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The Journal of Neuroscience, May 15, 1998, 18(10):3699-3707
Upregulation of Pleiotrophin Gene Expression in Developing
Microvasculature, Macrophages, and Astrocytes after Acute Ischemic
Brain Injury
Hsiu-Jeng
Yeh1,
Yong Y.
He2,
Jan
Xu2,
Chung Y.
Hsu2, and
Thomas
F.
Deuel1
1 Department of Medicine, Division of Growth
Regulation, Beth Israel Deaconess Medical Center, Boston, Massachusetts
02215, and 2 Department of Neurology, Washington University
School of Medicine, St. Louis, Missouri 63310
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ABSTRACT |
Pleiotrophin (PTN) is a heparin-binding, 18 kDa secretory protein
that functions to induce mitogenesis, angiogenesis, differentiation, and transformation in vitro. PTN gene
(Ptn) expression is highly regulated during development
and is highest at sites in which mitogenesis, angiogenesis, and
differentiation are active. In striking contrast, with the exception of
the neuron, the Ptn gene is only minimally expressed in
adults. We now demonstrate that Ptn gene expression is
strikingly upregulated within 3 d in OX42-positive macrophages, astrocytes, and endothelial cells in areas of developing neovasculature after focal cerebral ischemia in adult rat.
Ptn gene expression remains upregulated in these same
cells and sites 7 and 14 d after ischemic injury. However,
expression of the Ptn gene is significantly decreased in
cortical neurons 6 and 24 hr after injury and is undetectable in
degenerating neurons at day 3. Neurons in contralateral cortex continue
to express Ptn in levels equal to control, uninjured
brain. It is suggested that PTN may have a vital role in neovascular
formation in postischemic brain and that postischemic brain is an
important model in which to analyze sequential gene expression in
developing neovasculature. In contrast, Ptn gene
expression in injured neurons destined not to recover is strikingly
reduced, and potentially its absence may contribute to the failure of
the neuron to survive.
Key words:
pleiotrophin gene expression; ischemia; neovasculature; macrophage; astrocytes
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INTRODUCTION |
Trophic factors are required for
growth, differentiation, and maintenance of viability during
development and after injury. Pleiotrophin (PTN) is a member of a newly
identified family of developmentally regulated, secreted
heparin-binding proteins (Milner et. al., 1989 ; Rauvala, 1989; Li et
al., 1990); it is an 18 kDa protein that stimulates mitogenesis,
angiogenesis, and neurite and glial process outgrowth guidance
activities in vitro. In vivo, Ptn gene
expression peaks during late embryogenesis and in perinatal growth.
Because these are times of active proliferation and differentiation in
both mesenchyme and the nervous system, it has been suggested that PTN
signals these functions during development as well (Li et al., 1990;
Raulo et al., 1992; Wanaka et al., 1993; Matsumoto et al., 1994;
Rauvala et al., 1994; Silos-Santiago et al., 1996). In contrast, with
the exception of subpopulations of neurons, levels of Ptn
gene expression are very much lower in adult animals (Li et al., 1990;
Garver et al., 1994; Kurtz et al., 1995; Nakagawara et al., 1995;
Silos-Santiago et al., 1996), suggesting that activation of the
Ptn gene may occur and activate PTN signaling in responsive cells important in new tissue formation during recovery from
injury.
Cerebral ischemia and infarction lead to death of both neurons and
glial elements. However, because recovery of brain function is
frequently noted in patients with stroke even in the absence of
neuronal regeneration, the postischemic expression of trophic factors
has been analyzed to identify which factors may be upregulated and to
correlate the expression of these factors with tissue recovery. Different neurotrophins (Lindvall et al., 1992; Hsu et al., 1993) and
basic fibroblast growth factor (Speliotes et al., 1996; Lin et al.,
1997) are expressed after global or focal cerebral ischemia. Previously, the expression pattern of the Ptn gene was
analyzed and found to be similar to a number of the neurotrophin genes (Li et al., 1990; Silos-Santiago et al., 1996). Remarkably, its expression is also significantly increased in neurons of the
hippocampus, piriform cortex, and parietal cortex after chemically
induced seizures (Wanaka et al., 1993), indicating its potential for
activation in cells of the CNS. However, the significance of its
increased expression levels in cortex after chemically induced seizures is unclear, and its expression after other forms of brain injury has
not been studied. Because PTN stimulates proliferation of endothelial
cells (Courty et al., 1991; Fang et al., 1992) and has been implicated
in tumor angiogenesis (Chauhan et al., 1993; Czubayko et al., 1996;
Choudhuri et al., 1997; Relf et al., 1997), the formation of new blood
vessels with the increased vascular density frequently noted after
cerebral ischemia (Lin et al., 1998) offered the potential to seek
contribution of PTN to recovery of brain function after injury through
its angiogenic properties. To seek evidence that PTN may contribute to
tissue recovery, we have now examined its expression pattern after
focal cerebral ischemia in rats.
