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The Journal of Neuroscience, May 15, 2001, 21(10):3457-3475
Neurotrophic Factors and Receptors in the Immature and Adult
Spinal Cord after Mechanical Injury or Kainic Acid
Johan
Widenfalk,
Karin
Lundströmer,
Marie
Jubran,
Stefan
Brené, and
Lars
Olson
Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden
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ABSTRACT |
Delivery of neurotrophic factors to the injured spinal cord has
been shown to stimulate neuronal survival and regeneration. This
indicates that a lack of sufficient trophic support is one factor
contributing to the absence of spontaneous regeneration in the
mammalian spinal cord. Regulation of the expression of neurotrophic
factors and receptors after spinal cord injury has not been studied in
detail. We investigated levels of mRNA-encoding neurotrophins, glial
cell line-derived neurotrophic factor (GDNF) family members and
related receptors, ciliary neurotrophic factor (CNTF), and c-fos
in normal and injured spinal cord. Injuries in adult rats included
weight-drop, transection, and excitotoxic kainic acid delivery; in
newborn rats, partial transection was performed. The regulation of
expression patterns in the adult spinal cord was compared with that in
the PNS and the neonate spinal cord. After mechanical injury of the
adult rat spinal cord, upregulations of NGF and GDNF mRNA occurred in
meningeal cells adjacent to the lesion. BDNF and p75 mRNA increased in
neurons, GDNF mRNA increased in astrocytes close to the lesion, and
GFR -1 and truncated TrkB mRNA increased in astrocytes of
degenerating white matter. The relatively limited upregulation of
neurotrophic factors in the spinal cord contrasted with the response of
affected nerve roots, in which marked increases of NGF and GDNF mRNA
levels were observed in Schwann cells. The difference between the
ability of the PNS and CNS to provide trophic support correlates with their different abilities to regenerate. Kainic acid delivery led to
only weak upregulations of BDNF and CNTF mRNA. Compared with several
brain regions, the overall response of the spinal cord tissue to kainic
acid was weak. The relative sparseness of upregulations of endogenous
neurotrophic factors after injury strengthens the hypothesis that lack
of regeneration in the spinal cord is attributable at least partly to
lack of trophic support.
Key words:
rat; GFR; NGF; BDNF; NT3; NT4; GDNF; NTN; CNTF; PSP; TrkA; TrkB; truncated TrkB; TrkC; p75; in situ
hybridization; spinal cord injury; kainic acid; weight-drop; motoneuron
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INTRODUCTION |
The lack of regenerative properties
of the mammalian CNS is probably attributable to a combination of
factors, such as the inhibitory character of glial scars and CNS white
matter (Hoke and Silver, 1996 ; Chen et al., 2000) and lack of trophic
support (Skene, 1989; Doster et al., 1991; Schnell, 1994; Tetzlaff et al., 1994; Fournier and McKerracher, 1997). Neurotrophic factors, including members of the NGF and GDNF families, are potent
endogenous stimulators of neuron survival and nerve fiber growth. In
addition, they have been reported to elicit beneficial effects when
delivered after spinal cord injury (SCI) (Schnell, 1994; Grill et al.,
1997; Kobayashi et al., 1997; Menei et al., 1998; Liu et al., 1999). However, the expression levels of mRNA-encoding neurotrophic factors and their receptors after SCI have not been thoroughly studied previously. Therefore, in the present study we have characterized the
expression patterns of mRNA-encoding neurotrophic factors in the NGF
and GDNF families and related receptors, focusing on normal spinal cord
and regulations of mRNA levels in response to injury. These
investigations should help clarify whether there is a lack of trophic
support after SCI and which cells, if any, are responsible for
neurotrophic factor synthesis. We also address the question of whether
the spinal cord differs from the PNS, where successful regeneration
occurs, in terms of injury-induced mRNA patterns. Moreover we ask
whether the adult spinal cord differs from the neonate spinal cord in
these respects, because a degree of regeneration or rerouting, or
both, is known to take place after SCI in the neonate (Bernstein
and Stelzner, 1983; Bregman and Goldberger, 1983). Finally, we have
used the excitotoxin kainic acid, known to induce profound upregulation
of neurotrophic factors and receptors in the brain, to determine
whether any of these upregulations are shared by the spinal cord.
In addition to the NGF and GDNF families and their receptors, we also
investigated expression levels of ciliary neurotrophic factor (CNTF).
Glial fibrillary acidic protein (GFAP), myelin-associated glycoprotein
(MAG), and c-fos were used to monitor the degree of lesion and the
astrocytes and oligodendrocytes.
NGF, BDNF, NT3, and NT4 constitute the NGF family of neurotrophic
factors (the neurotrophins), which act through the high-affinity receptor tyrosine kinases (TrkA, TrkB, TrkC) and the low-affinity p75
receptor (Lindsay, 1994). GDNF, neurturin (NTN), persephin (PSP), and
artemin (ART) constitute the GDNF family of ligands, which
belongs to the TGF superfamily, and signal by binding to GFR-1-4
(Ibáñez, 1998; Baloh et al., 2000) followed by activation of the signal-transducing tyrosine kinase RET (Martin-Zanca et al., 1986; Treanor et al., 1996; Trupp et al., 1996). During
development, the neurotrophic factors support survival of and neurite
outgrowth from various neuron populations. Limited amounts of
neurotrophic factors are produced in the adult CNS, and the roles of
neurotrophic factors in the adult brain and spinal cord are less well
understood. They may increase neuronal survival (Tomac et al., 1995;
Novikova et al., 1996), induce regeneration after injury (Schnell,
1994; Tuszynski et al., 1996; Grill et al., 1997; Kobayashi et al., 1997; Houweling et al., 1998; Menei et al., 1998; Liu et al., 1999),
and be involved in synapse plasticity (McAllister et al., 1999).
Neurotrophic factors have additionally been shown to prevent neurodegeneration after excitotoxic damage to the CNS (Tomac et al.,
1995; Horger et al., 1998; Milbrandt et al., 1998; Rosenblad et al.,
1999, 2000).
There are few reports dealing with regulation of neurotrophins or their
receptors after spinal cord perturbations. Frisén and colleagues
(1992, 1993) demonstrated upregulation of truncated TrkB mRNA in cells
in the glial scar 3 weeks after longitudinal dorsal funiculus cuts.
After SCI, the low-affinity receptor p75 upregulates in cells
associated with blood vessels (Reynolds et al., 1991). A massive glial
BDNF and NT3 expression close to the lesion shortly after SCI was
reported recently (Hayashi et al., 2000). There are no previous studies
of regulation of these molecules after weight-drop injury produced
using the NYU impactor (Gruner, 1992). Dramatic upregulations of BDNF
and NT4 mRNA in glial cells and neurons of the spinal cord after kainic
acid delivery have been reported (Scarisbrick et al., 1999). The
expression of GDNF family ligands and related receptors after SCI has
not been studied previously.
We describe widespread expression of different neurotrophic factor
receptors in the normal adult spinal cord, whereas expressions of
ligands were low and spatially restricted. Upregulations of neurotrophic factors in gray and white matter of the spinal cord after
injury were limited, indicating a relative lack of trophic support in
the spinal cord. Strong neurotrophic factor expression was found in
meningeal cells, whereas neurons and astrocytes presented only low
levels. In contrast to the spinal cord glia, Schwann cells of affected
nerve roots readily increased their neurotrophic factor synthesis,
demonstrating a different ability of the PNS to provide trophic
support. The regulation of neurotrophic factors in the spinal cord of
newborn rats was almost identical to that found in adults. Kainic acid
delivery led to only minor increases of neurotrophic factor levels and
c-fos in the spinal cord. Brain regions of these animals exhibited more
robust increases of c-fos, neurotrophic factors, and their receptors.
