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The Journal of Neuroscience, August 1, 1998, 18(15):5714-5722
Nerve Growth Factor Induces Process Formation in Meningeal Cells:
Implications for Scar Formation in the Injured CNS
Jonas
Frisén1,
Mårten
Risling2,
Laura
Korhonen3,
Ute
Zirrgiebel4,
Clas B.
Johansson1, 2,
Staffan
Cullheim2, and
Dan
Lindholm3
1 Department of Cell and Molecular Biology, Medical
Nobel Institute, and 2 Department of Neuroscience,
Karolinska Institute, S-171 77 Stockholm, Sweden,
3 Department of Developmental Neuroscience, Biomedical
Center, S-751 23 Uppsala, Sweden, and 4 Brain Tumor
Research Centre, Montreal Neurological Institute, Montreal, PQ, Canada,
H3A 2B4
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ABSTRACT |
Nerve growth factor (NGF) induces the differentiation and supports
the survival of subpopulations of neurons in the PNS and CNS. Here we
report that meningeal cells in the pia mater express immunoreactivity
and mRNA for both known NGF receptors, the low-affinity receptor p75
and the tyrosine kinase receptor trkA. NGF induces rapid tyrosine
phosphorylation of trkA in meningeal cells in vitro. NGF
does not stimulate proliferation of primary meningeal cells but induces
process outgrowth. p75- and trkA-immunoreactive meningeal cells with
long processes, resembling NGF-treated cells in vitro, are abundant in the scar tissue that forms at spinal cord lesions in
rat and cat. These data suggest that NGF, which is expressed at
increased levels in the brain and spinal cord after lesions, may be
involved in scar formation in the injured CNS.
Key words:
NGF; trkA; p75; injury; regeneration; meningeal cells
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INTRODUCTION |
Nerve growth factor (NGF) is
required for the survival of sympathetic neurons and some sensory
neurons in the peripheral nervous system during development
(Levi-Montalcini, 1987 ; Crowley et al., 1994; Smeyne et al., 1994). In
the CNS, NGF regulates the phenotype of septal cholinergic neurons
(Thoenen, 1991; Sofroniew et al., 1993). p75, also known as the
low-affinity neurotrophin receptor, was the first identified
NGF-binding molecule that has a relative molecular mass of 75 kDa
(Johnson et al., 1986; Radeke et al., 1987) and in addition to NGF,
binds the structurally and functionally related neurotrophic factors in
the neurotrophin family (Ernfors et al., 1990; Rodriguez-Tébar et
al., 1990, 1992; Hallböök et al., 1991; Squinto et al.,
1991). trkA is a signal-transducing tyrosine kinase receptor for NGF
(Kaplan et al., 1991; Klein et al., 1991; Barbacid, 1995). NGF can
induce responses in fibroblasts expressing trkA in the absence of p75
(Cordon-Cardo et al., 1991), and mutant NGF molecules that do not bind
p75 can still induce effects in neuronal cells in vitro
(Ibáñez et al., 1992). The role of p75 has been difficult
to establish, but some data suggest that p75 may increase the neuronal
sensitivity to NGF (Davies et al., 1993; Barker and Shooter, 1994; Lee
et al., 1994; Maliartchouk and Saragovi, 1997; Rydén et al.,
1997). There are also reports that p75 may affect the signal
transduction pathways for NGF (Hantzopoulos et al., 1994; Verdi et al.,
1994). Furthermore, there is increasing evidence that p75 may induce
cell death during NGF binding in cells that do not express trkA
(Dechant and Barde, 1997).
In contrast to the restricted expression of trkA, the structurally and
functionally related neurotrophin receptors trkB and trkC are more
widespread in neurons in the PNS and CNS (Merlio et al., 1992). The
trkB and trkC loci encode, in addition to full-length tyrosine kinase
receptors, truncated receptors lacking the signal transducing domain
that are expressed at high levels by glial cells (Klein et al., 1990;
Middlemas et al., 1991; Tsoulfas et al., 1993; Valenzuela et al.,
1993). The function of glial trkB and trkC receptors is poorly
understood, although developmental regulation and altered expression by
injury may indicate important functions (Frisén et al., 1992,
1993; Funakoshi et al., 1993). After brain and spinal cord injuries,
astrocytes express truncated trkB-receptors at highly elevated levels
(Frisén et al., 1992, 1993; Beck et al., 1993). Additionally,
both trkB and p75 are expressed at high levels by meningeal cells
invading the injured CNS (Risling et al., 1992; Frisén et al.,
1992).