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MATERIALS AND METHODS |
Focal ischemia model. Long-Evans male rats (body
weight, 300-350 gm) were used in this study. Housing and anesthesia
concurred with guidelines established by the Institutional Animal
Welfare Committee, in accordance with the Public Health Services
Guide for the Care and Use of Laboratory Animals, of the
United States Department of Agriculture regulations, and the Guidelines
of Panel on Euthanasia of the American Veterinary Association. Rats
were allowed free access to water and rat chow until surgery. Rats were
anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg,
i.m.). The left femoral artery was cannulated for monitoring arterial
blood pressure and heart rate and for arterial blood gas analysis. Mean
arterial pressure was maintained at >80, and blood gas was maintained
at pH 7.4 ± 0.1, PaO2 >80, and
PaCO2 37 ± 7. The rectal temperature was
monitored and maintained at 37.0 ± 0.5°C via an electronic
temperature controller linked to a heating lamp. The right middle
cerebral artery (MCA) was exposed as described previously using
microsurgical techniques (Liu et al., 1989; He et al., 1993). Briefly,
after a 2 cm vertical skin incision midway between the right eye and
ear and splitting of the temporalis muscle, a 2 mm burr hole was
drilled at the junction of the zygomatic arch and the squamous bone.
The right MCA was ligated with a 10-0 suture under an operating
microscope. Complete interruption of blood flow at the MCA occlusion
site was confirmed under the microscope. Both common carotid arteries
(CCAs) that had been isolated previously and freed of soft tissues and
nerves were then occluded using nontraumatic aneurysm clips. Temporary occlusion of the right MCA and both CCAs resulted in severe focal ischemia (reduction of blood flow by 88-92%) confined to the cerebral cortex in the right MCA territory. Ischemia was mild (reduction of flow
by <20%) in the right subcortical structures including the striatum
and in the left hemisphere, which showed no morphological evidence of
ischemic injury. The occlusion was removed at 90 min followed by
reperfusion and at 6 hr and 1, 3, 7, and 14 d after occlusion.
Ischemia for 90 min resulted in a persistent infarction in the right
MCA cortex that was readily identified between 6 hr and 7 d after
ischemia (Lin et al., 1993). At the end of reperfusion, rats were
killed after an overdose of sodium pentobarbital (150 mg/kg) followed
by intracardiac perfusion with 200 ml of 0.9% saline and 200 ml of 4%
paraformaldehyde. Fixed cerebrum was removed, and coronal sections were
prepared.
Tissue fixation, embedding, and sectioning. After perfusion
fixation, brains from each time point (three animals per group) and one
brain from a sham-operated animal were post-fixed of 15 times the
tissue volume of 10% neutral formalin, dehydrated through a series of
ethanol baths, and embedded in paraffin, and serial sections of 5 µm
thickness were mounted on slides. Adjacent slides were used for
comparison of in situ hybridization and
immunohistochemistry.
Additional brains were removed and fixed in 4% paraformaldehyde
overnight at 4°C and cryoprotected with 30% buffered sucrose. Another set of brains were prepared for frozen sections, which were
used also for immunostaining.
In situ hybridization. The mouse Ptn cDNA (c
clone; Li et al., 1990) was linearized and transcribed in
vitro with 35S-UTP (1250 Ci/mmol; DuPont NEN, Boston,
MA). Details of preparation of 35S-labeled Ptn
RNA probes and conditions of hybridization were as described previously
(Yeh et al., 1991). In control experiments, sections adjacent to those
hybridized with the 35S-labeled Ptn antisense
RNA probe were treated as follows: (1) hybridization with a
35S-labeled Ptn sense RNA probe, and (2)
digestion with RNase A (50 ng/ml) for 1 hr at 37°C before
hybridization with 35S-labeled Ptn antisense RNA
probe to confirm the specificity of the 35S-labeled
Ptn antisense RNA probe (data not shown).
