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The Journal of Neuroscience, September 1, 2001, 21(17):6617-6625
Tumor Necrosis Factor Receptor Deletion Reduces Nuclear
Factor- B Activation, Cellular Inhibitor of Apoptosis Protein 2 Expression, and Functional Recovery after Traumatic Spinal Cord
Injury
Gyeong-Moon
Kim1,
Jan
Xu1,
Jinming
Xu1,
Sheng-Kwei
Song2,
Ping
Yan3,
Grace
Ku1,
Xiao Ming
Xu3, and
Chung Y.
Hsu1
Departments of 1 Neurology and Center for the Study of
Nervous System Injury, and 2 Radiology, Washington
University School of Medicine, St. Louis, Missouri 63110, and
3 Department of Anatomy and Neurobiology, St. Louis
University School of Medicine, St. Louis, Missouri 63104
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ABSTRACT |
Tumor necrosis factor- (TNF-) expression has been documented
extensively in animal models of traumatic spinal cord injury (SCI).
However, the pathophysiological significance of TNF- expression in
the injured cord remains to be delineated. The TNF receptor (TNFR)-nuclear factor- B (NF-B) signal transduction pathway is important for maintaining cell viability. NF-B exerts anti-apoptotic effects via an endogenous caspase inhibitory system mediated by cellular inhibitor of apoptosis protein 2 (c-IAP2). NF-B
transactivates c-IAP2 to inhibit caspase-3 activation. Progressive cell
death, including morphological and biochemical features suggestive of apoptosis, has been noted after SCI. We explored the effects of TNFR1
or TNFR2 deletion on the apoptotic events downstream of NF-B in
relation to SCI pathology and functional recovery. Nuclear proteins
from the injured cords of the TNFR1 / mice had a
reduced NF-B binding activity compared with the wild-type controls.
This decrease in NF-B activation was accompanied by a reduction in
c-IAP2 expression and an increase in the active form of caspase-3
protein. After SCI the TNFR1/ mice had greater
numbers of apoptotic cells, a larger lesion size, and worse functional
recovery than wild-type mice. TNFR2-deficient mice had a similar,
although not as pronounced, consequence as the
TNFR1/ mice. These findings support the argument
that the TNFR-NF-B pathway is beneficial for limiting apoptotic
cell death after SCI and that a defective TNFR-NF-B pathway results
in a poorer neurological outcome. A worse functional outcome in
TNFR/ mice suggests that an endogenous apoptosis
inhibitory mechanism mediated by TNFR activation, NF-B, and c-IAP2
may be of pathophysiological importance.
Key words:
apoptosis; caspase-3; tumor necrosis factor; TNFR1; TNFR2; cytokine
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INTRODUCTION |
Extensive demyelination and axonal
destruction in traumatic spinal cord injury (SCI) are caused by a
complex series of cellular and molecular events (Tator, 1995 ; Crowe et
al., 1997; Rosenberg and Wrathall, 1997; Rosenbluth et al., 1997). In
addition to neuronal and glial cell death, delayed waves of
oligodendrocyte degeneration with morphological features suggestive of
apoptosis have been described in the secondary white matter damage
after SCI (Katoh et al., 1996; Li et al., 1996; Crowe et al., 1997; Liu
et al., 1997; Springer et al., 1999). Tumor necrosis factor-
(TNF-), a proinflammatory cytokine, is cytotoxic to oligodendrocytes
(Louis et al., 1993; Mayer and Noble, 1994; D'Souza et al., 1995) and may contribute to white matter injury in CNS disorders (Probert et al.,
1995). Increases in TNF- expression after SCI have been documented
extensively (Yakovlev and Faden, 1994; Wang et al., 1996;
Bartholdi and Schwab, 1997; Xu et al., 1998). TNF- has been
implicated in the pathogenesis of CNS injury after traumatic or
ischemic insults (Shohami et al., 1996; Barone et al., 1997).
TNF- induces a wide spectrum of biological responses by interacting
with two cell surface receptors, TNF receptor 1 (TNFR1) and 2 (TNFR2)
(Smith et al., 1994). Recent studies have shown that TNFR activation is
not necessarily deleterious. TNF- and TNFR may prevent neuronal
apoptosis induced by excitotoxin in vitro (Glazner and
Mattson, 2000) and in vivo (Bruce et al., 1996), cerebral
ischemia (Nawashiro et al., 1997), experimental allergic encephalomyelitis (Liu et al., 1998), and traumatic brain injury (TBI)
(Scherbel et al., 1999). An important intermediate step in the
TNFR-mediated signaling process is nuclear factor-B (NF-B) activation (Lenardo and Baltimore, 1989; Baeuerle and Henkel, 1994).
