K5
photo-emulsion (Ilford); exposure times were 14-60 d.
Combination of immunohistochemistry and in
situ hybridization
Immunocytochemistry for von Willebrand factor (vWF), an
established marker of cerebral vascular endothelial cells (Yamamoto et
al., 1998), was combined with radioactive in situ
hybridization for SDF-1 and SDF-1 mRNAs. Before hybridization, the
formalin-fixed sections were heated to 92-95°C for 15 min in 0.01 M citrate buffer, pH 6.0. Hybridization and
washing were performed as described above. After washing, sections were
blocked by incubation in PBS containing 5% bovine serum albumin (BSA)
for 30 min. A rabbit anti-human vWF antibody (Dako, Glostrup, Denmark)
was diluted 1:1000 in PBS containing 2% BSA and applied overnight. For
negative controls, the primary antibody was omitted. After several
washes in distilled water followed by rinsing in PBS, a biotinylated secondary antibody (Dianova, Hamburg, Germany) was applied for 1 hr.
After another series of washes, sections were incubated for 45 min with
the ABC reagents (Vectastain ABC Kit, Vector) followed by
diaminobenzidine reaction (0.125 µg/ml diaminobenzidine) for 10 min
at room temperature. Autoradiographic detection of 35S was performed with NTB-2 nuclear
emulsion (Eastman Kodak).
Mapping of SDF-1, SDF-1, SDF-1, and CXCR4 mRNA expression in
the brain
Coronal brain sections of two male NMRI mice were cut at 14 µm. At intervals of 500 µm, cresyl violet staining and in
situ hybridization analysis of the SDF-1, SDF-1, SDF-1, and
CXCR4 mRNAs was performed on adjacent sections. Cerebral structures were identified, and abbreviations were assigned according to the mouse
brain atlas (Franklin and Paxinos, 1997).
Animal experiments
For all animal procedures, ethical approval was sought before
the experiments according to the requirements of the German National
Act on the Use of Experimental Animals. Male virus- and pathogen-free
NMRI mice (30 gm, Tierzucht Schönwalde or Charles River, Germany)
and male Wistar rats (300 gm, Tierzucht Schönwalde) were used.
Lipopolysaccharide challenge. NMRI mice were
intraperitoneally injected (8:30-9:30 A.M.) with lipopolysaccharide
(LPS) from Escherichia coli (50 µg, total volume 500 µl;
Sigma-Aldrich L3129, Schnelldorf, Germany) suspended in sterile saline.
Five groups of three LPS-injected mice each were killed by cervical
dislocation and decapitation after 0.5, 1, 3, 6, and 24 hr. As a
control group, four mice were injected with 500 µl sterile saline and
killed after 3 hr. Brains and spleens were removed and processed for quantitative in situ hybridization. Changes in cerebral
SDF-1 and CXCR4 mRNA expression were analyzed by comparison of the five LPS-treated groups with the control group. To verify the efficacy of
the LPS stimulus, we analyzed IL-1 mRNA levels in the spleens of all
groups by in situ hybridization. Expression of IL-1 was dramatically elevated in all animals 0.5-3 hr after LPS and was virtually absent 24 hr after LPS as well as in the control group, which
is in excellent agreement with previous studies.
Animal model of focal cerebral ischemia. Permanent middle
cerebral artery occlusion (MCAO) was performed in male NMRI mice according to Welsh et al. (1987). After the mice were anesthetized by
intraperitoneal injection of tribromoethanol (600 mg/kg), a hole was
drilled into the skull to expose the middle cerebral artery. The stem
of the middle cerebral artery and both branches were permanently
occluded by electrocoagulation. Body temperature was maintained at
37 ± 1°C with a heating lamp during the surgical procedure.
After MCAO, mice were kept at an environmental temperature of 30°C
for 2 hr and then transferred to their home cages. At different time
points after ischemia, the animals were deeply anesthetized with
halothane, and the brains were quickly removed and frozen in isopentane
at 30°C.
Three independent experiments were performed to determine
ischemia-related changes in SDF-1 and CXCR4 mRNA expression by
quantitative in situ hybridization. In experiment 1, three
naive animals were compared with the MCAO groups, which consisted of
three animals at 6 hr and 1 d as well as four animals at 2 and
4 d. In experiments 2 and 3, the possible effects of surgery
(skull trepanation) were analyzed 1 and 2 d after treatment. At
either time point three MCAO-treated, three sham-operated, and three
naive animals were compared with each other. For double in
situ hybridization, two animals were prepared 2 d after MCAO
and after sham operation, respectively.
Image analysis
Film autoradiograms of three sections from each animal were
digitized on an illumination screen using NIH Image 1.62. For calibration, density measurements of the film background and the [14C] standards were obtained and
plotted against the tissue radioactivity equivalents (nanoCuries per
gram tissue; American Radiolabeled Chemicals, St. Louis, MO). A
threshold level was set to exclude pixel values below background
density from the measurement. The density measurements were expressed
as integrated optical density (IOD) (i.e., average optical density × proportional area). The regions of interest were the entire brain
section in the LPS experiment and three manually selected areas in the
ischemia experiments: the infarct including the directly adjacent area,
the ipsilateral area, in which neurodegeneration was not observed, and
the entire contralateral hemisphere. To obtain the mean value of a
region of interest in a single animal, measurements of three sections were averaged.
Statistical analysis
One-way ANOVA followed by Dunnett's multiple comparison test
was used in the LPS experiment and in the ischemia-experiment 1. In
Ischemia-experiments 2 and 3, the groups were compared with each other
using ANOVA followed by Newman-Keuls multiple comparison test.
p values <0.05 were considered statistically significant.