Taken together, these results support the hypothesis that lack of
regeneration in the spinal cord is attributable, at least partly, to
lack of trophic support.
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MATERIALS AND METHODS |
Animals
A total of 154 adult female and 45 newborn Sprague Dawley rats
(B&K Universal, Sollentuna, Sweden) were used. Adults, weighing between
200 and 300 gm, were kept under standardized conditions and given food
and water ad libitum. Sixty-one adult rats were subjected to
spinal cord weight-drop injury, and 27 were subjected to spinal cord
transection. Fifty adult rats were included in a kainic acid experiment
(35 were kainic acid treated and 15 were Ringer's solution treated),
and 45 newborns were subjected to partial spinal cord transection. In
Table 1, the number of animals killed at each time point for each individual experiment is
displayed. In the SCI study of adult animals, no animals were lost
before these determined time points. In the kainic acid study, two
animals were lost. In the SCI study of newborn animals, one pup was
lost. Animals were killed from 2 hr to 6 months after injury. All
experiments had been approved by the Stockholm Animal Ethics
Committee.
Surgery
Dorsal laminectomies at the level of the ninth thoracic
vertebra were carried out under general halothane (Fluothane,
AstraZeneca) anesthesia.
Adults. The exposed spinal cord was either left untouched
(sham), contused (weight-drop), or completely transected. Contusion injuries were performed using the NYU impactor (Gruner, 1992) and a 25 mm drop. Sterile 3/0 and 2/0 sutures (PDSII and Ethilon II, Ethicon)
were used to suture muscle and skin, respectively. Animals were given
antibiotics (Borgal, Hoechst) the first week after injury and
thereafter if needed. Animals were allowed to recover from anesthesia
in warmed cages. A heating pad was placed under a part of each cage
during the first 3 d after the operation. Urinary bladders were
emptied manually three times every day the first week and twice daily
thereafter to prevent urinary tract infections.
Newborns. Under anesthesia, inspections of respiratory rate
and skin color were made at close intervals. The depth of analgesia was
assessed by the pedal withdrawal reflex. Laminectomies were followed by
partial spinal cord transections of approximately two-thirds of the
spinal cord width, cutting one side completely. The skin was sutured
using sterile 6/0 sutures (Vicryl, Ethicon). Pups were allowed to
recover from anesthesia before being reunited with the dam. Delivery of
halothane anesthesia to the newborn rats was according to Park and
colleagues (1992).
Kainic acid treatment
Thirty-five adult rats were injected with kainic acid at a dose
of 10 mg/kg, i.p.; 15 animals were given saline as controls. All
animals were observed for development of seizures. Kainic acid-injected
animals included in this study (n = 24) developed grade
V seizures (repeated incidences of rising on the hindlimbs and falling
over) (Sperk et al., 1985). Control animals had no such symptoms.
Animals were killed 2 hr (three kainic acid-treated, three controls), 4 hr (nine kainic acid-treated, four controls), 1 d (six kainic
acid-treated, four controls), 3 d (three kainic acid-treated, two
controls), and 7 d (three kainic acid-treated, three controls)
after injection. Seizure activity was still evident 4 hr after
treatment. Twenty-four hours after treatment, animals had no overt
signs of seizures.
In situ hybridization
Animals were decapitated, and brains and spinal cords were
dissected at 2 hr (weight-drop, n = 4; adult sham,
n = 2), 6 hr (weight-drop, n = 4; adult
sham, n = 2; newborn partial transection, n = 5; newborn sham, n = 1), 12 hr
(newborn partial transection, n = 5, newborn sham,
n = 1), 1 d (transection, n = 4;
weight-drop, n = 6; adult sham, n = 1;
newborn partial transection, n = 5; newborn sham,
n = 1), 3 d (transection, n = 2;
weight-drop, n = 4), 7 d (transection,
n = 4), 10 d (transection, n = 4),
and 6 weeks (transection, n = 5; weight-drop,
n = 8) after surgery. In addition, kainic acid-treated
(see above), normal postnatal day 0 (P0) (n = 5),
normal P1 (n = 2), and normal adult (n = 5) rats were included. Spinal cords and brains were rapidly dissected and frozen on dry ice. Although our efforts were focused on the spinal
cord tissue, excluding dorsal root ganglia from quantification assessments, dorsal root ganglia served as a positive control tissue
for many of the mRNA species not normally present in the spinal cord,
such as p75. Cryostat sections (14 µm) were collected from seven to
eight levels of the spinal cords, ranging from cervical to sacral, and
with a distance of ~7 mm between adjacent levels. Sections were
thawed to slides (ProbeOn, Fisher Biotech, Pittsburgh, PA). In
situ hybridization was performed using radiolabeled
oligonucleotide probes (Dagerlind et al., 1992). Probes complementary
to GDNF (nucleotides 540-589) (Lin et al., 1993), NTN (970-1019)
(Kotzbauer et al., 1996), PSP (261-310, 383-432) (Milbrandt et al.,
1998), GFR-1 (1067-1112) (Sanicola et al., 1997), GFR-2
(1051-1094) (Baloh et al., 1997), RET (2527-2576) (Iwamoto et al.,
1993), NGF (398-447) (Whittemore et al., 1988), BDNF (250-298)
(Leibrock et al., 1989), NT3 (558-568) (Maisonpierre et al., 1990),
NT4 (549-598) (Ip et al., 1992), TrkA (942-992) (Indo et al., 1997), TrkB (2567-2617) (Middlemas et al., 1991), truncated TrkB (167-211) (Middlemas et al., 1991), TrkC (1209-1259) (Merlio et al., 1992), p75
(873-920) (Radeke et al., 1987), GFAP (1397-1446) (Feinstein et al.,
1992), and c-fos (489-538) (Curran et al., 1987) were used. All
probes, except the ones complementary to GFR-3, have been
characterized previously [GDNF (Nosrat et al., 1996); NTN and GFR-2
(Widenfalk et al., 1997); GFR-1 and RET (Nosrat et al., 1997a); NGF
and NT3 (Wetmore and Olson, 1995); BDNF (Wetmore et al., 1990); NT4
(Nosrat et al., 1997b)] and do not match any known sequence in
GenBank except those of the intended genes. A control 50-mer random
probe (Nosrat and Olson, 1995) not complementary to any sequence
deposited in GenBank was also used. After 3' end-labeling with
[35S]dATP (NEN DuPont) by terminal
deoxynucleotidyl transferase (Amersham Pharmacia Biotech, Uppsala,
Sweden), probes were purified (QIAquick Nucleotide Removal Kit
Protocol, Qiagen, Hilden, Germany). Slides were incubated overnight
(42°C) with 0.1 ml of hybridization mixture containing 50%
formamide, 4× SSC (0.15 M NaCl, 0.015 sodium
citrate, pH 7.0), 1× Denhardt's solution, 1% Sarcosyl, 0.02 M
Na3PO4, pH 7.0, 10%
dextransulfate, 0.06 M DTT, 0.1 mg/ml sheared
salmon sperm DNA, and hot probe. Slides were then rinsed four times (45 min) in 1× SSC at 60°C. During a fifth rinse in 1× SSC, the bath was allowed to adjust to room temperature. Further rinsing was performed in distilled water and increasing concentrations of ethanol.