NF-B activation may prevent or enhance apoptosis under different
experimental paradigms (Mattson et al., 2000). An anti-apoptotic role
for the TNFR-NF-B cascade has been reported (Wang et al., 1998;
Tamatani et al., 1999; Chen et al., 2000). The cytoprotective-signaling events downstream of TNFR-NF-B may involve cellular mammalian homologs corresponding to baculovirus inhibitors of apoptosis protein
(IAPs). IAPs inhibit caspase activation (Xue and Horvitz, 1995; Urn et
al., 1996; Roy et al., 1997; Deveraux et al., 1998; Wang et al., 1998).
Among the IAPs, cellular inhibitor of apoptosis protein 2 (c-IAP2) is
expressed after the activation of TNFR. c-IAP2 is transactivated by
NF-B (Chu et al., 1997) and forms a complex with TNFR-associated
factor 2 (Rothe et al., 1995) to facilitate an anti-apoptotic action.
The TNFR-NF-B cascade is activated in rat SCI models (Bethea et
al., 1998; Xu et al., 1998). How this pathway regulates apoptosis in
the injured spinal cord remains to be fully explored. Mice with a
homozygous deletion of TNFR1 or TNFR2 have been generated and exhibit a
defect in TNF--mediated signaling processes (Rothe et al., 1993;
Erickson et al., 1994). In this study we examined the cellular events
and morphological and functional outcome after SCI in mice with a TNFR1
or TNFR2 deficiency, in comparison to wild-type mice, to explore the
pathophysiological significance of the TNFR-NF-B cascade.
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MATERIALS AND METHODS |
Transgenic animals. TNFR1 (Rothe et al., 1993) and
TNFR2 (Erickson et al., 1994) null-mutation mice on a C57BL/6 (B6, The Jackson Laboratory, Bar Harbor, ME) background were back-crossed with
B6 mice for >12 generations and were maintained in the Washington University School of Medicine animal facility. B6 mice were used as the
wild-type controls.
Spinal cord injury. Female mice, 8-12 weeks old, were
anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). During surgery the rectal temperature was
monitored and maintained at 37.0 ± 0.5°C by a heating pad. The
skin above the vertebral column was shaved and cleaned with antiseptic.
A 15 mm midline skin incision was made, and the vertebral column was
exposed. After the spinal thoracic region was exposed by separation of
dorsal muscles to the side, the spinous processes of T8-T13 vertebrae
were exposed. A laminectomy was performed at vertebral level T10 and
one-half of T9, exposing the dorsal cord surface with the dura
remaining intact. The vertebral column was stabilized with angled
clamps on the T8 and T12 transverse processes. SCI was induced by a
modified NYU Impactor (Gruner, 1992; Basso et al., 1996a,b; Liu et al.,
1997). A 10 gm weight (1.5 mm tip in diameter) was allowed to drop from
3 mm onto the T10 segment. The impact rod was removed immediately, and
the wound was irrigated. Muscles and incision were closed in layers,
and the animals were placed in a temperature- and humidity-controlled chamber (ThermoCare, Incline Village, NV) for 3 hr. Sham-operated animals received the same surgical procedures but sustained no impact
injury. Manual bladder expression was performed twice a day until
reflex bladder emptying was established. All surgical procedures,
interventions, and presurgical and postsurgical care followed the
Laboratory Animal Welfare Act, the Guide for the Care and Use of
Laboratory Animals, and the Guidelines and Policies for
Rodent Survival Surgery provided by the Animal Studies Committee of Washington University School of Medicine.
Behavioral tests. To evaluate the functional consequence of
SCI, we performed the locomotor rating test with the
Basso-Beattie-Bresnahan (BBB) scale, as described previously (Basso
et al., 1996a). The BBB scale is readily applicable to mice without
modifications (Jakeman et al., 2000) especially when the score is below
16 (G. M. Kim and C. Y. Hsu, unpublished observations).
Before surgery the mice were placed individually in a molded plastic
open field for 4 min to assure that all subjects consistently obtained
a maximum score of 21 (n = 10-11 per group). For
28 d, beginning 1 d after injury, the mice were placed daily
in an open field for 4 min, and well trained investigators scored the
BBB scale in a blinded manner.