We here report that leptomeningeal cells in the pia mater coexpress p75
and trkA, and that NGF induces rapid phosphorylation of trkA on
tyrosine residues in these cells in vitro. NGF is not mitogenic for leptomeningeal cells but stimulates process outgrowth in vitro. In the injured rat and cat spinal cord, trkA- and
p75-immunoreactive leptomeningeal cells with long processes are
abundant in the scar tissue formed at the lesion, implicating NGF in
CNS scar formation.
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MATERIALS AND METHODS |
Animals and surgery. Adult female Sprague Dawley rats
(200 gm) were anesthetized with chloral hydrate (300 mg/kg), and adult cats were anesthetized with pentobarbitone sodium (30 mg/kg) and xylazine chloride (0.4 mg/kg). A longitudinal incision was made in the
left ventral funiculus of the L5 (rats, n = 10) or L7
(cats, n = 6) segment as described (Risling et al.,
1983), resulting in intramedullary axotomy of motoneurons. In other
rats (n = 5) an incision was made in the lateral
funiculus in the L4/L5 segment. The rats were allowed to survive for
7 d (n = 8) or 3 weeks (n = 7),
and the cats were allowed to survive for 4 d (n = 1), 10 d (n = 2), 3 weeks (n = 2),
or 2 months (n = 1). The use of animals for this study
was approved by the Swedish ethical committee.
Immunofluorescence histochemistry. Anesthetized animals were
perfused through the ascending aorta with Tyrode's solution followed by 4% formaldehyde with 0.4% picric acid in 300 mOsm phosphate buffer. Tissues were rapidly dissected out and post-fixed in the fixative for 90 min, and subsequently rinsed overnight in 300 mOsm
phosphate buffer with 10% sucrose. Sections (14 µm) were cut on a
cryostat, thawed onto chrome alum-coated object slides, and incubated
in a humid atmosphere at 4°C for 18-24 hr with rabbit anti-trkA
antiserum (trk763, diluted 1:100, Santa Cruz Biotechnology, Santa Cruz,
CA; or OA-11-790A, diluted 1:200, Cambridge Research Biochemicals).
Other sections were incubated with mouse monoclonal antibodies against
p75 (diluted 1:100) (Ross et al., 1984). The sections were then rinsed
in 0.01 M PBS, pH 7.4, and incubated for 45 min at 20°C
with rhodamine-conjugated or fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit antiserum (diluted 1:10; Dako, Glostrup, Denmark), Cy3-conjugated affiniPure donkey anti-rabbit IgG
(diluted 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA),
or rhodamine-conjugated goat anti-mouse antiserum (diluted 1:100;
Boehringer Mannheim, Mannheim, Germany). All antisera were diluted in
0.01 M PBS containing 0.3% Triton X-100, 0.5% bovine
serum albumin (BSA), and 0.1% sodium azide. After the sections were
rinsed in PBS, they were mounted in a mixture of glycerol and PBS (1:9)
and coverslipped. The specificity of the antibody labeling was tested
by incubation with the secondary antibody in the absence of primary
antibody. In other sections, the trk763 antiserum was preincubated with
the peptide (20× excess) against which the antisera were raised. All
specific labeling was abolished in both of these control
experiments.
Immunoelectron microscopy. Anesthetized cats were perfused
as described above, with the exception that 0.1% glutaraldehyde was
used instead of picric acid in the fixative. Spinal cords were cut in
60-µm-thick sections on a cryostat. The sections were treated in
sodium borohydride, transferred through graded steps of ethanol, and
then rehydrated (Priestley, 1984). The sections were then incubated for
18-24 hr with rabbit anti-trkA antiserum and/or mouse monoclonal
antibodies to human p75 as above. After they were rinsed, the sections
were incubated with 0.8 nM gold particle-conjugated goat
anti-rabbit antiserum (diluted 1:50; Aurion) or 1 nM goat
anti-mouse antiserum (diluted 1:50; Auroprobe One, Amersham, Arlington
Heights, IL). In double-labeled sections, the gold particle-conjugated
goat anti-rabbit antiserum was combined with biotin-conjugated goat
anti-mouse antiserum (diluted 1:200; Vector Laboratories, Burlingame,
CA) for 2 hr at 20°C. All antisera were diluted in 0.01 M
PBS containing 0.5% gelatin (Janssen Biochimica, Berse, Belgium). The
sections were osmicated for 30 min, and gold labeling was intensified
with a silver enhancement reaction (Intense M, Amersham).