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RESULTS |
Cell-specific expression of SDF-1 splice variants
To analyze the differential cellular expression of SDF-1 splice
variants in the brain, two riboprobes were used for in situ hybridization that were specific for either the SDF-1 or the SDF-1 mRNA. As shown in x-ray autoradiograms of the septal area, SDF-1 mRNA was expressed by distinct neuronal structures in the cortex and claustrum (Fig.
1A, arrows).
In contrast, SDF-1 was expressed in numerous cells that exhibited a
homogenous distribution throughout the brain (Fig.
1B). A riboprobe that recognized all SDF-1 isoforms
(SDF-1 mRNA) showed a combined expression pattern of the SDF-1 and
SDF-1 mRNAs (Fig. 1C). Low-power analysis of emulsion-coated serial sections revealed that SDF-1 mRNA-expressing cells were not selectively localized in any specific structures of the
white or gray matter (Fig. 1D-G). At high-power
magnification it was apparent that SDF-1 mRNA-expressing cells
contained small round or small oval-shaped nuclei (Fig.
2D, inset).
These cells had a rod-like shape without processes and were aligned in
clusters of three to five cells, which suggests that SDF-1 is
expressed in the cerebral endothelium (Fig. 2E). To
test this hypothesis, a combination of immunohistochemistry for
vWF, which is an established marker for cerebral endothelial
cells, and in situ hybridization for SDF-1 mRNA was used.
This approach clearly revealed that SDF-1 was expressed by
vWF-immunoreactive (ir) endothelial cells of small but not large
cerebral blood vessels (Fig. 2F). SDF-1 transcripts were not detected in neurons (Fig. 2D).
The possible expression of the SDF-1 gene in glia was analyzed by
combining radioactive in situ hybridization for all SDF-1
mRNAs and nonisotopic in situ hybridization for GFAP and
C1q, established markers for astrocytes and microglial cells. However,
SDF-1 expression was not observed in these glial populations (Fig.
2B,C). The use of the riboprobe for
all SDF-1 transcripts allowed the visualization of a SDF-1 splice
variant in neurons (Fig. 2A). Hybridization with the
SDF-1-selective probe revealed that the neuronally generated SDF-1
transcripts consisted of the -isoform. SDF-1 mRNA was also
detected in meningeal cells and occasionally in the endothelium of
large parenchymal blood vessels (data not shown). These findings provide evidence for cell-specific splicing of SDF-1 transcripts in the
mouse brain. Although SDF-1 mRNA is selectively generated in
endothelial cells, SDF-1 mRNA is expressed predominantly by neurons.
SDF-1 expression in astrocytes and microglial cells was below the
detection limit.
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Figure 1.
Cerebral expression patterns of SDF-1
isoforms. In situ hybridization was performed with
riboprobes specific for SDF-1 (A), SDF-1
(B, D-G), and a riboprobe detecting all
SDF-1 isoforms (C). A-C, X-ray
autoradiograms of coronal brain sections at the striatal level.
A, Arrows, SDF-1 mRNA is expressed in
neuronal structures of the cingulate (Cg) and the
secondary somatosensory cortex (S2) as well as the
claustrum (Cl). Specific hybridization signals
for SDF-1 are undetectable in other structures of the
brain parenchyma. Note that some SDF-1 expression occurs in the
meninges. B, SDF-1 mRNA is evenly expressed
throughout the brain and not related to specific structures.
C, The probe for all SDF-1 isoforms shows an expression
pattern that reflects the simultaneous detection of SDF-1
(C, arrows) and SDF-1 mRNAs.
D-G, The left hemisphere of coronal sections after
hybridization with the SDF-1-selective probe is shown at the level
of the olfactory bulb (D), the medial septal
nucleus (MS) (E), the
ventromedial hypothalamic nucleus (VMH)
(F), and the facial nucleus
(7) (G).
D-G, Note that SDF-1 mRNA-expressing cells do not
show any specific clustering in structures of gray or white matter.
Gl, Glomerular layer of the olfactory bulb;
GrO, granule layer of the olfactory bulb;
ON, olfactory nerve layer; M2, secondary
motor cortex; LV, lateral ventricle; CPu,
caudate putamen; DEn, dorsal endopiriform nucleus;
RS, retrosplenial cortex; GrDG, granule
layer of the dentate gyrus; CP, choroid plexus;
VP, ventral posterolateral and posteromedial thalamic
nuclei; Ent, entorhinal cortex; BLA,
basolateral amygdaloid nucleus, anterior part; CBL,
cerebellum; 4V, fourth ventricle.
D-G, Exposure time, 21 d. Scale
bars: A-C, 3 mm; G, 2 mm.
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Figure 2.
Demonstration of neuronal SDF-1 expression and
endothelial SDF-1 expression by in situ hybridization
with a riboprobe detecting all SDF-1 splice variants
(A-C) and a riboprobe specific for SDF-1 mRNA
(D-F). A, D,
Dark-field micrographs of coronal sections of the cingulate cortex
reveal the mRNA expression of SDF-1 isoforms by neuronal cells in
laminas V and VI (A, arrows) and the
absence of SDF-1 mRNA from neurons in the cingulate cortex
(D). The corpus callosum (cc) and
the meningeal surface (dotted line) are indicated for
orientation. SDF-1 and SDF-1 mRNAs are expressed in vascular
structures (A, D,
arrowheads) running perpendicularly to the meningeal
surface. A, Inset, High-power
bright-field magnifications showing SDF-1 mRNA expression by neuronal
cells with large round nuclei (arrows) as well as
endothelial cells with small round and small oval nuclei
(arrowheads). D, Inset,
SDF-1 mRNA is present in endothelial cells with small round and
small oval nuclei (arrowheads) but not in a neuron with
large round nucleus (arrow). E,
Nonradioactive in situ hybridization showing the
rod-shaped morphology of SDF-1-expressing endothelial cells of a
microvessel. F, Combination of immunohistochemistry for
vWF (detected as black reaction product) and in
situ hybridization for SDF-1 mRNA (detected as
grains) identifies specific SDF-1 expression by
vWF-ir endothelial cells (arrowheads). B,
C, Black reaction products, microglial
cells (B), and astrocytes
(C) are identified by nonradioactive in
situ hybridization for C1q and GFAP, respectively.