Histopathology. After receiving behavioral testing for 4 weeks, animals (n = 6-7 per group) were given a lethal
overdose of pentobarbital and perfused intracardially with normal
saline, followed by 4% paraformaldehyde in 0.1 M
PBS, pH 7.4. For histological evaluation a 10 mm cord segment
centered at the injury site was blocked from the vertebral column,
placed in the same fixative overnight, and embedded in paraffin. Serial
10 µm cross sections were stained with hematoxylin and eosin. The
presence of inflammatory cells, loss of neurons, and the existence of
degenerating neurons, microcyst formation, and gliosis defined the
rostrocaudal boundaries of the tissue damage. Total lesion length was
determined by calculating the distance between the rostral and caudal
boundary of the lesion area as determined by light microscopy (Kuhn and
Wrathall, 1998).
Isolation of nuclear proteins. Nuclear protein extracts were
prepared from sham-operated or injured spinal cord segments at 1, 4, and 24 hr after injury (n = 3 per time point) as
described previously (An et al., 1993; Xu et al., 1998). An 8 mm cord
segment (4 mm rostral and caudal from the epicenter) was dissected and frozen immediately after the animal was killed under anesthesia. Nuclear proteins were extracted according to a high-salt method (Dignam
et al., 1983) with modifications (An et al., 1993; Yan et al., 1999; Xu
et al., 2001a).
Electrophoretic mobility shift assay. NF-B binding
activity was assessed by electrophoretic mobility shift assay (EMSA) as described previously (X. Xu et al., 1998; J. Xu et al., 2001a), using
the NF-B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Life Technologies, Rockville, MD). The NF-B oligonucleotide was
labeled with  -32P-ATP according to
Promega (Madison, WI) technical bulletin 106. The binding reaction was
performed in a total volume of 20 µl containing the binding buffer
[with (in mM) 10 Tris-HCl, 20 NaCl, 1 DTT, 1 EDTA, and 5%
glycerol, pH 7.6], 0.0175 pmol of labeled probe (>10,000 cpm), 20 µg of nuclear protein, and 1 µg of poly(dI-dC). After incubation
for 20 min at room temperature, the mixture was electrophoresed on a
nondenaturing 6% polyacrylamide gel at 180 V for 2 hr under
low-ionic-strength conditions. The gel was dried and subjected to autoradiography.
Western blot analysis. At 1, 4, and 24 hr after injury
(n = 3 per time point), an 8 mm spinal cord segment (4 mm rostral and 4 mm caudal from the lesion epicenter) was dissected
from control and injured mice and frozen immediately in liquid
N2. The cord segment was homogenized in a buffer
containing (in mM) 10 HEPES, 1.5 MgCl2, 10 KCl, and 0.5 DTT plus 1% NP-40, 1 µg/ml leupeptin, and 1 µg/ml aprotinin, pH 7.9, and then
centrifuged at 14,000 × g. Protein (40 µg) from the
supernatant of each sample was separated by SDS-PAGE and transferred to
polyvinylidene difluoride membranes by electrophoresis. The membranes
were blocked in TBST buffer containing 20 mM
Tris-HCl, 5% nonfat milk, 150 mM NaCl, and
0.05% Tween 20, pH 7.5, for 1 hr at room temperature. Then the blots were incubated with either a primary polyclonal rabbit anti-c-IAP2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit
anti-caspase-3 antibody (1:200; Santa Cruz Biotechnology), followed by
a secondary alkaline phosphatase-conjugated anti-rabbit IgG antibody
(1:5000; Promega). The Western blots were visualized with the Blot AP
System (Promega).
Immunohistochemical staining. At 1 d after injury, mice
(n = 3 per group) were overdosed by an intraperitoneal
injection of 100 mg/kg sodium pentobarbital. Intracardial perfusion
fixation was done with physiological saline, followed by 4%
paraformaldehyde in 0.1 M PBS, pH 7.4. The spinal
cord was dissected out carefully, and a 10 mm segment containing the
injured epicenter was blocked, post-fixed for 2 hr in the same fixative
solution, and transferred to a solution containing 30% sucrose in 0.1 M PBS, pH 7.4, for diaminobenzidine (DAB;
Sigma-Aldrich, St. Louis, MO) or immunofluorescence staining. Then the
spinal cord segments were embedded in tissue-freezing medium (Sakura
Finetek, Torrance, CA), cut horizontally or longitudinally at 10 µm
on a cryostat into sections, and mounted on Superfrost/Plus slides
(Fisher Scientific, Pittsburgh, PA). Sections were permeabilized with
0.3% Triton X-100 and 4% normal goat serum in 0.01 M PBS, pH 7.4, for 20 min and incubated overnight
at 4°C with either a polyclonal rabbit anti-c-IAP2 antibody (1:250;
Santa Cruz Biotechnology) or a rabbit polyclonal anti-caspase-3
antibody (1:100; PharMingen, San Diego, CA), followed by a
biotin-labeled goat anti-rabbit IgG (Vector Laboratories, Burlingame,
CA). After washing, the sections were incubated further with ABC Elite
complex (Vector Laboratories). The staining was visualized with DAB.