Biotin-conjugated antibodies were visualized by adding
avidin-conjugated horseradish peroxidase (Vector) and processing with
DAB as chromogen. The sections were then dehydrated in graded steps of
acetone and embedded in Vestopal W between transparent acetate foils.
Selected areas were thin-sectioned by the use of an Ultratome III (LKB)
and contrasted with uranyl acetate and lead citrate. Pieces of pia
mater stripped from cervical spinal cord were processed exactly as the
spinal cord sections. A Philips CM12 electron microscope was used. No
specific labeling was seen when the primary antibody was omitted.
Cell cultures. The leptomeninges were carefully removed from
the convexities of the cerebral hemispheres of newborn rats and enzymatically digested with papain (Sigma, St. Louis, MO) (2.5 mg in 5 ml of PBS containing 1 mg/ml BSA, 10 mM glucose, and 100 µg/ml DNase I) for 30 min at 37°C. The cells were triturated, collected by centrifugation, and resuspended in cell culture medium. Cells were maintained in DMEM (Life Technologies, Gaithersburg, MD)
containing 10% fetal calf serum (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin. The medium was changed the
next day and thereafter every fourth day. Cell cultures used for
immunohistochemistry were fixed for 10 min with 4% formaldehyde in PBS
and processed as above. Mouse monoclonal antibodies against p75
(Chandler et al., 1984) (final concentration 1.5 µg/ml) and trkA
(described above) were used. The secondary FITC-conjugated anti-mouse
antibody was from Sigma, and the Cy3-conjugated anti-rabbit antibody
was from Jackson ImmunoResearch Laboratories. NGF was prepared from
male mouse submandibular glands, and recombinant brain-derived
neurotrophic factor (BDNF) was produced using vaccinia virus-infected
cultured kidney cells (Götz et al., 1992). Bovine basic
fibroblast growth factor (bFGF) was purchased from Boehringer Mannheim.
RT-PCR and Northern blot. Total RNA was extracted by the
acid guanidine isothiocyanate/phenol-chloroform method of Chomczynski and Sacchi (1987). The recovery of RNA was measured
spectrophotometrically. Reverse transcription was performed using
oligo-dT and the avian myeloblastosis virus reverse transcriptase (Life
Sciences, Hialeah, FL). For the subsequent PCR reaction for trkA, the
following primers were used: sense primer (5'-GCTGGTATGGTGTACCTAGCC-3')
corresponding to nucleotides 1914-1935 in the rat trkA sequence, and
one of the two antisense primers A1 (5'-CAGCACCACCCGAAGCTCC-3') or A2 (5'-GCGGTAGAGGATGCTCTCTGG-3') corresponding to nucleotides 2140-2160 and 2084-2105, respectively. The sizes of the amplified products of
trkA were 246 bp for trkA-A1 and 191 bp for trkA-A2. The following primers were used to amplify p75: sense (5'-TTCAAGAGGTGGAACAG-3') and antisense (5'-GGGGTCACACTTGAGTG-3') corresponding to nucleotides 816-834 and 1266-1283 in the rat p75 sequence, resulting in a 476 bp
band. GAPDH primers, sense (5'-GGACTCCTCAGCAACTGAGGG-3') and
antisense (5'-GGCTGTGGGCAAGGTCATCCC-3'), were used to estimate that
equal amounts of cDNA were used for the assay. PCR reactions were
performed in a thermocycler (Perkin-Elmer, Emeryville, CA) passing 35 cycles (1 min 94°C, 1 min 53°C, and 2 min 72°C) using Taq-Polymerase (Perkin-Elmer) in the delivered buffer plus
4% DMSO. PCR products were separated on Agarose gels (3% NuSieve Agarose; FMC Bio Products, Rockland, ME) and blotted onto
nitrocellulose membranes (Hybond N+, Amersham). The amplified bands
were cut out of the gel, cloned into pBluescript, and sequenced using
the dideoxy method to verify the molecular identity. No amplification was seen in samples processed without reverse transcriptase.