Co-hybridized 35S-labeled riboprobes to SDF-1 (detected as
grains) reveal SDF-1 expression by endothelial cells
(arrowheads in B, C) and the absence of
SDF-1 expression from microglial cells (arrows in
B) and astrocytes (arrows in
C). B, Double arrow, Note
the juxtavascular localization of the SDF-1 mRNA-negative microglial
cell. Exposure times: A, 42 d; D,
21 d; B, C, F,
28 d. Scale bars: A, D, 400 µm;
insets, 25 µm; B, C,
F, 30 µm; E, 100 µm.
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In a brain-mapping study using the probe detecting all SDF-1 isoforms,
we clearly identified neurons expressing SDF-1 mRNA in the cerebral
cortex (Fig. 3B-D), claustrum
(Fig. 3B), granular layer of the dentate gyrus (Fig.
3C,D), basolateral nucleus of the amygdala (Fig.
3C,D), and the amygdalohippocampal area (Fig. 3D). In the cortical areas, high neuronal SDF-1 mRNA levels
were localized to the deep laminae of the prelimbic and infralimbic cortex and areas 1 and 2 of the cingulate cortex (laminas V and VI), as
well as in the insular and entorhinal cortex. In the secondary motor,
secondary somatosensory, and secondary visual as well as the ventral
part of the auditory cortex, SDF-1 mRNA-expressing neurons were seen in
the deepest layer.
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Figure 3.
Cerebral expression patterns of CXCR4 mRNA and
neuronal and non-neuronal SDF-1 isoforms. Dark-field micrographs of
coronal sections hybridized with riboprobes detecting all SDF-1
transcripts (A-F) or CXCR4
(G-L). A-F, In addition to
moderate SDF-1 mRNA levels in endothelial cells throughout the brain,
high SDF-1 mRNA levels are present in neuronal structures of the
forebrain (B-D). G, In the
olfactory bulb, CXCR4 mRNA expression is abundant in the glomerular
layer (Gl). B-D,
H-J, SDF-1 and CXCR4 mRNA-expressing neurons are
located in areas 1 and 2 (Cg1, Cg2) of
the cingulate cortex (B, H), in
the insular cortex (In) (B,
H), in the lateral entorhinal and ectorhinal
cortex (LEnt, Ect) (C,
D, I, J), and in the
claustrum (Cl) (B,
H). B-D, In the secondary motor
cortex (M2) (B, C) and
secondary somatosensory cortex (S2) (B,
C), the medial aspect of the secondary visual cortex
(V2) (D) and the ventral auditory
cortex (AuV) (D), cortex
neurons expressing SDF-1 mRNA are present, whereas CXCR4
mRNA-expressing neurons are mostly absent (H-J).
Lower neuronal SDF-1 mRNA levels and fewer CXCR4 mRNA-expressing
neurons are present in the retrosplenial (RS)
(C, I) than in the cingulate
cortex (B, H). C,
D, Cerebrocortical neurons expressing SDF-1 mRNA are
frequently located in the deep cortical layers. B,
H, Neurons expressing CXCR4 mRNA but none expressing SDF-1 mRNAs are present in the dorsal endopiriform nucleus
(DEn) (H). Note SDF-1 mRNA
expression in the choroid plexus (CP) of the lateral
ventricle (B, C) and CXCR4 mRNA
expression in the subfornical organ (SFO)
(H) as well as the ependymal layers of the
lateral ventricle (LV)
(H-J), the dorsal and ventral parts of the third
ventricle (D3V, 3V)
(H-J), and the fourth ventricle
(4V) (K). In the amygdala,
neuronal SDF-1 mRNA is selectively located in the anterior
(BLA) (C) and posterior
(BLP) (D) parts of the basolateral
nucleus and in the amygdalohippocampal area (AHi)
(D), whereas only few CXCR4 mRNA-expressing
neurons without any clear assignment to subnuclei are detectable
(I, J). In the hippocampal
formation, numerous CXCR4 mRNA-expressing neurons are present in the
moleculare lacunosum and molecular layers (Mol)
(I, J) as well as the polymorphic
layer (PoDG) (I,
J), whereas SDF-1 mRNA is expressed only
in the granular layer (GrDG) (C,
D). K, L, In the
cerebellum (CBL), CXCR4 mRNA is expressed in the
Purkinje cell layer. K, L, In the
brainstem, neuronal CXCR4 mRNA expression cannot be identified.
Exposure time, 42 d. 7n, Facial nerve. Scale bar
(shown in A for A-L): 2 mm.
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Expression of the CXCR4 receptor in mouse brain
To examine the distribution of CXCR4-expressing cells in the
brain, we first performed in situ hybridization (Fig.