Immunohistochemistry. Two types of anti-PTN antibody were
used for immunoperoxidase staining. A monoclonal anti-PTN antibody was
generated as described previously (Silos-Santiago et al., 1996). A
chicken polyclonal anti-human PTN antibody was commercially prepared
(Pocono Rabbit Farm and Laboratory, Canadensis, PA). Polyclonal
anti-GFAP antibodies (Sigma, St. Louis, MO) and monoclonal anti-OX42 antibodies (mouse anti-rat CD11b/c equivalent;
Harlan Sera Laboratories Ltd., Indianapolis, IN) were used for
identification of astrocytes and macrophages, respectively. Endogenous
peroxidase activity was blocked by treatment with 0.75%
H2O2 in PBS for 10 min, and sections were
incubated with 10% normal goat serum or 1% chicken serum from
nonimmunized animals (in PBS) for 10 min. The sections were
subsequently incubated overnight at 4°C with anti-PTN antibody (1:100
dilution in 1% BSA-PBS) or anti-GFAP antibody (1:50 dilution in 1%
BSA-PBS). The sections were then incubated with affinity-purified,
biotinylated anti-mouse IgG (1:100 dilution; Life Technologies,
Gaithersburg, MD), anti-chicken IgG (1:200 dilution; Sigma), or
anti-rabbit IgG (1:100 dilution; Life Technologies), respectively, for
45 min followed by a 30 min incubation with streptavidin-horseradish
peroxidase (1:100 dilution, Life Technologies). Finally, the sections
were developed in DAB solution (Sigma)
Frozen sections were used to stain the macrophage using the monoclonal
anti-OX42 antibody. The anti-OX42 antibody
recognizes a common epitope shared by CD11b/c specific for macrophage
(Robinson et al., 1986). Briefly, sections were pretreated with 1%
H2O2 in methanol for 30 min at  20°C to
block endogenous peroxidase, followed by 0.1% trypsin in 0.1%
CaCl2-PBS for 30 min at 37°C and blocking of nonspecific
binding sites with 10% goat serum and 0.4% Triton X-100 in PBS.
Sections were then incubated with anti-OX42 (1:100 dilution
in blocking solution) for 48 hr at 4°C, followed by the ABC procedure
as described for immunostaining of PTN. Controls were performed in the
adjacent sections to those reacted with antibodies by incubating
sections with normal goat serum, rabbit serum, nonimmune chicken serum
or 1% BSA-PBS in place of primary antibodies.
Histochemistry. The macrophage and microglial cells were
also identified using D-galactosyl-specific B4
isolectin (GSA-IB4) conjugated with horseradish
peroxidase followed by DAB in the presence of hydrogen peroxidase,
according to methods described by Streit (1990) and Menon and
Landerholm, (1994). The GSA-IB4 derived from
Griffonia simplicifolia seeds has been shown to stain selectively rat microglial cells in normal as well as pathologically altered brain (Streit and Kreutzberg, 1987). Sections adjacent to those
immunostained with anti-PTN antibodies were incubated overnight at
4°C or for 2 hr at room temperature with GSA-IB4 (10 µg/ml in HBSS with 0.1% Triton X-100), followed by development in
DAB, and counterstained with hematoxylin.
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RESULTS |
The pleiotrophin (Ptn) gene is predominantly expressed
in cortical neurons in adult rat and mouse brain (Li et al., 1990; Wanaka et al., 1993; Silos-Santiago et al., 1996). In control sections
from sham-operated brain and the left (contralateral to the ischemic
lesion) hemisphere, the pattern of PTN expression was similar to that
described previously (Wanaka et al., 1993; Silos-Santiago et al.,
1996). Ptn mRNA and immunoreactivity of the PTN protein are
readily seen in cortical neurons, but little or no expression of
Ptn was observed in glia or the endothelial cells of the
blood vessels in the normal brain or uninjured hemisphere (Fig.