Immunoprecipitation and Western blotting. Meningeal cells
were cultured for 10 d in 3.5 cm Petri dishes. Cells were
stimulated for 5 min with 50 ng/ml NGF followed by cell lysis in 500 µl lysis buffer (50 mM HEPES, pH 7.5, 150 mM
NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM
MgCl2, 10 mM EDTA, 10 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 250 µM p-nitrophenol phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). Lysates were centrifuged, and the
supernatants were used for immunoprecipitation with anti-pan Trk 203 antibody (Verdi et al., 1994) as described by Moran et al. (1991). The
Sepharose A (Pharmacia, Uppsala, Sweden)-bound immunoprecipitates were
washed, run on 6% SDS-PAGE, and transferred to Immobilon-P membranes
(Millipore, Bedford, MA), which were incubated with mouse monoclonal
anti-phosphotyrosine antibodies (P469; Santa Cruz Biotechnology) or
rabbit anti-trkA antibodies (trk763; Santa Cruz Biotechnology).
Detection was performed with enhanced chemiluminescence substrate
solution (Amersham) after incubation with horseradish
peroxidase-conjugated anti-mouse antiserum (Dako).
Cell proliferation assay. The mitogenic activity of NGF,
BDNF (50 ng/ml), and bFGF (10 ng/ml) was determined by incubating leptomeningeal cells with the respective factor for 2 d. No factor was added to control cultures. During the last 6 hr of incubation, 3[H]thymidine (2 µCi/ml) was present, and the amount of
TCA-insoluble radioactivity in the cells was subsequently
determined.
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RESULTS |
Meningeal cells express p75 and trkA
Two different antisera were used to analyze the expression of
trkA-like immunoreactivity (LI). Both antisera gave the same labeling
pattern in all analyzed regions of the nervous system (data not shown),
which corresponded to that observed in previous trkA localization
studies (Verge et al., 1992). No specific labeling was seen in sections
incubated with the secondary antiserum in the absence of primary
antiserum or in sections incubated with trkA antiserum preincubated
with the peptide against which the antiserum was raised (data not shown
and see Fig. 9). Except for trkA-LI in thin nerve fibers in the dorsal
horn, no labeling of neurons or glial cells was seen within the spinal
cord (data not shown). Strong trkA-LI was seen in the pia mater
covering the spinal cord (Fig. 1). Thin
cell processes extending from the pia mater into the superficial spinal
cord, in most cases in association with blood vessels, showed trkA-LI
(Fig. 1). Silver-enhanced immunogold electron microscopy was used to
study p75- and trkA-immunoreactive structures in meninges stripped from
the spinal cord at the ultrastructural level. Cells with morphological
characteristics of leptomeningeal cells (Peters et al., 1976; Risling
et al., 1992) showed strong p75-LI and trkA-LI localized to the cell
membrane (Fig. 2). p75-LI was also seen
in Schwann cells associated with axons in the meninges (data not
shown).
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Figure 1.
trkA-like immunoreactivity in the pia mater. The
micrographs show immunohistochemical detection of trkA at the surface
of the uninjured adult cat (A) and rat (B,
C) lumbar spinal cord. The arrows indicate
labeling in the pia mater. Thin cell process associated with blood
vessels extend into the spinal cord (arrowheads). In
C, trkA-LI is seen in the pia mater in the ventral
fissure and in the pia mater surrounding a blood vessel. Scale bars, 50 µm.
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Figure 2.
Ultrastructural localization of p75- and trkA-like
immunoreactivity. Silver-enhanced immunogold electron microscopy of pia
mater covering the uninjured spinal cord is shown. Leptomeningeal cells
with long processes (arrows in A) show
strong p75-LI (A) and trkA-LI (B)
at the cell surface. Scale bars, 1 µm.
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To confirm the immunohistochemical results we analyzed p75 and trkA
mRNA expression. RT-PCR demonstrated p75 and trkA mRNA expression in
cultured leptomeningeal cells, as well as in septum and PC12 cells
(Fig. 3).
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Figure 3.
3 RT-PCR detection of p75 and trkA mRNA. mRNA was
extracted from PC12 cells (lanes 1-3), septum
(lanes 4-6), and pia mater (lanes
7-9) and subjected to RT-PCR amplification with primers for
p75 and two different sets of primers for trkA. No amplification from
pia mater mRNA was seen in the absence of reverse transcriptase
(lanes 10-12).