3G-L). In contrast to the pancerebral endothelial SDF-1
mRNA expression, the expression of CXCR4 mRNA was restricted to
distinct neuronal cell groups, the ependymal layer of the four
ventricles, and cells in the Purkinje cell layer of the cerebellum
(Fig. 3G-L). Neuronal populations expressing CXCR4 mRNA
were localized to the cerebral cortex, hippocampal formation, and
amygdala. In the cerebral cortex, scattered CXCR4 mRNA-expressing
neurons were found in the prelimbic and infralimbic areas as well as
the cingulate, insular, and entorhinal areas (Fig.
3H-J). Thus, neuronal CXCR4 and neuronal SDF-1
revealed a closely overlapping distribution in these regions. The
highest levels of CXCR4 mRNA were detected in the glomerular layer of the olfactory bulb and in the hippocampal formation (Fig.
3G,I,J).
To further characterize the subcellular distribution of the CXCR4
receptor protein, we generated an anti-peptide antibody directed
against the cytoplasmic tail of the CXCR4 receptor. Antibodies were
affinity purified and further characterized using immunofluorescent staining of stably transfected HEK 293 cells. When wild-type HEK 293 cells or T7-tagged CXCR4-transfected cells were stained, the anti-CXCR4
antibodies yielded prominent immunofluorescence at the plasma membrane
only in HEK 293 cells bearing the CXCR4 receptor but not in wild-type
cells (Fig.
4A,B).
This staining was virtually identical to that seen with anti-T7 tag
antibodies and completely blocked by preincubation of the antiserum
with homologous peptide (Fig. 4C,D). After
exposure of CXCR4-expressing cells with SDF-1, a proportion of
immunoreactive CXCR4 receptors underwent a dose-dependent redistribution from the plasma membrane into vesicle-like structures within the cytoplasm (Fig.
4E,F). In Western blot
assays, the anti-CXCR4 antibody detected a broad band migrating at 45 kDa only in membrane extracts from CXCR4-expressing cells but not in
wild-type cells (Fig. 4G). Essentially identical results
were obtained when CXCR4 receptors were first immunoprecipitated with our C-terminal rabbit anti-CXCR4 antibody (2144) and then immunoblotted with N-terminal rat monoclonal anti-CXCR4 antibody (2B11) (Fig. 4H). The anti-CXCR4 antibody (2144) also detected a
single 45 kDa band in membrane extracts from mouse brain (Fig.
4I). The CXCR4-ir bands were no longer detected when
the antiserum was preincubated with its immunizing peptide (Fig.
4G,I).
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Figure 4.
Characterization of the anti-CXCR4 antiserum using
stably transfected HEK 293 cells. Immunofluorescent labeling and
confocal imaging of wild-type HEK 293 cells (B)
and HEK 293 cells transfected with a construct coding for a T7-tagged
CXCR4 receptor (A, C-F) are
shown. Cells were either not treated (A-D) or
exposed to 10 or 100 ng/ml SDF-1 for 30 min (E,
F), fixed, and stained with the anti-CXCR4
antiserum (2144) (A-C, E,
F) or anti-T7 antibody (D).
G-I, Western blot analysis of CXCR4-LIR in transfected
HEK 293 cells and mouse brain. G, I,
Membrane preparations from wild-type HEK 293 cells, T7CXCR4-expressing
HEK 293 cells, or mouse brain were immunoblotted (IB)
with the anti-CXCR4 antibody (2144).
H, CXCR4 receptors were immunoprecipitated
(IP) using the C-terminal rabbit anti-CXCR4 antibody
(2144) and immunoblotted with the N-terminal rat
anti-CXCR4 antibody (2B11) as described in Materials and
Methods. For absorption controls, the anti-CXCR4 antiserum was
preincubated with 10 µg/ml of its immunizing peptide
(C, G, I). Note
that anti-CXCR4 antiserum yielded prominent immunofluorescence
localized at the level of the plasma membrane only in CXCR4
T7tag-expressing HEK 293 cells but not in wild-type cells. This
staining was essentially identical to that seen with the anti-T7
antibody and completely abolished by preincubation with homologous
peptide. SDF-1 induced a dose-dependent internalization of the CXCR4
receptor. Note that anti-CXCR4 antiserum detected a single 45 kDa band
in both transfected cells and mouse brain. WT,
Wild-type. Ordinate represents migration of protein
molecular weight markers (Mr × 10 3). Scale bar
(A-F): 15 µm.
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In brain sections of adult mice, high levels of
CXCR4-like-immunoreactivity (LIR) were detected in brain regions
expressing high levels of CXCR4 mRNA, including the olfactory bulb, the
hippocampal formation, and the ependymal layer of the four ventricles
(Fig. 5). CXCR4-ir axon terminals were
occasionally detected in regions where CXCR4 mRNA was not
detectable, e.g., in the accumbens shell (Fig. 5B). In
contrast to in situ hybridization, immunocytochemistry did
not label neurons in the claustrum and dorsal endopiriform nucleus. All
immunostainings were completely abolished by preabsorption of the CXCR4
antibody with homologous but not with heterologous peptides (Fig.
5C).
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Figure 5.
Distribution of CXCR4-LIR in the forebrain.
Low-power confocal images of coronal brain sections stained by
fluorescent immunohistochemistry. Strong CXCR4-LIR is present in the
glomerular layer of the olfactory bulb (Gl)
(A) and the ependymal layer of the lateral
ventricle (LV) (B,
D) as well as the ventral and dorsal parts of the third
ventricle (3V, D3V)
(D). The molecular layer of the dentate gyrus and
the lacunosum moleculare layer of the hippocampus
(Mol) (D) exhibit strong
CXCR4-LIR. Note the presence of CXCR4-ir fibers in the shell region of
the accumbens nucleus (ACbSh) (B) and the virtual
absence of CXCR4-ir perikarya from the claustrum and the dorsal
endopiriform nucleus (Cl, DEn)
(B). C, CXCR4-LIR is completely
neutralized by preabsorption with the peptide used for immunization.