Slides were then air dried and dipped in emulsion (Kodak NTB2, diluted
1:1 with water) or exposed for 3 weeks on x-ray films
(Hyperfilm--max, Amersham) for quantification (see below). After 6 weeks of exposure, slides were developed, counterstained with cresyl
violet, and mounted (Entellan, Merck). All hybridization was performed
under high-stringency conditions. The control probe was hybridized and
processed together with the other probes and gave rise to no specific
pattern of hybridization signals.
Detection of positive autoradiographic signals was based on serial
observations of adjacent sections from each tissue specimen, and
accumulation of silver grains in the emulsion above specific cells and
tissues was identified by the staining procedures. Only cells over
which silver grain accumulation was clearly above surrounding background levels and could be confirmed by both dark-field and bright-field high magnification were regarded as positive.
Astrocytes and oligodendrocytes were distinguished by morphology and
localization. Comparisons were made with adjacent sections hybridized
with GFAP (astrocytes) and MAG (oligodendrocytes) mRNA probes. Clear
distinction between the two glial cell types was sometimes difficult
but could be accomplished, e.g., for reactive astrocytes near lesions
and oligodendrocytes in horizontally sectioned spinal cord white
matter, where oligodendrocytes typically line up in rows.
Quantification of x-ray films was performed blindly for all probes and
treatments with specific labeling confirmed by microscopic observations
on emulsion-dipped slides. Exceptions were BDNF and p75 mRNA probes,
where the signals were restricted to a very small number of cells,
making quantification from x-ray film exposures unsuitable. BDNF and
p75 mRNA in situ hybridization signals instead were
quantified using emulsion-dipped slides by counting the number of
positively labeled cells per section. Two sections per spinal cord
level (cervical ~2 cm above lesion, thoracic immediately below the
lesion, and lumbar ~2.5 cm below the lesion) and animal were
averaged, resulting in one value per level and animal. For probes
analyzed using x-ray-films, treatment groups to be compared were
exposed together on one x-ray film, enabling reliable between-group comparisons under standardized conditions. Occasionally artifacts precluded quantification of certain sections, resulting in different numbers of animals for different probes. No group included in quantification had fewer than four individuals. Hence, sham-operated, normal P2, kainic acid-treated animals killed 3 and 7 d after treatment were not included in quantifications. Only sections clearly
devoid of artifacts were measured. X-ray films were developed and
computer scanned, and quantification of the optical density was
performed using image analysis software (NIH Image 1.62, National Institutes of Health, Bethesda, MD). Background levels were set to 0 and complete blackening of the film was set to 100, and calibration was
determined as a straight line between the two control values. The areas measured for each probe and experiment were as follows: c-fos
and GFAP in the whole cross section; NGF in the meninges of adults at
thoracic level (SCI); and the whole cross section including meninges in
newborn; NT3 and TrkA in the whole cross section including meninges in
newborn; truncated TrkB in both white and gray matter of adults and
whole cross section in newborn; TrkB and TrkC in spinal cord gray
matter (kainic acid and SCI experiments); GDNF in the meninges of
adults at thoracic level (SCI) and in the whole cross section area
including meninges in newborn; GFR-1 in spinal cord gray and white
matter in adults and spinal cord gray matter in newborn; GFR-2 in
the dorsal horns of adult spinal cords (kainic acid and SCI
experiments) and in spinal cord gray matter in newborn; and RET in
motoneurons of the ventral horns (kainic acid and SCI
experiments). Spinal cords from adult normal and spinal
cord-injured animals were analyzed at cervical, thoracic, and lumbar
levels. When measurements were to be performed in gray matter, which
was often absent at the level of the injury, a thoracic level
was chosen immediately below the level of the lesion. Kainic
acid-treated animals were quantified at the lumbar level. Spinal cords
of newborn animals were analyzed at the level of the injury (thoracic)
and below (lumbar).
Ribonuclease protection assay
Six spinal cords each were collected from normal newborn (P0)
rats, as well as 12 hr and 1 d after partial spinal cord
transection in newborn rats. Eight spinal cords each were collected
from normal adult rats, 1 d after complete transection and 6 weeks
after weight-drop injury. Spinal cords of adults and spinal cords with
adjacent parts of the vertebral column of newborn animals were rapidly dissected and frozen on dry ice. Pieces of adult spinal cord (~10 mm
long) were collected starting from the lesion center and reaching 1 cm
caudally. From neonates, 7-mm-long tissue pieces, with the lesion
located centrally and thus containing the whole spinal cord lesion,
were collected. RNA was extracted by phenol-chloroform extraction
(RNAgents Total RNA Isolation System, Promega) followed by purification
(RNeasy Mini kit, Qiagen). The ribonuclease protection assay (RPA)
protocol was according to instructions (Ambion RPAIII, modified
protocol for PharMingen Riboquant multi-probe template sets) and
[32P]-labeled probes (Riboprobe in vitro
Transcription System, Promega). Equal amounts of total RNA (6 µg)
were hybridized, using probes complementary to rat NGF, BDNF, GDNF,
CNTF, NT3, NT4, L32, and GADPH (Rat multi-probe template set rNT-1,
Riboquant, PharMingen). Protected cRNA fragments were separated by
denaturing 5% polyacrylamide gel electrophoresis. X-ray films were
exposed to the gels overnight (for pictures) and to phosphoimager
plates for 3 hr (for quantifications). Quantification using computer
software connected to a phosphoimaging system (IBAS3000, BAS reader,
Raytest Isotopenmessgeräte) was performed by measuring the
average radioactivity per square millimeter for each band. The
housekeeping gene L32 was used as an internal standard, ensuring RNA
integrity and equal RNA loading.
Immunohistochemistry and counterstaining
Normal adult animals (n = 8), weight-drop
injured (1 week, n = 8; 6 weeks, n = 8;
6 months n = 1) animals, and adult animals subjected to
partial spinal cord transection as newborn (n = 3) were
anesthetized deeply by intraperitoneal injections of pentobarbital and
intracardially perfused with 50 ml of Tyrode's solution, containing 0.1 ml of 5000 IOU/ml heparin (Løvens), followed by 200 ml
fixative (4% paraformaldehyde and 0.4% picric acid in PBS). Spinal
cords were carefully dissected and post-fixed for 1 hr in fixative (4% paraformaldehyde and 0.4% picric acid in PBS), rinsed several times in
10% sucrose in PBS, and stored at 4°C until sectioned. Cryostat
sections (14 µm thick) were taken at seven to eight spinal cord
levels with 7 mm intervals, and primary antibodies were applied overnight at room temperature. The primary antibodies that were used
are listed in Table 2. Secondary antisera
were conjugated with FITC or rhodamine (Jackson ImmunoResearch) and
usually diluted 1:50. For each spinal cord and antibody, ~100
sections collected from cervical to lumbar level and mounted on five to
seven slides, were microscopically examined regarding immunoreactivity.
Control slides treated with secondary antibodies were examined in
parallel. Double-labeling with GFAP and GFR-1 antibodies was used to
confirm astrocyte labeling.