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NGF induces tyrosine phosphorylation of trkA in
leptomeningeal cells
NGF binding to trkA receptors induces receptor dimerization and
transphosphorylation, initiating the signal transduction by trkA
(Kaplan et al., 1991; Klein et al., 1991; Jing et al., 1992). Cultured
leptomeningeal cells were lysed, and the lysates were immunoprecipitated with anti-trk antiserum. Western blots were then
made, and activated trkA was detected using anti-phosphotyrosine antibodies. There was a low degree of phosphorylation of trkA in
untreated cultures (Fig. 4). When NGF (50 ng/ml) was added to the cultures, an increase of trkA phosphorylation
was seen within 5 min (Fig. 4).
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Figure 4.
Activation of trkA by NGF in leptomeningeal cells.
The cells were lysed, immunoprecipitated with trk-antiserum, separated
on SDS-PAGE, transferred to a membrane, and incubated with trkA
(left panel) or phosphotyrosine (right
panel) antibodies. Cells were grown in the absence of
NGF ( ) or in the presence of NGF for 5 min before lysis (+).
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NGF is not mitogenic for leptomeningeal cells
Previous studies have demonstrated that NGF stimulates
proliferation of NIH3T3 cells ectopically expressing trkA (Cordon-Cardo et al., 1991). This prompted us to test wether NGF may be mitogenic for
leptomeningeal cells. To this end, we studied
3[H]thymidine and bromodeoxyuridine (BrdU) incorporation
in leptomeningeal cells grown in the presence or absence of various
factors. As shown in Figure 5, bFGF
strongly stimulated proliferation of leptomeningeal cells. In contrast,
no change in thymidine incorporation was detected in NGF- or
BDNF-treated cells compared with controls (Fig. 5). No difference in
the number of labeled nuclei could be detected after BrdU incorporation
between NGF-treated leptomeningeal cells and controls (data not
shown).
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Figure 5.
NGF does not stimulate proliferation of
leptomeningeal cells. Leptomeningeal cells were cultured in the
presence of NGF (50 ng/ml), bFGF (10 ng/ml), or BDNF (50 ng/ml) for
2 d. 3[H]thymidine was added to the medium for the
last 6 hr, and incorporation was analyzed. Error bars represent the
mean + SD from four independent experiments.
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NGF induces process formation in leptomeningeal cells
Leptomeningeal cells grown in medium supplemented with 10% fetal
calf serum had a flat polyhedral shape. Leptomeningeal cells could
easily be distinguished from rare contaminating astrocytes by their
coexpression of p75-LI and trkA-LI (Fig.
6) and their lack of glial fibrillary
acidic protein-LI. Schwann cells, which also express p75 but not trkA,
did not proliferate in these cultures. After the cells had been grown
in the presence of NGF for 3 d, the morphology of the
leptomeningeal cell was drastically changed: long thin processes
extended from the cell bodies (Fig.
7).
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Figure 6.
Colocalization of trkA and p75 immunoreactivity in
leptomeningeal cells. Meningeal cells kept in culture for 5 d were
fixed and incubated with antibodies against p75 and trkA. Fluorescent
secondary antibodies were used to visualize p75 (A,
FITC, green) or trkA (B, Cy3,
red). There is a colocalization of p75 and trkA in most
of the immunoreactive cells (arrows).
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Figure 7.
NGF induces process outgrowth in leptomeningeal
cells. Leptomeningeal cells positive for p75 have short processes
in vitro (A). When 50 ng/ml NGF is
added to the cultures (B, C) the cells extend long
processes. Scale bars: A, B, 25 µm; C,
50 µm.
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Leptomeningeal cells in the injured spinal cord express p75-LI
and trkA-LI
Leptomeningeal cells migrate into the CNS at penetrating injuries.