Scale bars: A, C, 700 µm;
B, D, 1.2 mm.
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The potential hippocampal targets of the SDF-1-producing dentate gyrus
granule cells and entorhinal neurons were then characterized in detail.
In the hippocampus, CXCR4 mRNA-expressing neurons were detected most
frequently in the lacunosum moleculare layer, sparsely in the oriens
layer, and very rarely in the pyramidal cell layer and the stratum
radiatum (Fig. 6A). In
the dentate gyrus, numerous CXCR4 mRNA-expressing neurons were
localized in the molecular layer (Fig. 6A). In
addition, numerous CXCR4-expressing neurons, some of which coexpressed
glutamic acid decarboxylase (data not shown), were detected in the
polymorphic layer as well as at the border of the granular and
polymorphic layers (Fig. 6A). The distribution patterns of CXCR4-ir neuronal perikarya and CXCR4 mRNA-expressing neurons in the hippocampal formation were virtually identical (Fig.
6A,B). The only exception were
several neurons in the polymorphic layer (hilar neurons) that exhibited
high CXCR4 mRNA levels, coexpressed the mRNA of pre-prosomatostatin
(data not shown), but exhibited no detectable CXCR4-LIR at their
somatodendritic domain (Fig. 6A,B).
Because hilar somatostatinergic neurons are known to project to the
outer molecular layer, this discrepancy could be explained by the
selective targeting of the CXCR4 receptor to their axonal compartment
in the molecular layer. In fact, CXCR4-ir axons running perpendicularly
to the granular layer were observed in the granular layer and in the
inner molecular layer (Fig. 6C, arrowheads). These axons may contribute to the dense network of CXCR4-ir fibers in
the outer molecular layer (Fig. 6B). In the CXCR4-ir
neurons at the border of the granular and polymorphic layers, CXCR4
receptors were restricted to the somata and proximal dendrites (Fig.
6C, asterisk). In neurons of the lacunosum
moleculare layer and the molecular layer, CXCR4-LIR was present at the
plasma membrane of somata and dendrites (Fig.
6C,D, arrows) as well as axons (Fig. 6C,D,G,
arrowheads). In the outer molecular layer, CXCR4-ir neurons were frequently orientated parallel to the hippocampal fissure. In both
the molecular and the lacunosum moleculare layers, immunoreactive CXCR4
receptors were found in spine-free neurons (Fig. 6F)
and neurons with spine-like protrusions at their cell bodies and
dendrites (Fig. 6E,G,
arrows).
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Figure 6.
CXCR4 mRNA expression and cellular
localization of CXCR4-LIR in the hippocampal formation. Coronal
sections are shown in a dark-field micrograph after in
situ hybridization (A) and in confocal
images after fluorescent immunohistochemistry
(B-G). A, B,
Low-power micrographs comparing the distribution of CXCR4 mRNA
(A) and CXCR4-LIR (B); note
corpus callosum (cc) for orientation. In the oriens layer
(Or), few CXCR4 mRNA-expressing and CXCR4-LIR neurons
are detectable (shown at higher magnification in B), but
none are detectable in the pyramidal cell layer (Py),
stratum radiatum (Rad), or granular layer
(GrDG). In the lacunosum moleculare layer
(LMol), in the molecular layer
(Mol), and at the border of the granular and
polymorphic layers (PoDG), numerous CXCR4
mRNA-expressing and CXCR4-ir neurons are detected. Within the
polymorphic layer, neurons express high CXCR4 mRNA levels but exhibit
no detectable somatodendritic CXCR4-LIR.
C-G, High-power confocal images demonstrating the
subcellular targeting of CXCR4-LIR. C, In neurons of the
molecular layer, CXCR4-LIR is somatodendritic (C,
arrows) and axonal (C,
arrowheads). Note the perpendicular orientation of
several CXCR4-ir axons to the granular layer (C,
arrowheads in GrDG and
Mol). At the border of the granular and
polymorphic layers, CXCR4-LIR is restricted to neuronal perikarya and
their proximal dendrites (C, asterisk).
D, In the lacunosum moleculare layer, CXCR4-LIR
typically exhibits somatodendritic (D,
arrows) and axonal targeting (D,
arrowhead) in parallel orientation to the hippocampal
fissure. E-G, CXCR4-ir neurons in the molecular and
lacunosum moleculare layers either exhibit CXCR4-ir spine-like
protrusions at their cell bodies and dendrites (E,
G, arrows) or exhibit a spine-free
morphology (F). G, Note the
CXCR4-ir axon (arrowhead). Scale bars: A,
400 µm; B, 300 µm; inset in
B, 30 µm; C, 40 µm; D,
20 µm; E, F, 10 µm; G,
15 µm.
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Cell-specific regulation of SDF-1 expression after systemic
LPS administration
To test whether endothelial and neuronal SDF-1 isoforms may be
differently regulated in the brain, we applied LPS systemically at a
dose known to activate the cerebral endothelium and analyzed expression
levels of endothelial SDF-1 and neuronal SDF-1 transcripts. Quantitative analysis of x-ray autoradiograms of hybridized brain sections (Fig.