Quantification of the intensity of GFR-1 immunoreactivity was
performed on a blind basis using computer image analysis equipment (Nikon, Microphot-FXA; ZVS-47EC CCD video camera). The dorsal roots of
16 weight-drop-injured (1 week, n = 8; 2 weeks,
n = 8) and 8 normal animals were included. The
weight-drop-injured animals were analyzed at two levels: the cervical
level and the level of the injury. The intensities of GFR-1
immunoreactivity in one cervical and one thoracic dorsal root were
measured in two sections for each animal and averaged, resulting in one
value per animal and level. When GFR-1 immunoreactivity was
quantified in spinal cord white matter of cervical dorsal funiculi,
normal, degenerating (the portion hosting ascending sensory fibers),
and nondegenerating white matter (the portion hosting descending fibers
such as the corticospinal tract) of the cervical dorsal funiculi were
measured separately in two sections per animal, yielding one value for each tissue and animal. Comparisons were made between dorsal roots at
cervical and thoracic levels 1 week after injury, and
normal/degenerating/nondegenerating spinal cord white matter tissue at
cervical levels 6 weeks after injury.
Behavior tests
Behavior analyses of adult animals subjected to a partial spinal
cord transection as newborns were performed by BBB scoring (Basso et al., 1995) and a grid pathway test (Grill et al., 1997). The
BBB score for evaluation of hindlimb function after SCI ranges from 0, which corresponds to flaccid paralysis, to 21, which is normal gait.
Animals were allowed to walk around freely in a circular field (1.2 m
in diameter) for 4 min while movements of the hindlimbs were closely
observed. Ranking according to the scoring system described by Basso
and colleagues (1995) includes frequency and quality of hindlimb
movement as well as forelimb/hindlimb coordination. In the grid pathway
test, animals are allowed to walk three times voluntarily across a
1.2-m-long grid pathway. The number of errors (hindlimbs stepping
through the grid) was counted for each crossing. The three values were
averaged and resulted in one value per animal. Four adult animals,
which were partially spinal cord transected as neonates, were examined
with respect to their hindlimb function 6 months after injury. For
comparison in the grid pathway test, 20 normal animals were also
examined. Six months after injury, animals were tested on two occasions
2 d apart. Although both sessions gave rise to similar results,
the statistics of only one session are presented in Results.
Statistical analyses and image processing
Statistical analyses used for comparison of different in
situ hybridization probe signals and RPA autoradiograms included factorial ANOVA followed by a Fisher's post hoc test. Data
from the grid pathway test were analyzed using an unpaired t
test (confidence interval 95%). For statistical analyses of the
GFR-1 immunohistochemistry, an unpaired t test
(confidence interval 95%) and ANOVA followed by a Fisher's post
hoc test were used. Significance levels were defined as follows:
*p < 0.05; **p < 0.01;
***p < 0.001. Photomicrographs were scanned, digitally
processed, and compiled using computer image software. Occasional
particles of dust and other obvious artifacts were digitally retouched.
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RESULTS |
Mechanical injury of rat spinal cord
Lesion characteristics
Adult. Complete transections lead to severe damage of
the spinal cord restricted to the level of injury, whereas weight-drop injuries lead to the characteristic egg-shaped zone of necrosis (Fig.
1A) extending several
spinal cord segments rostrocaudally. One important difference between
the two injury models is that in weight-drop injuries the dura is left
mechanically intact, whereas in the transection model the dura is cut,
exposing the spinal cord. Cutting the dura has severe consequences for
the cerebrospinal flow and may aggravate secondary degenerative
processes. It could also induce repair responses in meningeal cells
other than those seen after contusion injury.
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Figure 1.
Dark-field photomicrograph depicting GFAP mRNA
in situ hybridization signals in normal and
weight-drop-injured adult spinal cord. A, Horizontal
section showing the lesion area 1 d after injury. The primary
lesion zone with the shape of an ellipse is characterized by
markedly reduced mRNA synthesis, whereas cells surrounding the lesion
are strongly positive for the GFAP mRNA probe. B,
E, H, GFAP mRNA hybridization signals in
normal uninjured spinal cord at cervical, thoracic, and lumbar levels.
C, F, I, One day after
SCI, GFAP mRNA signals have increased at the thoracic level close to
the lesion (F) but also at cervical
(C) and lumbar levels
(I). Note increased signals in cells
around the central canal at cervical and lumbar levels.
D, G, J, Six weeks after
injury, strong GFAP mRNA expression is present in degenerated white
matter, especially at the level of injury (G).
Degenerating white matter fiber tracts, such as the ascending sensory
tract in the dorsal funiculus above the lesion
(D), and the descending corticospinal tract below
the lesion in the dorsal funiculus (J), are also
strongly labeled. Scale bar (shown in B):
A, 650 µm; B-J, 1 mm.
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In the necrosis zone of contused animals, mRNA signals were generally
lost because of absence of living spinal cord neurons and glia (Fig.
1A). This loss of signal was seen for all
investigated neurotrophic factor and neurotrophic factor receptor mRNA
species and is not described below for each individual mRNA species.
Likewise, the absence of hybridization signals in the lesion area after transections caused by necrosis is not detailed below. The astroglial response, monitored by probes for GFAP mRNA after weight-drop and
transection injury, is shown in Figure 1. GFAP mRNA hybridization signals increased gradually up to the latest time point investigated, 6 weeks after injury (Fig. 1B-J)
(quantification data not shown), and the upregulation was observed
primarily in degenerating fiber tracts at cervical, thoracic, and
lumbar levels (Fig.
1D,G,J). The mRNA
expression of the immediate-early gene c-fos was also investigated to
further characterize the glial and neuronal response to injury. In
normal intact spinal cord, c-fos mRNA was restricted to a few cells in
the dorsal root entry zone. After mechanical SCI, c-fos mRNA signals
were upregulated at all times investigated, the strongest for
weight-drop injury being 1 d after injury (Figs. 2A,B,
3A,F,K).
One day after injury, strong c-fos mRNA signals were found close to the
injury in glial cells, primarily astrocytes, and in neurons. Strong
c-fos labeling was also observed in ependymal cells of the central
canal. At cervical levels 1 d after weight-drop injury,
c-fos-labeled neurons were found in laminas VII-VIII and at
lumbar levels IV-VII and X. Six weeks after injury, glial cells, primarily astrocytes, and neurons close to the lesion expressed low
levels of c-fos mRNA. At cervical and lumbar levels 6 weeks after
injury only low levels of c-fos were found, restricted to a few cells
located in the same laminas as 1 d after injury.
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Figure 2.
Alterations of c-fos mRNA expression in response
to SCI and kainic acid delivery. A, Two hours after
weight-drop injury of the adult spinal cord, glial cells and neurons
present strong c-fos mRNA signals close to the lesion.
B, Six weeks after injury, glial cells and some neurons
close to the injury are still positively labeled by the c-fos probe.
C, A weak c-fos mRNA expression is found in newborn
(P0) spinal cord. D, One day after
partial transection of the newborn spinal cord (SCI 1 d), robust c-fos mRNA upregulations occur primarily in cells of
the gray matter and in nerve roots. E, In normal adult
rat spinal cord, very low levels of c-fos mRNA were found.
F, One day after kainic acid delivery, scattered neurons
in Rexed's laminas III-VII present strong c-fos mRNA signals.
G, In a normal brain (the brain of the animal depicted
in E), no robust c-fos mRNA signals can be found.