We used a well established spinal cord injury model in the adult rat
and cat to study leptomeningeal cells in the injured spinal cord. In
this lesion model a longitudinal incision is made unilaterally in the
lumbar ventral funiculus, resulting in proximal axotomy of spinal
motoneurons. Many of the surviving motoneurons send axonal sprouts into
the scar tissue that forms at the site of the injury, and the axons
often reinnervate the ventral root (Risling et al., 1983). Cells with
long processes projecting through the scar showed p75-LI and trkA-LI
(Figs. 8,
9). The scar tissue labeling was
continuous with the pia mater (Fig. 9). The levels of p75-LI and
trkA-LI in the cells in the scar tissue appeared similar to that in
leptomeningeal cells in the pia mater. The time course for the invasion
of cells showing trkA-LI and the extension of processes in these cells
mimicked that described previously for p75-expressing leptomeningeal
cells after injury (Risling et al., 1992). Thus, there was an
increasing number of cells showing trkA-LI in the scar tissue from 4 to
10 d after the injury. During this time the staining pattern
became more dense as more cells entered the scar tissue and the cells
projected elaborate processes. The labeling pattern remained
essentially the same at the later survival times. A similar pattern of
p75-LI and trkA-LI was seen after an incision in the lateral funiculus in adult rats (data not shown). Immunoelectron microscopy demonstrated that the cells showing p75-LI and trkA-LI displayed ultrastructural characteristics of reactive leptomeningeal cells (Fig.
10). Double-labeling was performed with
antibodies to p75 and trkA to study whether both NGF receptors are
coexpressed in the same leptomeningeal cell. Visualization of trkA and
p75 was performed with silver-enhanced immunogold and peroxidase
methodology, respectively, enabling ultrastructural examination of
double-labeled cells. p75-LI and trkA-LI were coexpressed in most, if
not all, leptomeningeal cells (Fig. 10B). Schwann
cells, which often were seen on the surface of the spinal cord scar
tissue, showed strong p75-LI at the cell membrane but no trkA-LI (Fig.
10C).
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Figure 8.
p75- and trkA-like immunoreactivity in the injured
cat spinal cord. Immunofluorescence detection of p75-LI and trkA-LI in
the scar tissue formed at a ventral funiculus lesion in adult cat.
Scattered cells showing p75-LI (A) and trkA-LI
(B) are seen in the scar tissue 10 d after
the injury. Scale bars, 100 µm.
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Figure 9.
trkA-like immunoreactivity in the injured rat
spinal cord. Immunofluorescence showing trkA-LI in the rat spinal cord
7 d after a ventral funiculus incision. The labeling in the scar
tissue is continuous with the pia mater (A). The
cells in the scar have long slender processes
(B). All labeling is abolished in an adjacent
section when the primary antiserum was preincubated with the peptide
against which the antiserum was raised
(C). Note that the exposure is lighter in
C compared with A and B.
Scale bars: A, 200 µm; B, 25 µm;
C, 100 µm.
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Figure 10.
Coexpression of p75- and trkA-like
immunoreactivity in leptomeningeal cells in the injured spinal cord.
Silver-enhanced immunogold and ABC electron microscopy was used to
localize p75-LI and trkA-LI in the spinal cord after ventral funiculus
lesions in adult cats. In A a leptomeningeal cell
(LMC) with a long process shows strong p75-LI as
revealed by immunogold methodology. The adjacent macrophage
(M) is unlabeled
(A). In B and C,
p75-LI is visualized with ABC, and trkA-LI is visualized with
silver-enhanced immunogold methodology. Leptomeningeal cells with
elongated processes coexpress p75-LI (dark peroxidase product by the
cell membrane, arrows) and trkA-LI
(B). In the superficial part of the scar tissue
Schwann cells showing p75-LI (arrows) ensheath axons
(A). The Schwann cells do not show trkA-LI in
contrast to leptomeningeal cells (LMC), which show dense
p75-LI and trkA-LI. All micrographs are from 3 weeks after the injury.
Scale bars, 1 µm.
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DISCUSSION |
In this study we have characterized the expression of NGF
receptors in leptomeningeal cells in the normal animal and after spinal
cord injury and have analyzed the effects of NGF on these cells
in vitro. Leptomeningeal cells express both known types of
NGF receptors, p75 and trkA, and the presence of functional signal
transducing NGF receptors is indicated by rapid tyrosine phosphorylation of trkA in response to NGF. NGF does not affect the
proliferation of leptomeningeal cells, but induces process outgrowth
in vitro. In animals that have undergone spinal cord injuries, p75-LI and trkA-LI leptomeningeal cells with long processes, morphologically resembling NGF-stimulated leptomeningeal cells in
vitro, were scattered throughout the scar formed at the
injury.