1A-C). However,
remarkable changes were demonstrated in the intensity and the regional
distribution of Ptn gene expression in the right (injured)
hemisphere. At 6 hr and 1 and 7 d after reperfusion, expression of
the Ptn gene in cortical neurons in the ischemic brain was
drastically reduced. The decrease in Ptn gene expression was
more rapid and to a greater degree in the ischemic core than in the
peri-infarct region. Ptn mRNA signals were low in
degenerating neurons in the outer layers of the infarct but not
detectable in the ischemic core 6 hr after induction of the lesion
(Fig. 1D).
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Figure 1.
Top. Sections of a brain from a
sham-operated rat hybridized with 35S-Ptn
antisense cRNA probe (A, dark field; B,
bright field) or 35S-Ptn sense cRNA probe
(C, bright field). Ptn hybridization
signal was highly expressed in neurons of cortex (large
arrow). A little signal was found in glial cells (small
arrow) and microvascular endothelium (medium
arrow). D (bright field), Section hybridized
with 35S-PTN antisense cRNA shows that PTN mRNA decreases
to a greater degree in degenerating neurons in the ischemic core
(C) then in the periphery
(P) of the infarct compared with normal neurons
(N) 6 hr after reperfusions. Magnification:
A-C, 200×; D, 100×.
Figure 2.
Coronal sections of a brain 3 d after
ischemia hybridized with 35S-Ptn antisense
cRNA probe (dark field). A strong hybridization signal is shown at the
border of the infarct (arrows) and microvasculature in
the infarct (B, arrows). Magnification:
A, 10×; B, 40×.
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In contrast, transcripts of Ptn and PTN immunoreactivity
were readily detected in glial cells in ischemic cortex, especially in
the periphery of the infarct and the regions surrounding the injured
area when examined 1-3 d after induction of the ischemic lesion (Figs.
2,
3A-C). Using an adjacent
section immunostained with the anti-GFAP antibody, it was established
that expression of the Ptn gene was localized to GFAP(+)
astrocytes (Fig. 3D). The astrocytes were hypertrophic in
appearance and exhibited strong GFAP(+) staining in the cell bodies and
in the thick processes of these cells in the areas immediately adjacent
to the border of injury area (Fig. 3D). At 1 and 3 d
after ischemia, the astrocytes were increased in number in the white
matter adjacent to the infarct (data not shown). In contrast, the
contralateral side had little GFAP(+) staining in cortical astrocytes
(data not shown). The morphology and distribution of the
anti-PTN-immunostained cells bordering the injured area and
peri-infarct region corresponded to that of GFAP(+) astrocytes in the
same area, suggesting that the Ptn and GFAP(+)
gene-expressing cells in sites immediately surrounding the ischemic
lesion are astrocytes.
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Figure 3.
Sections of a brain 3 d after ischemia
hybridized with 35S-Ptn antisense cRNA
(A, B) and immunostaining with anti-PTN antibody
(C) or anti-GFAP antibody
(D). Ptn hybridization signal was
predominantly expressed in the neurons (large arrow) and
glial cells (small arrow) along the border of the
infarct and blood vessels (arrowhead) immediately
adjacent to the infarct (top right in A,
dark field, B, bright field). PTN immunoreactivity was
detected in glial cells (large arrow) at the border of
the infarct (C), which corresponded to the
hypertrophic anti-GFAP(+) astrocytes in the similar area on day 3 (D, large arrow); the small arrow denotes
neurons under degeneration. Magnification, 200×.
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At day 3, transcripts of the Ptn gene were not seen in the
area of the infarct. However, both transcripts and PTN protein were
expressed at striking levels in the microvasculature and macrophages
(Figs. 4, 5, 6B).
Virtually all of the cells that expressed detectable levels of
Ptn transcripts were immunoreactive with anti-PTN antibodies
as well. Of particular importance, a large number of macrophages were
found near the vessels (Fig. 5)
within the area of the infarct. These cells express high levels of
Ptn mRNA (Fig.
6A,C) and PTN
immunoreactivity (Figs. 5A,B, 6B) but did not
stain positively with anti-GFAP antibody (data not shown). The cells
were confirmed as macrophages, because they were recognized by the
anti-OX42 antibody (Fig. 5C) and histochemical staining with GSA-IB4 conjugated peroxidase (Figs.