7E,F)
revealed that the expression levels of SDF-1 mRNA at 3, 6, and 24 hr
after LPS administration were decreased to 34, 36, and 53%
(p < 0.05) of the levels seen in
saline-injected animals, respectively (Table
1). The downregulation was of similar intensity in cortex, caudate putamen, basal forebrain, and choroid plexus. Qualitative analysis at high power confirmed that at 3 hr after
LPS stimulation the endothelial SDF-1 mRNA expression was decreased
to levels near the detection limit (Fig.
7G,H). Hybridization with the probe
detecting all SDF-1 isoforms showed a LPS-induced decrease in
endothelial SDF-1 expression similar to the SDF-1-selective probe
(Fig. 7A-D). Hybridization with the probe to SDF-1 or
the probe to all SDF-1 isoforms revealed that LPS application did not
affect SDF-1 mRNA expression in neurons of cortex or claustrum (Fig.
7A-D). Thus, systemic LPS stimulation selectively affects
endothelial SDF-1 expression but does not alter neuronal SDF-1
levels.
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Figure 7.
LPS-induced decrease in endothelial but not
neuronal SDF-1 mRNA expression revealed by in situ
hybridization with a riboprobe detecting all SDF-1 isoforms
(A-D) and a riboprobe specific for SDF-1 mRNA
(E-H). Coronal brain sections 3 hr after LPS
(LPS) (B, D,
F, H) or saline application
(Sal) (A, C,
E, G) are shown as x-ray autoradiograms
of the whole brain (A, B,
E, F) and as dark-field micrographs of
the cingulate cortex (C, D,
G, H). A,
B, After LPS application, SDF-1 mRNA expression is
dramatically reduced as compared with saline-injected animals. Note
that the LPS-induced decrease in SDF-1 mRNA levels is most obvious
where neuronally expressed SDF-1 isoforms are absent, e.g., in the
caudate putamen (CPu) and choroid plexus
(CP). SDF-1 expression in neuronal structures
(A, B, arrows) of the
cingulate and secondary somatosensory cortex (Cg, S2) as
well as the claustrum (Cl) is unchanged after LPS
stimulation as compared with the saline-injected control.
C, D, SDF-1 expression in the cingulate
cortex is decreased by LPS in endothelial cells
(arrowheads) but not neurons (arrows).
E, F, Throughout the brain, SDF-1 mRNA
levels are strongly reduced after LPS stimulation as compared with the
saline-injected control. Note the pronounced downregulation of SDF-1
in endothelial cells after LPS injection
(H) as compared with the control
(G, arrowheads). Exposure times:
A, B, 3 d; E,
F, 2 d; C, D,
G, H, 21 d. Scale bars:
A, B, E, F,
3 mm; C, D, G,
H, 550 µm.
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CXCR4 mRNA levels analyzed in the ependymal layer of the lateral
ventricles and neurons in the cingulate cortex were not changed after
LPS stimulation as compared with the saline-injected group. There was
no evidence for an infiltration of the brain by CXCR4 mRNA-expressing
leukocytes in any of the investigated regions (data not shown).
Adaptive plasticity of the SDF-1/CXCR4 receptor system after
focal ischemia
We then investigated adaptive changes of
endothelial and neuronal SDF-1 mRNA expression in relation to the
presence of CXCR4 mRNA-expressing infiltrates and regulation of
neuronal CXCR4 mRNA levels after focal cerebral ischemia. A comparative
analysis of the primary infarcted area, the area at the border of the
infarcted and nonlesioned tissues (border zone), and the ipsilateral
area without detectable neurodegeneration (ipsilateral nonlesioned area) as well as the contralateral hemisphere was performed (Fig. 8G). For simplicity during
quantitative image analysis, the border zone and the primary infarct
were treated as one region.
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Figure 8.
Patterns of middle cerebral artery occlusion
(MCAO)-induced changes in SDF-1
(A-C) and CXCR4 (D-F)
mRNA expression. A-F, Low-power micrographs of x-ray
autoradiograms of hybridized coronal sections from naive mice
(CTRL) (A, D) and mice
1 d after sham operation (B, E) or
MCAO (C, F). The lateral borders
of the infarct are indicated by arrowheads
(C, F). C, After
MCAO, SDF-1 mRNA levels are decreased throughout the brain as
compared with the sham operation (B) or the naive
control (A). The decrease in SDF-1 mRNA
expression is most pronounced within the infarct. C, In
the nonlesioned brain areas, SDF-1 expression is decreased more
strongly in the ipsilateral than in the contralateral hemisphere.
B, Note that sham operation induced a decrease in
SDF-1 mRNA expression throughout the brain, which increases with
proximity to the trepanation site (B,
arrow). F, In the hemisphere ipsilateral
to MCAO, there is a strong increase in CXCR4 mRNA expression in lamina
VI of the cingulate cortex and the dorsal endopiriform nucleus
(F, arrows) as compared with naive
animals or sham operations (D, E). A
similar but less pronounced increase is observed in cingulate cortex
and dorsal endopiriform nucleus of the contralateral hemisphere.
G, Low-power micrograph of a cresyl violet
(nissl)-stained coronal section through a mouse
forebrain 2 d after MCAO at bregma +0.14 mm. Delineated by
dotted lines are the infarcted area, which includes the
primary (S1) and secondary (S2)
somatosensory cortex, the insular cortex
(I), and the dorsal parts of the piriform
cortex (Pir). The cingulate (Cg) and
secondary motor (M2) cortices are nonlesioned
cerebrocortical areas. The primary motor cortex (M1),
the lateral aspect of the caudate putamen (CPu), and
parts of the piriform cortex contribute to an area at the border of
nonlesioned and infarcted tissue. The largest part of the caudate
putamen (medial aspect) is not infarcted. Exposure times:
A-C, 2 d; D-F, 3 d. Scale bar
(shown in F for A-F): 3 mm.