NGF induces differentiation of postmitotic trkA-expressing neurons
(Levi-Montalcini, 1987). In contrast, NGF is mitogenic for some
proliferating cells, such as adrenal chromaffin cells, neuronal stem
cells, and fibroblasts ectopically expressing trkA (Lillien and Claude,
1985; Cattaneo and McKay, 1990; Cordon-Cardo et al., 1991).
Interestingly, we found that NGF was not mitogenic for proliferating
leptomeningeal cells in vitro. Instead, NGF induced process
outgrowth in these cells. It is not fully understood how tyrosine
kinase receptor signaling in some situations can lead to proliferation
and in other cases can lead to differentiation. The temporal dynamics
of receptor activation may be important because sustained receptor
activation over a longer period of time seems to be correlated with
differentiation. Other studies suggest that activation of alternative
signal transduction pathways may be more important for the outcome of
receptor activation (for review, see Marshall, 1995; Kaplan and Miller,
1997). There is increasing evidence that p75 can facilitate signaling
by trkA (Barker and Shooter, 1994; Hantzopoulos et al., 1994; Verdi et al., 1994; Maliartchouk and Saragovi, 1997; Rydén et al., 1997), although the mechanism is not known. Interestingly, MAH neuronal progenitor cells expressing p75 differentiate more rapidly in response
to NGF compared with MAH cells lacking p75 (Verdi et al., 1994).
Moreover, a pancreatic  cell line (RINm5F), which expresses p75 and
trkA, differentiates in response to NGF (Polak et al., 1993). Further
characterization of NGF signal transduction pathways in leptomeningeal
cells may provide information regarding the role of p75 in trkA
signaling.
Leptomeningeal cells in the normal animal and in the injured spinal
cord show strong p75-LI and trkA-LI and have long thin processes. This
morphology resembles that of NGF-treated meningeal cells in
vitro, indicating that NGF may induce this phenotype in
vivo. We do not know whether leptomeningeal cells are exposed to
NGF in the uninjured animal. However, we see a low degree of trkA
phosphorylation in cultured leptomeningeal cells in the absence of
exogenously added NGF (Fig. 4), indicating endogenous NGF production and autocrine stimulation in these cells. At CNS injuries, NGF synthesis by astrocytes is increased from very low or undetectable levels to rather high levels (Bakhit et al., 1991; Ishikawa et al.,
1991; Lindholm et al., 1992b), which may affect meningeal cells
invading the lesion site. The NGF levels are highest during the first
week after a lesion (Bakhit et al., 1991; Ishikawa et al., 1991;
Lindholm et al., 1992b), corresponding to the time course for the
invasion and process extension of leptomeningeal cells in the scar
tissue. Interestingly, astrocyte conditioned medium can induce process
formation by leptomeningeal cells but not by skin fibroblasts in
vitro (Colombo et al., 1994). The present data suggest that NGF
may be one astrocyte-derived factor that induces this morphological
transformation of meningeal cells.
Glucocorticoids have been demonstrated to improve functional recovery
to some extent after spinal cord injury in man (Bracken et al., 1990)
and are now used in clinical practice. This effect may be somewhat
unexpected because glucocorticoids downregulate NGF synthesis in
astrocytes (Lindholm et al., 1992a) and thus may lead to less
neurotrophic support for severed neurons. However, the data presented
here suggests that NGF may participate in the formation of scar tissue
at the site of injury, and decreased NGF levels at the lesion may
modify the properties of leptomeningeal cells and affect CNS scarring
and axonal regeneration.
| |
FOOTNOTES |
Received March 25, 1998; revised May 13, 1998; accepted May 13, 1998.
This study was supported by grants from the Swedish Medical Research
Council, the Swedish Medical Society, Stiftelsen Sigurd och Elsa Goljes
minne, Stiftelsen Lars Hiertas Minne, Jeanssons stiftelse, Ostermans
stiftelse, Magnus Bergvalls stiftelse, Tore Nilssons stiftelse, the
Wenner-Gren Center Foundations, Björklunds fond, Marcus
Borgströms stiftelse, Åke Wibergs stiftelse, Schweizer Paraplegiker Stiftung (SPS), and Kapten Arthur Erikssons fond. We
gratefully acknowledge Ms. M. Meier and Mr. J. Richter for expert
technical assistance. We thank Dr. David Kaplan for kindly providing
trk antiserum.
Correspondence should be addressed to Dr. Jonas Frisén,
Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18155714-09$05.00/0
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Abstract
Introduction