5D, 6D, brown in
cytoplasm).
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Figure 4.
Sections of an ischemic brain on day 3 hybridized
with 35S-Ptn antisense cRNA (A,
C, dark field; B, D, bright field). PTN
transcripts were found in the endothelium of blood vessels
(large arrow) and glial cells (small
arrow). Endothelial sprouts were seen in A
and B (large arrow). Magnification,
200×.
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Figure 5.
Sections of an ischemic brain on day 3. A,
B, PTN immunoreactivity was found in endothelial cells
(small arrow) and macrophages (large
arrow) in the infarcted region after staining with anti-PTN
antibody. C, D, Macrophages surrounding blood vessel
(large arrow) were identified by immunostaining with
anti-OX42 antibody (C, arrow, frozen
section) and histochemistry staining with GSA-IB4
(D, arrow). Magnification: A, B, 400×;
C, D, 200×.
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Figure 6.
Sections of ischemic brain on days 3, 7, and 14. A, 35S-Ptn cRNA hybridization
signals were detected in macrophages (arrow) near the
infarcted area (top) on day 3 (bright field).
B, A number of macrophages with PTN immunoreactivity
(small arrow) with variable morphology in the infarcted
area on day 7 (large arrow denotes a blood vessel).
C, 35S-Ptn antisense cRNA
hybridization signals were detected in numerous macrophages
(arrow) surrounding residual necrotic tissue at the
infarcted region on day 14 (bright field). D, A number
of macrophages stained with GSA-IB4 with various
morphological features in the infarct on day 14. Magnification:
A, B, 400×; C, D, 100×.
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Seven days after ischemia, the injured area consisted of large numbers
of PTN-positive macrophages and hyperplastic blood vessels (Fig.
6B). The macrophages exhibited a wide range of
morphological appearances, with round, oval, triangular, or square
shapes and with the majority lacking identifiable processes. PTN
immunoreactivity was readily identified in the cytoplasm of these
macrophages and in the endothelial cells of the hyperplastic blood
vessels (Fig. 6B).
Fourteen days later, the infarct contained numerous macrophages with
various morphological features (Fig. 6D), and
residual necrotic tissue was usually surrounded by macrophages (Fig.
6C). GFAP(+) astrocytes were no longer seen. There was very
little PTN immunoreactivity in the microvascular endothelium or the
cells surrounding the vessels.
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DISCUSSION |
In this work, it is demonstrated that levels of the Ptn
gene are differentially expressed in different cell types in ischemic rat brain. A very striking increase in the levels of expression of the
Ptn gene was found in microglia and macrophages within areas
of the exuberant neovasculature that formed at the margins of the
infarct and in the endothelial cells of the newly formed vessels
themselves. As described previously, a remarkable angiogenic response
is seen after severe focal cerebral ischemia in this rat model (Lin et
al., 1998). In the present study, it was observed that both the
endothelial cells in neovasculature and the cells identified by the
different specific macrophage and microglial markers that associate
with the sites of angiogenesis exhibit intense Ptn mRNA
signals, initially at 3 d and continuously through day 14. Because
PTN is a potent angiogenic agent in vitro, and tumors that
derive from Ptn-transformed cells have striking new vessel
formation (Chauhan et al., 1993), it is highly likely that PTN
signaling is a very important contributor to the neovascularization in
postischemic brain. It is also highly likely that the differential regulation of the Ptn gene in recovery from ischemic injury
results from a specific set of "angiogenic" signals that are
responsible for coordination of gene expression needed for the
development of new blood vessels characteristic of ischemic injury.
PDGF may be a candidate to initiate Ptn gene activation
locally. Ptn gene expression is increased by PDGF (Li et
al., 1992a,b). PDGF is released by platelets, and the expression of
PDGF-A is upregulated within 24 hr in different cells at sites of
injury. The neuron fails to express the PDGF- receptor (Yeh et al.,
1993). For this reason, it is possible that the neuron cannot respond
to the same PDGF-A signal potentially responsible for upregulation of
the Ptn gene in the context of ischemic brain injury.