H, One day after kainic acid delivery (the brain of the
animal depicted in F), dramatic c-fos
hybridization signals appear in areas such as the neocortex, the
hippocampal formation, and amygdala. Scale bar (shown in
A): A, B,
G, H, 1 mm; C,
D, 2 mm; E, F, 300 µm.
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Figure 3.
Regulation of different mRNA species in the spinal
cord after weight-drop injury as monitored by in situ
hybridization. Quantifications of c-fos, truncated TrkB, and GFR-1
were performed by measuring the relative optical density on x-ray films
exposed to hybridized sections. Quantifications of BDNF and p75 were
performed by counting the number of positively labeled cells per
section. All statistical comparisons were made to normal animals
(N). A, At the cervical
level c-fos was significantly increased at 2 hr and 1 d after
injury (F(4,20) = 4.9;
p < 0.01). B, Significant increase
of truncated TrkB in white matter at cervical levels was found 6 weeks
after surgery (F(3,18) = 48;
p < 0.001) C, At the cervical level
significant upregulation of GFR-1 mRNA in white matter was found 6 weeks after injury (F(4,21) = 3.3;
p < 0.05). D, The number of BDNF
mRNA-labeled cells was significantly increased 6 hr after injury
(F(4,24) = 3.3;
p < 0.05). E, There was an increase
in the number of p75 cells 6 hr, 1 d, and 6 weeks after injury at
the cervical level, but the difference compared with normal was not
statistically significant. F, At the thoracic level
c-fos was significantly increased at 2 hr, 6 hr, and 1 d after
injury (F(4,20) = 6.6;
p < 0.01). G, A significant
increase of truncated TrkB in white matter at the thoracic level was
found 6 weeks after surgery (F(3,18) = 108; p < 0.001). H, At the thoracic
level significant upregulation of GFR-1 mRNA in white matter was
found 6 weeks after injury (F(4,21) = 28; p < 0.001). I, The number of
BDNF mRNA-labeled cells was significantly increased at the thoracic
level 2 hr, 6 hr, and 1 d after injury
(F(4,24) = 11; p < 0.001). J, The number of p75 probe-labeled cells was
elevated at 6 hr, 1 d, and 6 weeks after injury at the thoracic
level. The difference was significant after 1 d
(F(3,17) = 21; p < 0.001). K, c-fos mRNA expression levels were
significantly increased 2 hr, 6 hr, and 1 d after injury
(F(4,20) = 5.1; p < 0.01). L, A significant increase of truncated TrkB in
white matter at lumbar levels was found 6 weeks after surgery
(F(3,18) = 54; p < 0.001). M, At the lumbar level significant upregulation
of GFR-1 mRNA in white matter was found 6 weeks after injury
(F(4,21) = 14; p < 0.001). N, The number of BDNF mRNA-labeled cells was
significantly increased at the lumbar level 6 hr after injury
(F(4,30) = 3.1; p < 0.05). O, The number of p75 mRNA-labeled cells was
elevated at 6 hr, 1 d, and 6 weeks after injury at the thoracic
level. The increase was significant after 1 d
(F(3,17) = 8; p < 0.01).
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Newborn. Partial transection injuries penetrating the dura
in newborn rat spinal cord led to severe local injury. Moderate increases of GFAP mRNA (Fig.
4A-F)
and strong increases of c-fos mRNA (Fig. 2C,D)
were observed 1 d after the operation. In the necrosis zone, mRNA
signals were generally lost because of the absence of living spinal
cord cells (Fig. 4D). This loss of signal was seen
for all investigated neurotrophic factor and neurotrophic factor
receptor mRNA species and is not described below for each individual
mRNA species. A few of the animals that were allowed to grow to
adulthood were investigated regarding behavior and general histology.
Compared with normal uninjured controls, animals receiving a partial
transection as newborns showed only minor behavior differences. No
difference could be found using the BBB score for hindlimb function;
all animals scored the highest possible value, 21. However, when we
used the grid pathway test used previously to assess corticospinal
tract regeneration functionally (Grill et al., 1997), animals receiving
a partial transection as newborns were found to make a higher number of
errors, 2.83 ± 0.30 (mean ± SEM), compared with controls
0.97 ± 0.15 (unpaired t test; p < 0.001; df = 22; t = 5.2). Spinal cords of treated
animals displayed a markedly decreased cross-section area at the level
of injury compared with normal spinal cords. The white matter of the
lesioned side was reduced at the level of the lesion and several
millimeters above and below (Fig. 5).
Examination of 5HT- and TH-positive fibers above and below lesion
revealed no gross anatomical disturbances of those systems. The dorsal
corticospinal tract can be labeled by GAP-43 and CAM kinaseII in the
normal intact adult rat spinal cord (Terashima, 1995). In animals
receiving a partial transection as newborns and studied in adulthood,
the dorsal corticospinal tract exhibited GAP-43 and CAM kinaseII
immunoreactivity above the lesion. Below the lesion these two
immunoreactivities were lacking in the expected position, supporting an
altered development of the corticospinal tract in neonatally injured
rats as shown previously for this kind of lesion (Bernstein and
Stelzner, 1983).
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Figure 4.
Dark-field photomicrograph depicting GFAP mRNA
in situ hybridization signals in normal and injured
neonatal spinal cord. A, C,
E, GFAP mRNA hybridization signals in normal uninjured
neonatal spinal cord. B, D,
F, One day after injury GFAP mRNA expression is
increased in tissue close to the lesion (B,
D) and slightly increased in ventral white matter at the
lumbar level (F). Scale bar (shown in
A): A, B, 600 µm;
C-F, 300 µm.
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Figure 5.
Appearance of the adult spinal cord of neonatally
injured animal. Five levels, cervical to lumbar, of a spinal cord
collected from an adult individual that underwent a partial spinal cord
transection as newborn. A cut spanning approximately two-thirds
of the width of the thoracic spinal cord beginning from the right side
was carried out on the first postnatal day, a time point when long
nerve fiber tracts, such as the corticospinal tract, are developing.
The white matter of the cut side is reduced in size at all levels, but
most prominently at thoracic levels close to the injury. This animal
scored 21 (= normal) on the BBB score as an adult. Luxol fast blue was
used for staining. Scale bar, 1 mm.
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Neurotrophins and related receptors
TrkA. TrkA mRNA hybridization signals were not found in
normal uninjured adult spinal cord. A subpopulation of dorsal root ganglia neurons was positively labeled in adults (Fig.
6D) as well asin
neonates (Fig. 7B). No robust
upregulations could be detected in the spinal cord white or gray matter
at any time point after SCI in either neonates or adults.
However, in neonatal dorsal roots, TrkA mRNA signals were upregulated
on the affected side 6, 12, and 24 hr after injury (data not
shown).
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Figure 6.
Expression of mRNA encoding neurotrophic factor
receptors in the intact normal spinal cord of the adult rat.
A, RET mRNA signals are seen in motoneurons.
B, p75 mRNA signals were not detectable in spinal cord
gray or white matter but were detectable in a subpopulation of dorsal
root ganglia neurons, which is shown here in a horizontal section of
cervical spinal cord. C, GFR-1 mRNA signals are
present in motoneurons and interneurons. D, TrkA mRNA
expression was not found in spinal cord gray or white matter but was
found in a subpopulation of dorsal root ganglia neurons, which is shown
here in a horizontal section of cervical spinal cord (section
adjacent to B). E, GFR-2 mRNA
signals are seen in the dorsal horns of gray matter and in a
subpopulation of dorsal root ganglia neurons. F, TrkB
mRNA synthesis occurs throughout the gray matter. Scale bar (shown in
A): A-F, 700 µm.