Slides were washed, dehydrated, cleared in xylene, and mounted.
For the immunofluorescence labeling for myelin basic protein (MBP),
mice were perfused at 7 d after injury (n = 3 per
group). Transverse sections 3 mm rostral to the lesion epicenter were incubated with polyclonal rabbit anti-MBP antibody (1:100; Zymed Laboratories, South San Francisco, CA) at 4°C overnight, followed by
fluorescein (FITC)-conjugated anti-rabbit IgG antibody (1:100; Vector
Laboratories). Slides were washed, wet-mounted, and examined with the
Olympus BX60 microscope (Olympus Optical, Tokyo, Japan). For negative
controls the primary antibodies were replaced by normal rabbit serum in
0.01 M PBS, pH 7.4.
For the immunofluorescence double-labeling experiment, spinal cord
sections were permeabilized with 0.3% Triton X-100 and 4% normal
goat serum in 0.01 M PBS, pH 7.4, for 20 min.
Polyclonal rabbit anti-c-IAP2 antibody (1:100; Santa Cruz
Biotechnology) or rabbit anti-caspase-3 antibody (1:100; PharMingen)
with either monoclonal mouse anti-NeuN antibody (1:500; Chemicon,
Temecula, CA) or mouse anti-RIP antibody (1:100; Chemicon) was
applied to the sections at 4°C overnight. Then the sections were
incubated with an FITC-conjugated goat anti-rabbit IgG antibody (1:100; Vector Laboratories) and Texas Red-conjugated goat anti-mouse antibody
(1:100; Vector Laboratories). Slides were washed, wet-mounted, and
examined under fluorescent microscopy.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling staining. Spinal cord tissue (n = 4-6 per group) from mice with a 7-d-after-SCI survival time was
examined via the terminal deoxynucleotidyl transferase (TdT)-mediated
dUTP-biotin nick end labeling (TUNEL) method (Liu et al., 1997) with
the Apoptag in situ kit (Oncor, Gaithersburg, MD). Negative
control sections were treated similarly, but the TdT enzyme was
omitted. TUNEL-positive nuclei were counted visually in five
different regions at sampling sites 0, 1, and 2.5 mm rostral and caudal
to the lesion center for each animal. Immunofluorescence double
labeling with TUNEL staining and an oligodendrocyte cell surface
marker, as detected with an anti-adenomatus polyposis coli
antibody (1:400; Oncogene, San Diego, CA), was used to assess
oligodendrocyte apoptosis after SCI.
Ex vivo magnetic resonance imaging magnetic resonance
imaging (MRI) analysis. At 1 d and 7 d after injury,
mice were perfused with saline; 2 cm of spinal cord with vertebral
column was removed immediately for magnetic resonance imaging (MRI).
Diffusion-weighted images (DWI) maps were acquired. MRI studies were
performed on an Oxford Instruments-200/400 (4.7 Tesla, 40 cm clear
bore; Oxford, UK) magnet equipped with a 10 cm in inner diameter
actively shielded gradient coil (60 G/cm, 200 µsec rise time;
Magnex). The magnet and gradient coil and power supply were interfaced
with a Varian UNITY-INOVA console controlled by a Sun
Microsystems (Palo Alto, CA) Ultra-170 Sparc workstation. Excised mouse
spinal columns were placed on a 2 × 3 cm plate for the imaging
plan alignment. A birdcage coil of 2 cm in inner diameter was used as
the transceiver. The DWIs were collected by using a conventional
multislice spin echo sequence modified by adding a pair of
Stejskal-Tanner diffusion-sensitizing gradients. The DWIs were
acquired with TR 1.5 sec, TE 35 msec, slice thickness 0.2 mm,
rectangular field of view 1.5 × 2.5 cm, and data matrix 128 ×128
(zero filled to 256 × 256). Diffusion-sensitizing gradients were
applied along six directions of [Gx,Gy,Gz] = [1,1,0], [1,0,1], [0,1,1], [ 1,1,0], [0,1,1], and [1,0,1].