During embryogenesis, Ptn mRNA is primarily expressed by
progenitor cells in the subependymal layer of the brain in developing neuroepithelium and in the ependymal cells themselves, suggesting roles
in cell division of both neural and vascular progenitors. During the
perinatal stage, Ptn mRNA is seen in cells of neural as well
as glial origins. In the adult brain, Ptn expression is restricted to selective neuronal subpopulations, including cerebral cortex (Li et al., 1990; Wanaka et al., 1993; Silos-Santiago et al.,
1996). It is interesting to note that after ischemia, expression of
Ptn in glia becomes evident again in the adult brain and is preferentially distributed in the regions surrounding the injured area.
The identification of Ptn expression in astrocytes with GFAP
immunoreactivity indicated that the injured brain reverts to a
perinatal pattern of glial Ptn expression. Because
Ptn is also expressed in the ependymal cells in
embryogenesis and therefore is a potentially important contributor to
early vasculogenesis in developing brain, its expression in neovascular
endothelial cells may also reflect a reversion to the perinatal pattern
of Ptn gene expression.
Ptn expression in cortical neurons is different; selected
populations of cortical neurons continue to exhibit basal levels of
Ptn gene expression in normal adult brain (Wanaka et al.,
1993; Silos-Santiago et al., 1996). After severe focal ischemia leading to infarction, a striking loss of expression of the Ptn gene
was observed in neurons that were irreversibly injured within the ischemic core. A substantial reduction was found in stressed or severely injured neurons at the periphery. Thus, in striking contrast to the glial elements, macrophages, and endothelial cells,
Ptn gene expression is not activated in neurons. As noted
above, it is likely that an important signal in recovery from ischemia
brain injury is PDGF-A; the absence of the PDGF- receptor in neurons may therefore underlie the failure of neurons to express PTN in ischemic injury. The neurons in which Ptn gene expression is
downregulated seem destined for cell death, in contrast to the neurons
distant from the site of injury and neurons on the contralateral side that retain the high levels the Ptn mRNA signal intensity
that is characteristic of uninjured neurons.
The significance of Ptn expression relative to the fate of
neurons under ischemic insult is not clear. However, expression of
selected genes may distinguish cell survival from cell death (States et
al., 1996). Heat shock protein 70 (HSP70), a stress gene with putative
cytoprotective action, is expressed only in neurons that survive
ischemic insult but not in those neurons that sustain irreversible
injury. Neurons dying of apoptosis can be separated from those that
survive and express HSP70 by the absence of expression of this marker
gene. Despite the neurotrophic properties of PTN, Ptn gene
expression was not enhanced in injured neurons in the periphery of the
ischemic core, suggesting that Ptn is not a stress gene for
cortical neurons in the context of focal cerebral ischemia. In
contrast, this failure of an increase in Ptn gene expression
contrasts with increased Ptn expression and enhanced
neuronal activity after chemical seizure (Wanaka et al., 1993),
suggesting that perhaps Ptn gene expression contributes to
selective maintenance of neuronal viability. Because the principle activities directed by PTN in neurons in culture are neurite outgrowth and perhaps axonal guidance (Rauvala et al., 1989, 1994; Li et al.,
1990; Nolo et al., 1996), it is likely that the major role of PTN in
neurons is related to differentiation and thus to promoting and
maintaining the differentiated state of cortical neurons.
The present study demonstrates an altered pattern of Ptn
expression affecting neuronal, glial, macrophage, and endothelial cell
populations in the brain after focal cerebral ischemia-reperfusion. The fact that PTN protein was expressed in parallel with its mRNA signals supports the view that Ptn expression probably plays
important pathophysiological roles in the restorative processes of the
brain in response to ischemic injury and that neovascularization may be
the principle role of PTN in this context.
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FOOTNOTES |
Received Dec. 30, 1997; revised Feb. 25, 1998; accepted March 4, 1998.
This work was supported in part by Office of Naval Research Grant
N00014-95-1-0582, National Institute of Neurological Diseases and
Stroke Grants NS25545, NS28995, and NS32636, and National Institutes of
Health Grants HL31102, CA66029, and CA49712 (T.F.D.).
Correspondence should be addressed to Thomas F. Deuel, Department of
Medicine, Division of Growth Regulation, Beth Israel Deaconess Medical
Center, 330 Brookline Avenue, Boston, Massachusetts 02215.
| |
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Top
Abstract
Introduction