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Figure 7.
Expression of mRNA encoding neurotrophic factor
receptors in the intact normal spinal cord of newborn rat.
A, GFR-1 mRNA signals are found throughout the gray
matter, with motoneurons displaying particularly strong signals, and in
a subpopulation of dorsal root ganglia neurons. B, TrkA
mRNA expression is present in dorsal root ganglia neurons.
C, GFR-2 mRNA signals are found throughout the gray
matter and in a subpopulation of dorsal root ganglia neurons.
D, TrkB mRNA synthesis occurs throughout the gray matter
and in some dorsal root ganglia neurons. E, RET mRNA
signals were confined to motoneurons and a subpopulation of dorsal root
ganglia neurons. F, The probe directed against truncated
TrkB plus full-length TrkB-labeled cells in dorsal root ganglia and
spinal cord gray and white matter. G, p75 mRNA signals
are seen in motoneurons and many dorsal root ganglia neurons.
H, TrkC mRNA expression is present throughout gray
matter and in many dorsal root ganglia neurons. Scale bar (shown in
A): A-H, 170 µm.
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TrkB. TrkB mRNA in spinal cords of normal uninjured adult
(Fig. 6F) and newborn (Fig. 7D) animals
was present in most neurons of the gray matter throughout the spinal
cord and in a subpopulation of dorsal root ganglia neurons. Expression
patterns were in agreement with previous findings (Klein et al., 1990;
Ernfors et al., 1992). No robust alteration of full-length TrkB message
could be detected in the spinal cord at any time point after injury in
either adult or newborn animals.
Truncated TrkB. Truncated TrkB mRNA signals, defined as
signals seen in areas where the full-length TrkB probe was negative, were fairly weak and found in white matter tissue of the normal uninjured adult spinal cord in agreement with Frisén et al.
(1992). Upregulation after SCI was found 6 hr after surgery, and
increased expression continued up to the latest time point
investigated, which was 6 weeks after injury. Figure
8 depicts truncated TrkB mRNA
hybridization signals in normal spinal cord and at selected time points
after injury. Six weeks after injury a strong truncated TrkB mRNA
hybridization signal was present in degenerating fiber tracts of the
white matter along the whole length of the spinal cord (Fig.
8C,F,I). For
instance, the ascending fiber tract of the dorsal funiculus was
strongly positive above the lesion (Fig. 8C) and weakly
labeled below (Fig. 8I). The expression pattern of
truncated TrkB resembles the distribution of GFAP mRNA expression (see
above) or vimentin expression (data not shown) at similar time points,
suggesting that cells were mainly reactive astrocytes (Eddleston and
Mucke, 1993). No specific robust truncated TrkB signals could be found
in oligodendrocytes, although we cannot rule out the possibility
that some oligodendrocytes were positive. Close to weight-drop
injury or transection injury signals were strong in both gray and white
matter. The upregulations were statistically significant in both white
and gray matter, although much more prominent in white matter (Fig.
3B,G,L). Microscopic
observations suggest that the increase was confined to glial cells,
although we cannot rule out a minor increase of truncated TrkB mRNA
expression in neurons. No robust truncated TrkB mRNA labeling could be
found in the scar tissue 6 weeks after injury (Fig.
8J). Ependymal cells of the central canal were
positively labeled (Fig. 8J). In normal newborn
animals, truncated TrkB mRNA signals were present in cells of the white
matter (Fig. 7F). No robust upregulation could be found during the postoperative period investigated.
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Figure 8.
Truncated TrkB and p75 mRNA are both upregulated
after injury to the adult spinal cord. Hybridization was performed with
a probe that recognizes both truncated and full-length TrkB. The
expression of truncated TrkB was deduced from comparisons with the
expression pattern of the full-length-specific TrkB probe. No robust
increases in hybridization signals could be detected when using a probe
only complementary to full-length TrkB. A,
D, G, The expression of full-length TrkB
and truncated TrkB mRNAs in normal uninjured spinal cord is found in
neurons of the gray matter and glial cells, respectively.
B, E, H, One day after
injury glial cells near the injury upregulated truncated TrkB mRNA
signals (E). The truncated TrkB mRNA expression
is also increased to some extent at cervical and lumbar levels
(B, H). C,
F, I, Six weeks after injury, white
matter tissue at the injury level is strongly positive for truncated
TrkB probe (F). In addition, the truncated TrkB
mRNA
expression is markedly increased in cells of
degenerated fiber tracts, such as the ascending sensory tract in the
dorsal funiculus above the lesion (C), and the
descending corticospinal tract below the lesion in the dorsal funiculus
(I). J, Horizontal section
of a spinal cord 6 weeks after complete transection. Cells strongly
positive for the truncated TrkB probe are found in both gray and white
matter tissue but not in the glial scar separating the rostral and
caudal stumps. Ependymal cells of the central canal are also strongly
positive, as seen in the rostral stump (left) along the
midline and reaching the glial scar in the middle. K,
Bright-field photomicrograph showing strong truncated TrkB mRNA signals
in a subpopulation of cells in the lateral funiculus of lumbar white
matter 6 weeks after the operation. L, Neurons close to
the lesion 1 d after injury labeled by p75 mRNA hybridization.
Scale bar (shown in A):
A-I, 1 mm; J, 300 µm;
K, L, 25 µm.
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TrkC. TrkC mRNA hybridization signals in normal uninjured
adult as well as newborn (Fig. 7H) spinal cord were
found in motoneurons and most other neurons in gray matter and a
subpopulation of dorsal root ganglia neurons, in agreement with
previous studies (Ernfors et al., 1992; Merlio et al., 1992; Elkabes et
al., 1994). No robust upregulation of TrkC mRNA could be detected in
the spinal cord at any time point after injury.
p75. p75 mRNA expression could not be detected in normal
uninjured adult spinal cord, in agreement with previous findings (Ernfors et al., 1989). A subpopulation of adult dorsal root ganglia neurons was positive (Fig. 6B). After both
weight-drop and transection injury, p75 mRNA hybridization signals
upregulated in neurons, especially in close proximity to the lesion,
where most neurons including motoneurons were labeled, but also at
cervical and lumbar levels (Figs.
3E,J,O,
8L). Positively labeled neurons at a distance from
the injury were present in Rexed's laminas V-IX, varying with time
after injury and level. Most neurons were found in lamina VII. No
robust upregulation of p75 mRNA could be found in pyramidal layer V
cells of the cerebral hindlimb motor cortex after injury in adult
animals (data not shown). In newborns, p75 mRNA signals were found in
ventral horns and a subpopulation of dorsal root ganglia neurons (Fig.
7G). No change in expression levels was found in newborns in
response to injury.
BDNF. BDNF mRNA expression in normal uninjured adult spinal
cord was detected only in a few neurons of gray matter restricted to
Rexed's lamina VII, in good agreement with previous findings (Conner
et al., 1997). Counting of positively labeled neurons revealed an
increase in the number of positively labeled cells in spinal cord gray
matter of injured animals (Fig.
3D,I,N). The BDNF
mRNA signals after injury were found in laminas IV, V, and VII. RPA
assays demonstrated an increase in BDNF mRNA in the adult spinal cord
1 d, but not 6 weeks, after injury (Figs.