Individual apparent diffusion coefficients of the diffusion tensor were
determined by nonlinear squares curve fitting of the voxel amplitudes,
A(b), of the six DWIs obtained at b
value of 764 sec/mm2, and a
T2-weighted image without diffusion-sensitizing
gradient, A(b = 0), as described by Equation 1:
|
(1)
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The diffusion-sensitizing factor, b, is defined as
Equation 2:
|
(2)
|
where is the gyromagnetic ratio, G is the
amplitude of diffusion-sensitizing gradient,  is the diffusion
gradient pulse duration, and  is the time separation of the
diffusion-sensitizing gradient pulse pairs.
Quantitation of spinal cord lesion area. At 7 d after
injury, mice (n = 3 per group) were perfused
intracardially. Frozen sections (16 µm thick) were permeabilized and
blocked with 0.3% Triton X-100 and 3% normal donkey serum in 0.01 M PBS, pH 7.4, for 15 min. To identify axons, we
incubated the sections with a monoclonal mouse anti-SMI-31 antibody
(1:1000; Sternberger Monoclonals, Lutherville, MD) at 4°C overnight,
followed by a tetra-rhodamine (TRITC)-conjugated donkey anti-mouse
antibody (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA).
Sections were washed, mounted, and examined with Olympus BX60
microscopy (Olympus Optical). Control sections were incubated with
normal mouse IgG as the primary antibody. The lesion area was
identified by the boundary formed by SMI-31-positive axons. Three
horizontal sections, one each at the central canal, 60 µm dorsally,
and 60 µm ventrally, were selected for measurement with a Neurolucida
system (MicroBrightField, Colchester, VT) attached to a Leica
DRMB microscope; the values that were obtained were averaged.
Statistical analysis. Data are presented as mean ± SEM
values. For BBB scores, histopathological analysis, TUNEL staining, and
lesion area measurement, one-way ANOVA with post hoc Tukey test was used to determine statistical significance. A p
value of <0.05 was considered statistically significant.
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RESULTS |
Functional assessment in TNFR-deficient mice
After traumatic SCI, all injured mice were paralyzed in both hind
limbs. The functional recovery score was followed for 28 d with
the BBB scoring system, a standard method for assessing the hind limb
motor function after SCI in both rats (Basso et al., 1996a,b) and mice
(Jakeman et al., 2000). Poor locomotor performance was identified in
TNFR1/ and
TNFR2/ mice (Fig.
1A). At 4 weeks of
injury, significantly lower BBB scores were noted in
TNFR1/ (8.6 ± 0.5;
n = 10; p < 0.001) and
TNFR2/ (11.6 ± 0.9;
n = 10; p < 0.01) mice compared with
wild-type B6 mice (15.0 ± 0.8; n = 11). At 4 weeks after injury most of the TNFR1/
mice were unable to bear weight on the hind limbs and had incomplete hind limb coordination.
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Figure 1.
Quantitative analysis of functional recovery and
lesion length after traumatic SCI. A,
TNFR1/ and TNFR2/ mice
showed worse functional outcomes than the wild-type
(WT) control B6 mice by BBB score at 4 weeks
after injury (*p < 0.01 and
**p < 0.001 vs wild-type). B,
Lesion length measurements demonstrated that
TNFR1/ mice had more extensive tissue injury
than B6 mice (*p < 0.02; n = 6-7 per group).
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Histological analysis
At 4 weeks after SCI the lesion epicenter showed marked gliosis
and white matter vacuolation as well as gross cord atrophy (data not
shown). The lesion lengths in TNFR1/
mice (3.71 ± 0.32 mm) were significantly longer than those in the
wild-type controls (2.38 ± 0.27 mm; p < 0.01).
The difference in lesion lengths between the
TNFR2/ mice (2.75 ± 0.26 mm) and
the wild-type controls was not statistically significant (Fig.
1B). Conventional spared tissue quantitation (Beattie
et al., 1997; Liu et al., 1997) could not be used for mice in these
experiments because of the lack of cavitation in the lesion center.