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Changes in endothelial SDF-1 mRNA expression
As shown for 1 d after MCAO (Fig.
8A,C), there was a downregulation
of SDF-1 mRNA expression to different extents in the four regions as
compared with controls. The decrease in SDF-1 mRNA levels was most
pronounced in the primary infarct, most likely due to degeneration of
cells. However, SDF-1 expression was also decreased in the
nonlesioned areas. This downregulation was more pronounced in the
ipsilateral than in the contralateral hemisphere (Fig. 8C).
Quantitative analysis of SDF-1 mRNA expression revealed that MCAO
induced a decrease from 6 hr to 4 d in all regions as compared
with control animals. In addition, the quantitative analysis revealed
an inverse relationship of lesion-distance and extent of SDF-1
downregulation (Table 2). All changes in
endothelial SDF-1 mRNA expression were similar, when examined with the
SDF-1selective probe or the probe detecting all SDF-1 isoforms
(Figs. 8, 9). Notably, also the sham
operation induced a decrease in SDF-1 mRNA expression 1 d and
2 d after treatment as compared with control animals (Table
3, Fig. 8A,B).
Comparison of ischemic and sham operated animals at 1 d and 2 d revealed significantly lower SDF-1 mRNA levels after MCAO than
after sham operation (Table 3). These findings strongly suggest, that
skull trepanation decreases SDF-1 expression in the brain and that
focal ischemia induces a reduction in endothelial SDF-1 mRNA
expression independently from trepanation.
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Figure 9.
Dark-field micrographs of the brain
hemisphere ipsilateral to middle cerebral artery occlusion at various
time points after surgery (B, D,
E-H) and of the corresponding brain hemisphere
of control mice (CRTL) (A,
C). In situ hybridization was performed
with a riboprobe detecting all SDF-1 isoforms (A,
B, E, F) and a
riboprobe for CXCR4 mRNA (C, D,
G, H). A,
B, E, F, SDF-1 mRNA
expression by neurons in the cingulate cortex is strongly decreased at
6 hr (B) and unchanged at 2 and 4 d after
MCAO (E, F) as compared with the
control (A). After 6 hr and 2 d, endothelial
SDF-1 mRNA expression is at the detection limit within the infarct and
strongly decreased in the nonlesioned area (B,
E) as compared with the control
(A). Note the very strong induction of SDF-1 mRNA
expression in the border zone of the infarct after 2 d
(E, arrowhead). F, After
4 d a moderate to strong SDF-1 mRNA expression is detectable
within the infarct and its border zone (F,
arrowhead), whereas non-neuronal SDF-1 mRNA expression
in the nonlesioned area is moderately decreased as compared with the
control (A). C, D,
G, H, After 6 hr and 2 d but not
after 4 d, neuronal CXCR4 mRNA expression is strongly increased in
the dorsal endopiriform nucleus (D, G,
asterisks) and lamina VI of the cingulate cortex
(D, G, arrows) as compared
with the control (C). G, Note the
induction of CXCR4 mRNA expression in the superficial layers of the
cingulate cortex after 2 d. G, H, In
the border zone of the infarct (G,
arrowhead), a massive accumulation of CXCR4
mRNA-expressing cells is detectable after 2 and 4 d.
H, After 4 d, numerous CXCR4 mRNA-expressing cells
are also present within the infarcted core. Exposure times:
A, B, E, F,
21 d; C, D, G,
H, 42 d. Scale bar (shown in H for
A-H): 3 mm.
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Recruitment of CXCR4-expressing cells to the lesioned brain
The spatial relationship of SDF-1- and CXCR4-expressing cells in
the ischemic brain was analyzed at low-power under dark-field illumination (Fig. 9). There were virtually no peripheral
CXCR4-expressing cells infiltrating the ischemic brain after 6 hr or
1 d (Fig. 9D, Fig. 8F), when
endothelial SDF-1 expression was strongly decreased in all analyzed
regions (Figs. 9A,B, 8A,C). Surprisingly,
after 2 d numerous CXCR4 mRNA-expressing cells were detected in
the border-zone of the infarct (Fig. 9G,
arrow-head). In close spatial and temporal overlap with the
accumulation of CXCR4-expressing cells a strong focal induction of
SDF-1 mRNA expression was observed in single vascular structures (Fig.
9E,G, arrow-heads). In
contrast, in the infarcted core and the nonlesioned areas SDF-1
expression was still strongly decreased at 2 d after MCAO as
compared with the control (Fig. 9A,E). After 4 d
numerous CXCR4-expressing cells were present in both the primary
infarct and the border-zone, which was in correspondence with a
moderate SDF-1 mRNA expression in both regions (Fig.
9F,H). It should be noted, that there was no evidence
for peripheral CXCR4-expressing cells infiltrating the nonlesioned
areas at all stages after cerebral ischemia.
Transient changes of neuronal SDF-1 and neuronal CXCR4
mRNA expression
In addition to the changes of non-neuronal SDF-1 and CXCR4
expression, there were pronounced changes of neuronal SDF-1 and CXCR4
expression in nonlesioned brain regions after focal ischemia (Figs.
8,9). Sham operation had no effect on neuronal SDF-1 (not shown) or
CXCR4 expression (Fig. 8D,E). An
ischemia-induced upregulation of neuronal CXCR4 mRNA expression was
seen in the dorsal endopiriform nucleus (Fig. 9D,G,
asterisks) and the cingulate cortex (Fig. 9D,G, arrows)
from 6 hr to 2 d after MCAO. Neuronal CXCR4 levels in both
structures were similar to controls after 4 d (Fig.