9I,
10B). In neonatal
spinal cord, weak BDNF mRNA signals were found in some gray matter
cells. A subpopulation of dorsal root ganglia cells of both neonatal
and adult animals was strongly positive (data not shown), in agreement
with previous findings (Ernfors et al., 1992). Partial transection of
the spinal cord in newborns led to no robust alteration of BDNF mRNA in
the spinal cord tissue at the level of the lesion or below (Fig.
10A).
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Figure 9.
Regulation of different mRNA species after SCI
measured by RPA (A, B, E,
F, I, J) and
in situ hybridization (C,
D, G, H, K,
L). Bars in RPA graphs represent average
radioactivity per area (detected by phosphoimaging) expressed in
arbitrary units and displayed as percentage of the housekeeping gene
L32. A, GDNF mRNA was increased at the level of injury
1 d after transection (T), but not 6 weeks
after weight-drop (WD) injury
(F(2,30) = 8; p < 0.01). B, GDNF mRNA expression levels were significantly
elevated at the level of injury 1 d after partial transection of
the neonatal spinal cord measured by RPA
(F(2,15) = 4.3; p < 0.05). C, Quantification of in situ
hybridization demonstrated increased GDNF mRNA expression at the level
of the injury, 6 hr, 12 hr, and 1 d after injury.
D, No significant difference in GDNF mRNA expression
could be found at the lumbar level. E, NGF mRNA probe
signals were upregulated 1 d after transection, but not 6 weeks
after weight-drop injury (F(2,30) = 11;
p < 0.001). F, NGF mRNA expression
levels were significantly elevated at the level of injury 12 hr and
1 d after partial transection of neonatal spinal cord
(F(2,15) = 4.3; p < 0.05). G, NGF mRNA expression increased at the
thoracic level 6 hr, 12 hr, and 1 d after injury.
H, No significant difference in NGF mRNA expression
could be found at the lumbar level. I, BDNF mRNA probe
signals were upregulated 1 d after transection but not 6 weeks
after weight-drop injury (F(2,30) = 9;
p < 0.001). J, No statistically
significant differences in NT3 mRNA expression were found after injury.
K, No statistically significant differences were found
in truncated TrkB mRNA expression at the thoracic level after injury.
L, Likewise, no statistically significant differences
were found in truncated TrkB mRNA expression at the lumbar level.
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Figure 10.
Neurotrophic factor expression in intact and
injured adult and neonatal spinal cord. Representative autoradiograms
from RPAs are shown. A riboprobe complementary to the housekeeping gene
L32 was used as an internal standard. A, At the newborn
stage, NGF and GDNF mRNA signals increased. B, GDNF,
NGF, and BDNF signals were upregulated 1 d after transection in
adults. C, CNTF mRNA levels were upregulated 1 d
after kainic acid delivery.
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NGF. NGF mRNA in situ hybridization signals could
not be detected in normal adult spinal cord (Fig.
11A), correlating
well with previous findings (Korsching and Thoenen, 1985). With use of
RPA, weak signals were found (Fig. 10B). After
injury, weight-drop as well as transection, meningeal cells and some
Schwann cells of nerve roots exhibited strong NGF mRNA signals (Fig.
11B,G). Positively labeled cells
were primarily found in arachnoidea and seen 6 hr, 1 d, and 3 d after injury, peaking at 1 d. RPAs confirmed the in
situ hybridization findings, with upregulated levels of NGF mRNA
1 d, but not 6 weeks, after injury (Figs. 9E,
10B). At the neonatal stage, neurons of the lateral
horn presented moderate levels of NGF mRNA (Fig.
12A,D),
and an area located centrally, close to the midline, presented low
levels. Meningeal cells and white matter were devoid of
significant signals (Fig. 12A,D).
After injury of the newborn spinal cord, strong NGF mRNA signals were found in the meningeal cells around the site of the lesion (Fig. 12B,C,H,I).
Most positively labeled cells resided in arachnoidea. Strong NGF mRNA
labeling in the meningeal cells was detected 6 hr, 12 hr, and 1 d
after injury (Fig. 9G). Significant increases were also
detected by RPA quantification (Fig. 9F,
10A). No robust alterations of NGF mRNA signals
could be detected in gray or white matter above or below the lesion
(Figs. 9H,
12E,F).
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Figure 11.
GDNF and NGF mRNA expression is increased after
injury of the adult spinal cord. A, No robust NGF mRNA
signals were found in normal uninjured spinal cord. B,
Six hours after weight-drop injury, strong NGF mRNA signals were found
in meningeal and Schwann cells close to the lesion. C,
In normal adult spinal cord, no robust GDNF mRNA expression could be
detected. D, Six hours after weight-drop injury, a
marked upregulation of GDNF mRNA signals was found in meningeal and
Schwann cells at the level of the lesion. Weak GDNF mRNA signals were
observed in glial cells, presumably astrocytes, in the immediate lesion
vicinity (arrow). E, Horizontal section
of a thoracic dorsal root in a normal animal. No specific GDNF mRNA
labeling is observable. F, Six hours after weight-drop
injury, intense GDNF mRNA labeling was found in dorsal root Schwann
cells at the lesion level. G, Bright-field images
depicting meningeal cells expressing NGF mRNA 6 hr after injury.
H, GDNF mRNA signals in cells of the dorsal meninges 6 hr after injury. Scale bar (shown in A): A-D, 400 µm;
E, F, 250 µm; G,
H, 60 µm.
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Figure 12.
NGF mRNA hybridization signals increase after
partial transection injury to the neonatal spinal cord.
A, D, NGF mRNA expression in normal
uninjured neonatal (P0) spinal cord is present in cells
of the gray matter lateral horn (A, D).
B, C, E, F,
Six and 12 hr after injury, NGF mRNA synthesis is markedly increased in
meningeal cells around the lesion. Schwann cells of dorsal roots on the
transected side (left side) also exhibit strong NGF mRNA
signals, whereas the dorsal roots of the opposite side are devoid of
signal or only weakly positive (B, C). At
lumbar levels no altered expression of NGF mRNA could be found
(E, F). G, Sagittal
section of normal uninjured neonatal spinal cord. No robust NGF mRNA
signals are seen in meningeal cells. H, Sagittal section
of neonatal spinal cord 1 d after injury exhibiting strong NGF
mRNA expression in meningeal cells close to the lesion.
I, Bright-field view depicting NGF probe-labeled
meningeal cells close to the lesion. Scale bar (shown in
A): A-F, 250 µm;
G, H, 400 µm; I, 40 µm.
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NT3. NT3 mRNA hybridization signals could not be detected by
in situ hybridization of either normal or injured adult
spinal cord. In good agreement with previous findings (Ernfors and
Persson, 1991; Ernfors et al., 1992), the neonatal spinal cord
exhibited moderate NT3 expression signals in cells of the ventral horn
(data not shown). RPAs did not detect any statistically significant alterations of NT3 mRNA after SCI (Figs. 9J,
10A,B).
NT4. NT4 mRNA hybridization signals could not be detected by
in situ hybridization of either adult or neonatal spinal
cord tissue. Measured by RPA, only very low NT4 mRNA levels were
detected in adult and neonatal spinal cord (Fig.
10A,B), which is in agreement with
previous findings (Timmusk et al., 1993). No statistically significant
alterations of NT4 mRNA after SCI could be detected by RPAs.