NF-B binding activity
EMSA, performed with nuclear proteins extracted from the spinal
cords of sham-operated mice at 1, 4, and 24 hr after injury, demonstrated a low basal level of NF-B activity in wild-type as well
as in TNFR1/ and
TNFR2/ mice. SCI resulted in a
significant time-dependent increase in NF-B activity at 1 hr, with
the activity reaching maximal levels at 4 hr and decreasing at 24 hr
after SCI. TNFR1/ and
TNFR2/ mice had a blunted NF-B
activation when compared with the wild-type controls, with the
TNFR1/ mice exhibiting the lowest
levels of NF-B binding activity (Fig. 2A).
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Figure 2.
Effects of TNFR deletion on the NF-B
anti-apoptotic pathway after SCI. Samples from sham-operated and
injured spinal cords were collected 1, 4, and 24 hr after SCI.
A, EMSA showed a decrease in NF-B binding activity in
TNFR1/ and TNFR2/ mice at
all of the time points that were tested. B, Western blot
analysis showed a decrease in c-IAP2 expression after SCI in
TNFR-deficient mice, especially TNFR1/.
C, Western blotting shows that the expression of the
active form of caspase-3 (17 kDa) was increased in TNFR-deficient mice,
especially TNFR1/. The  -actin Western blot
using the same spinal cord samples served as a control to demonstrate
equal protein loading in each lane.
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c-IAP2 and caspase-3 expression in TNFR-deficient mice
Because c-IAP2 is known to act downstream of the TNF--NF-B
cascade in preventing cell death (Chu et al., 1997), c-IAP2 protein expression in the spinal cord was examined by Western blot analysis. Basal levels of c-IAP2 in sham-operated wild-type controls were expressed at higher levels than in the
TNFR1/ and
TNFR2/ mice. After injury an increase
in c-IAP2 expression was noted but was substantially less pronounced in
the TNFR1 and TNFR2 mutants at 1 and 4 hr (Fig. 2B).
However, at 24 hr the c-IAP2 protein levels remained low in the
TNFR1/ mice, whereas the
TNFR2/ mice expressed more c-IAP2
protein when compared with the wild-type mice. The expression of the
active form of caspase-3 is a hallmark of apoptotic cell death (Kumar,
1997) and has been shown after SCI (Springer et al., 1999). The
expression of caspase-3 was increased after injury and more pronounced
in the TNFR1/ and
TNRF2/ mice than in the wild-type
controls (Fig. 2C). To ensure that the apparent increase in
caspase-3 immunoreactivity represented an increase in the active form
of caspase-3, we also did immunostaining with an antibody, CM1,
specific for the caspase-3 active form (Han et al., 2000). Similar
results were obtained with the CM1 antibody (data not shown).
Consistent with the c-IAP2 Western blot result, c-IAP2 immunoreactivity
was less intense in the injured cord in
TNFR2/ and especially in
TNFR1/ mice compared with the
wild-type controls (Fig.
3A-C). The immunoreactivity of caspase-3 was enhanced to a greater extent in
TNFR1/ mice compared with the
wild-type B6 mice, whereas TNFR2/ mice
showed a similar intensity of staining as the wild-type controls (Fig.
3D-F), confirming the results from the caspase-3 Western blot.
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Figure 3.
c-IAP2 and active caspase-3 immunoreactivity in
B6, TNFR1/, and TNFR2/
mice 24 hr after SCI. A-C, c-IAP2 expression was
decreased in TNFR1/ mice compared with B6 mice
in the white matter tract 1 mm rostral to the lesion epicenter.
D-F, Compared with B6 mice, caspase-3 expression was
increased in TNFR1/ mice in both the gray matter
(GM) and white matter (WM).
Longitudinal sections 1-2 mm rostral to the lesion epicenter are
shown. Scale bars: A-C, 20 µm, D-F,
100 µm.
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Cellular localization of c-IAP2 and caspase-3
To determine whether c-IAP2 and caspase-3 are present in neurons
and oligodendrocytes, we performed an immunofluorescence double-labeling experiment on wild-type controls 1 d after SCI. c-IAP2 and the oligodendrocyte marker, RIP, showed cellular
colocalization in the white matter tract in the injured cord (Fig.
4A). Additionally, c-IAP2 colocalized with the neuronal nuclear filament maker, NeuN, in
neurons in the gray matter (Fig. 4A) in the injured
cord. In similar experiments caspase-3 immunoreactivity also was
localized to neurons as well as to oligodendrocytes around the lesion
epicenter area (Fig. 4B).