9C,H). At 6 hr and 1 d the increased CXCR4
expression in the cingulate cortex was restricted to lamina VI (Figs.
9D, 8F). After 2 d CXCR4 mRNA
expression was induced in laminae II/III and VI (Fig. 9G). Neuronal SDF-1 mRNA expression in the cingulate cortex was decreased in
lamina V and VI 6 hr and 1 d after MCAO (Fig. 9A,B) but
virtually unchanged at later time-points (Fig.
9A,E,F). All described changes of neuronal SDF-1 and
CXCR4 mRNA expression were also seen in the contralateral hemisphere
but were less pronounced than in the ipsilateral hemisphere (Fig.
8F).
Characterization of SDF-1- and CXCR4-expressing cells in the
border zone
Combinations of radioactive in situ hybridization and
immunocytochemistry or nonisotopic in situ hybridization
were applied to further characterize SDF-1- and CXCR4-expressing cells
in the border zone after 2 d. The vascular SDF-1 upregulation was
detected in vWF-ir endothelial cells using the probe to all SDF-1
isoforms (data not shown) as well as the SDF-1selective probe
(Fig. 10A, arrowheads). SDF-1 isoforms were not detected by
either probe in reactive astrocytes (data not shown) and large
reactive microglial cells (Fig. 10B,
asterisks), which were identified by their strong GFAP and
C1q mRNA expression, respectively. CXCR4 mRNA was detectable in a small
subset of C1q-positive cells (~30 cells per section). These cells
were small, without processes, and restricted to the border zone (Fig.
10C, arrow). Most of the C1q-positive cells that were devoid of any CXCR4 labeling were larger, exhibited swollen processes, and were frequent in the entire ipsilateral hemisphere (Fig.
10C, asterisk). Because C1q detects both
macrophages and microglial cells in the brain, the smaller C1q-positive
cells expressing CXCR4 most likely represent infiltrating macrophages, whereas the larger CXCR4-negative cells are resident activated microglial cells. Notably, the cells coexpressing CXCR4 and C1q mRNAs
were only a subset (~10%) of CXCR4 mRNA-expressing cells in the
border zone. Double in situ hybridization and double
immunohistochemistry revealed that most of the CXCR4 mRNA-expressing
cells were negative for GFAP (Fig.
10D,E,E'). A minor
population (< 1%) of reactive astrocytes in the border zone expressed
CXCR4 mRNA (data not shown). These findings strongly suggest that
resident astrocytes and microglial cells do not upregulate CXCR4
expression during activation after cerebral ischemia. CXCR4-expressing
cells in the border zone 2 d after MCAO most likely represent
infiltrating peripheral blood cells, including macrophages.
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Figure 10.
High-resolution analysis of the cellular
expression of SDF-1 isoforms and CXCR4 mRNA in the border zone 2 d
after MCAO. Hybridized 35S-labeled riboprobes
(35S) to SDF-1 (A), SDF-1
(B), and CXCR4 (C,
D) mRNAs are detected as grains;
immunocytochemistry for vWF (A) and co-hybridized
DIG-labeled riboprobes (DIG) to C1q (B,
C) and GFAP (D) mRNAs are detected
as black reaction products. A,
Arrowheads, Strong SDF-1 mRNA expression is detected
specifically in vWF-ir endothelial cells. B, SDF-1 mRNA
is absent from activated C1q mRNA-expressing microglial
cells/macrophages (B, asterisks) but
expressed by endothelial cells (B,
arrowheads). Note the absence of SDF-1 mRNA expression
from a juxtavascular C1q mRNA-expressing cell (B,
arrow). C, CXCR4 mRNA is absent from a large C1q
mRNA-expressing activated microglial cell (C,
asterisk) but present in a small presumed C1q
mRNA-expressing macrophage (C, arrow). Note C1q
mRNA-negative CXCR4 mRNA-expressing cells (C,
arrowheads). D, Detection of numerous CXCR4
mRNA-expressing cells (D, arrows) in the
presence of CXCR4 mRNA-negative GFAP-positive activated astrocytes
(D, asterisks). E,
E', Double immunohistochemistry for CXCR4
(E) and GFAP (E') demonstrating
the absence of CXCR4-LIR (arrows) on activated
astrocytes (asterisks). Scale bar:
A-D, 40 µm; E,
E', 20 µm.
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DISCUSSION |
In the present study, we provide the first in
situ hybridization evidence for cell- and tissue-specific
expression of SDF-1 splice variants in the mouse brain using a
pan-SDF-1 probe, a SDF-1-selective probe, and a SDF-1selective
probe, which does not detect SDF-1 but may hybridize to a putative
mouse SDF-1 isoform as well. We show that in the adult mouse brain
SDF-1 is selectively generated in the endothelium of cerebral
microvessels but not in neurons, whereas SDF-1 is generated
predominantly in distinct neuronal populations and meningeal cells.
After systemic LPS administration, cerebro-endothelial SDF-1 was
selectively downregulated, whereas neuronal SDF-1 remained
unchanged. After focal ischemia, endothelial SDF-1 showed a
persistent downregulation in the nonlesioned brain, and neuronal
SDF-1 showed a transient downregulation in the nonlesioned brain. In
the penumbra, SDF-1 was selectively upregulated in the endothelium
of single blood vessels. Thus, alternative splicing may represent the
primary mechanism by which the expression of SDF-1 in the CNS is
regulated in a tissue- and stimulus-specific ma |
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ABSTRACT
INTRODUCTION