GDNF family ligands and related receptors
RET. RET mRNA expression was observed in motoneurons of
normal adult (Fig. 6A) and neonatal spinal cord and a
subpopulation of dorsal root ganglia cells (Fig. 7E), in
agreement with previous findings (Nosrat et al., 1997a; Golden et al.,
1999; Leitner et al., 1999; Widenfalk et al., 1999). No change of RET
mRNA levels could be detected after injury.
GFR-1. GFR-1 mRNA signals were found in most neurons
of the spinal cord gray matter and a subpopulation of dorsal root
ganglia cells in adults as well as newborns (Figs. 6C,
7A). Motoneurons in the ventral horn were strongly positive.
These findings were in agreement with previous studies (Nosrat et al.,
1997a; Golden et al., 1999; Leitner et al., 1999). Beginning 1 d
after injury, GFR-1 mRNA levels were upregulated in dorsal roots at
the lesion level. Three days after injury, weak GFR-1 mRNA signals
were found in glial cells close to the lesion. In adult spinal cord 6 weeks after impact injury, a few cells of the scar tissue were GFR-1
mRNA positive, suggesting that they might be Schwann cells. In
addition, moderate GFR-1 mRNA hybridization signals were found in
degenerating white matter, presumably in astrocytes (Figs. 3C,H,M,
13A). There was no
statistically significant increase in gray matter tissue.
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Figure 13.
GFR-1 protein immunoreactivity is increased in
nerve roots and degenerating white matter after SCI. A,
Dark-field photomicrograph depicting GFR-1 in situ
hybridization signals in degenerating white matter, close to the
lesion, 6 weeks after weight-drop. B, GFR-1
immunostaining in a dorsal horn and nerve root in a normal animal.
Robust immunoreactivity is seen in lamina II of the dorsal horn. Other
laminas of the gray matter are weakly positive, and in the nerve root
the GFR-1 antibody labels some nerve fibers and possibly weakly
Schwann cells. C, GFR-1 immunoreactivity in a
cervical dorsal root 6 weeks after weight-drop injury. Some nerve
fibers are positive, and a weak staining is possibly also present in
Schwann cells. D, One week after injury a dramatic
increase in GFR-1 immunoreactivity is seen in Schwann cells of a
dorsal root immediately caudal to the weight-drop site (thoracic
level). Meningeal cells are also GFR-1 positive. E,
Increased GFR-1 immunoreactivity (green
fluorescence), was found in degenerating white matter of the ascending
fiber tract in the dorsal funiculus 6 weeks after weight-drop injury.
Autofluorescence in infiltrating immune cells
(orange-yellow fluorescence) is also seen. Note that no
robust immunolabeling is seen in the white matter adjacent to the
ascending portion of the dorsal funiculus. F, Six weeks
after injury, robust GFR-1 antibody immunoreactivity
(green) is observed in the remaining white matter
at the level of injury. Numerous immune cells/phagocytes are present,
displaying an orange-yellow autofluorescence. Scale bar
(shown in A): A, 200 µm;
B, 400 µm; C-F, 100 µm.
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The GFR-1 antibody was tested on P0 rat kidney, and GFR-1
immunoreactivity correlated well with previous studies of GFR-1 mRNA
expression in P0 kidney (Nosrat et al., 1997a; Golden et al., 1999). In
the spinal cord, the most prominent GFR-1 immunoreactivity was found
in lamina II of the dorsal horn, labeling axon terminals of sensory
neurons. The rest of the spinal cord gray matter was only weakly
labeled (Fig. 13B). In dorsal root ganglia, a subpopulation of neurons was GFR-1 positive, correlating well with previous studies of GFR-1 mRNA (Bennett et al., 1998). The most striking difference 1 and 6 weeks after injury was an increased GFR-1 immunoreactivity in nerve roots at the level of the lesion (Figs. 13D, 14A).
Six weeks after injury an increased GFR-1 immunolabeling was found
in degenerating fiber tracts of the spinal cord white matter (Figs.
13E,F, 14B),
correlating well with in situ hybridization data. No
specific GFR-1 immunostaining could be found in nondegenerating white matter. Additionally, a few cells in the scar tissue 6 weeks after injury were moderately labeled. Six months after injury the
GFR-1 immunostaining of degenerated white matter still persisted but
had become somewhat weaker.
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Figure 14.
GFR-1 protein levels increase in injured nerve
roots and degenerating white matter of the spinal cord.
A, The intensity of GFR-1 immunoreactivity in
thoracic nerve roots in normal animals
(N) and animals subjected to SCI 1 week
earlier (WD 1 w). A marked increase is observed in
injured nerve roots (unpaired t test; df = 14;
t = 8.6; p < 0.001).
B, An increase in GFR-1 protein levels was also found
in degenerating white matter of the spinal cord 6 weeks after injury
(ANOVA; F(2,21) = 11;
p < 0.001). Measurements were performed in intact
white matter in normal animals (N), in
nondegenerated white matter in the dorsal funiculus at the cervical
level 6 weeks after injury (WD 6 w
non-deg), and in degenerated white matter in the dorsal
funiculus at the cervical level 6 weeks after injury (WD 6 w deg).
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GFR-2 mRNA expression was detected in adults as weak labeling of
laminas I, III, and IV of the dorsal horns and strong hybridization signals in a subpopulation of dorsal root ganglia cells (Fig. 6E). The spinal cords of uninjured newborn animals
exhibited a weak GFR-2 mRNA hybridization labeling throughout the
gray matter and in a subpopulation of dorsal root ganglia cells (Fig.
7C). Expression patterns were in agreement with previous
findings (Widenfalk et al., 1997; Golden et al., 1999; Leitner et al.,
1999). SCI led to no robust alteration in GFR-2 mRNA expression in
either adult or newborn rats.
GDNF. GDNF mRNA hybridization signals could not be detected
by in situ hybridization in normal adult spinal cord (Fig.
11C,E). After injury strong upregulation of GDNF
mRNA occurred in the meninges (Fig.
11E,H) and Schwann cells of
nerve roots (Fig. 11F). The time course of this
increased expression resembled that for NGF, starting 2 hr after injury
and continuing up to 3 d after injury. Analyses with RPA
demonstrated increased GDNF mRNA levels 1 d, but not 6 weeks,
after injury (Figs. 9A, 10B).
In neonatal spinal cord, strong GDNF mRNA signals were found in cells
located dorsolateral to the central canal, presumably Clark's nucleus
(thoracic nucleus), and weak signals were noted in the dorsal horns
(Fig.
15A,D),
as reported previously (Nosrat et al., 1996; Widenfalk et al., 1999).
After partial transection of neonatal spinal cord, a marked
upregulation was seen in meningeal cells in the lesion vicinity (Fig.
15B,C,I). Similar
to the upregulation of NGF mRNA, most GDNF-positive cells were found in
the arachnoid. GDNF mRNA in situ hybridization signals were
upregulated 6 hr, 12 hr, and 1 d after injury (Fig.
9C). No robust alteration could be found in gray or white
matter of the spinal cord below the lesion (Figs. 9D,
15E,F). RPAs revealed
that the upregulation was statistically significant 12 hr after the
operation (Figs. 9B, 10A).
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Figure 15.
GDNF mRNA hybridization signals increase after
partial transection injury to the neonatal spinal cord.
A, D, Normal uninjured neonatal
(P0) spinal cord exhibits strong |
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ABSTRACT
INTRODUCTION