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Figure 4.
c-IAP2 and caspase-3 expression in neurons
and oligodendrocytes 1 d after SCI in B6 mice. Longitudinal
sections 1 mm rostral to the lesion epicenter are shown. c-IAP2 and
caspase-3 were localized to neurons and oligodendrocytes as
demonstrated by immunofluorescence double staining for an
oligodendrocyte marker (Rip, red) or
neuronal nuclear protein (NeuN, red) with
either c-IAP2 (A, green) or caspase-3
(B, green). Scale bar, 20 µm.
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TUNEL-positive cells in TNFR-deficient mice
Apoptosis of neurons, oligodendrocytes, astrocytes, and microglia
has been demonstrated in the injured cord (Crowe et al., 1997; Liu et
al., 1997; Shuman et al., 1997). To test whether TNFR deletion
affected the extent of cell death, we counted TUNEL-positive cells at
five levels of the spinal cord (the lesion epicenter and 1 and 2.5 mm
rostral and caudal) in TNFR1/ and
TNFR2/ mice in comparison to the
wild-type controls. It should be noted that TUNEL stain is not specific
for apoptotic cells. Cells dying of necrosis also may be
TUNEL-positive. There were significantly more TUNEL-positive cells in
TNFR1/ mice than in the wild-type mice
at rostral 1 and 2.5 mm and caudal 2.5 mm levels
(p < 0.05) (Fig.
5A,B). Although the
TNFR2/ mice appeared to have a greater
number of TUNEL-positive cells than
TNRF1/ or wild-type mice in the
epicenter, the difference in the number of TUNEL-positive cells between
the TNFR1 and TNFR2 mutants was not significant. Despite this exception
in the epicenter, TNFR1/ mice had
significantly more TUNEL-positive cells than
TNFR2/ or wild-type mice. Across the
five spinal cord regions that were examined
(TNFR1/, 156.5 ± 23.2;
TNFR2/, 116.6 ± 8.5; wild type,
73.9 ± 8.4), the TNFR2/ mice
exhibited an intermediate number that corresponded to the intermediate
functional outcome after SCI, as shown in Figure 1. TUNEL-positive
cells were located mostly in the white matter and expressed APC, an
oligodendrocyte marker, indicating oligodendrocyte death (Fig.
5C).
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Figure 5.
Enhanced apoptotic cell death in TNFR-deficient
mice. A, Representative photomicrographs illustrating
more TUNEL-positive cells (arrowheads,
green) in TNFR1/ and
TNFR2/ mice compared with B6 mice at 2.5 mm from
the lesion center in the dorsal column area of the injured spinal cord
7 d after SCI. Note that nuclei from all of the cells are stained
with propidium iodide (red). B,
Quantitative analysis of TUNEL-positive cells 7 d after injury.
TNFR1/ mice showed a significantly larger number
of TUNEL-positive cells compared with wild-type mice at 2.5 and 1 mm
rostral and 2.5 mm caudal to the lesion epicenter
(*p < 0.05; n = 4-6 per
group). C, TUNEL (a), APC
oligodendrocyte marker (b), and Hoechst 33342 (c) immunofluorescence staining of a section of
the white matter from a wild-type mouse 1 mm rostral to the lesion
epicenter shows that TUNEL-positive cells are oligodendrocytes. This is
confirmed by Hoechst 33342 nuclear staining (c,
arrowheads). Morphologically normal oligodendrocytes
(APC, red) in the white matter 2.5 mm
rostral from the lesion epicenter are not TUNEL-positive and have
a normal nuclear chromatin Hoechst 33342 (blue) staining
pattern (d). The arrows in
a (TUNEL), b (APC), and
c (Hoechst) point to the same cells (triple
staining experiment). The arrows in d
(APC/Hoechst) show live/normal oligodendrocytes double
stained with APC and Hoechst. Scale bars: A, 100 µm;
C, 20 µm.
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Ex vivo MRI analysis after SCI
To examine the degree of spinal cord damage in the acute phase, we
performed ex vivo MRI study 1 and 7 d after SCI. The
sagittal MRI scan showed localized lesions in the spinal cord in the
wild-type, TNFR1/, and
TNFR2/ mice (Fig.
6A,B). The signal
changes in spinal cord lesion area represent edema, hemorrhage, and
tissue necrosis (Ohta et al., 1999; Bilgen et al., 2000). The lesion
length determined by MR images is a reliable indicator of traumatic SCI
severity (Hackney et al., 1994). A larger area of damage at the site of
impact is apparent in both TNFR1/ and
TNFR2/ mice. DWI measured at 7 d
after SCI showed that the rostrocaudal spreading of the lesion was more
extensive in both TNFR1/ and
TNFR2/ mice.
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|
Figure 6.
Representative MR images of damaged spinal cords
after traumatic SCI. A, Spinal cord lesions in
TNFR1/
(R1/),
TNFR2/
(R2/), and wild-type
(WT) mice as shown by DWI mapping 1 d after
SCI. B, Lesions shown at 7 d. Two slice sections
are shown, one from the center (Slice #3) and one that
is 200 µm from the center (Slice #2).
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Myelin and axonal damage in TNFR-deficient mice
The extent of myelin loss and axonal disruption was assessed by
immunostaining with the use of an anti-MBP antibody (Fig. 7) or axonal neurofilament marker, SMI-31
antibody (Fig. 8), respectively. Demyelination of white matter tract was observed in dorsal and lateral
funiculi in TNFR1/ mice (Fig.
7C). Tissue destruction and microcyst formation were seen in
the dorsal funiculus of TNFR1/ mice
(Fig. 7D) and TNFR2/ mice
to a lesser degree (Fig. 7F). The total area of
tissue disruption at 7 d after injury as quantified on the basis
of SMI-31 immunostaining showed more severe tissue damage in
TNFR1/ mice (3.21 ± 0.30 mm2) when compared with the wild-type
controls (1.48 ± 0.23 mm2;
p < 0.02) (Fig. 8B). The difference
in the area of axonal damage between
TNFR2/ mice (2.43 ± 0.39 mm2) and the wild-type controls was not
statistically significant.
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Figure 7.
White matter demyelination demonstrated by MBP
immunoreactivity counterstained with Hoechst 33342 at 7 d after
SCI. Sections shown are 3 mm rostral to the lesion epicenter. At low
magnification (A, C, E), white matter tract
demyelination is observed in dorsal and lateral white matter in
TNFR1/ mice (arrowheads,
C). At high magnification, tissue destruction and
microcyst formation are more pronounced in the dorsal white matter in
TNFR1/ mice (arrowheads,
D). Microcyst formation in the dorsal white matter
is not as extensive in TNFR2/ mice
(arrowheads, F). Scale bars:
A, C, E, 200 µm; B, D, F, 50 µm.
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Figure 8.
Axonal disruption in TNFR-deficient mice after
SCI. A, Representative photomicrographs illustrating
spinal cord lesion areas defined by neurofilament (SMI-31)
immunostaining in B6 (a),
TNFR1/ (b), and
TNFR2/ (c) mice 7 d
after SCI. All sections were cut longitudinally through the central
canal (c.c.) for comparison. Dotted lines
delineating the lesion areas are based on SMI-31 immunoreactivity.
d, High power view of the boxed area in
a that demarcates the border between intact cord tissue
(arrows) and the lesion area (asterisk).
Scale bars: a-c, 400 µm; d, 100 µm.
B, A histogram showing the mean lesion area among the
three experimental groups (n = 3 per group).
*Significant difference from the wild-type (WT)
control (p < 0.02).
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|
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DISCUSSION |
TNFR1/ and
TNFR2/ mice, compared with the
wild-type mice, demonstrated poor functional recovery and decreased
NF-B binding activity after SCI. In agreement with the decrease in
NF-B activation, c-IAP2 expression based on Western blot analysis
also was decreased in TNFR1/ mice
compared with the wild-type mice after SCI. Consequent to a reduced
c-IAP2 expression, active caspase-3 expression and the numbers of
TUNEL-positive cells increased in the damaged spinal cords of
TNFR1/ mice.
TNF- (Yakovlev and Faden, 1994; Wang et al., 1996; Bartholdi and
Schwab, 1997; Xu et al., 1998) and TNFR (Li et al., 1998) are expressed
in neurons and oligodendrocytes after traumatic SCI. A dual role can be
played by TNF- in CNS injury. TNF- is known to be cytotoxic to
oligodendrocytes in vitro (D'Souza et al., 1995). In
addition, TNF- has a detrimental role in TBI (Shohami et al., 1996)
and experimental ischemic stroke (Barone et al., 1997; Yang et al.,
1998), as shown by improved outcome measures that use anti-TNF-
strategies. Conversely, a protective role of TNF- (Nawashiro et al.,
1997) and a deleterious effect of TNFR deletion (Gary et al., 1998)
have been demonstrated in cerebral ischemia, excitotoxic neuronal
injury (Bruce et al., 1996), autoimmune-mediated demyelination (Liu et
al., 1998), and TBI (Scherbel et al., 1999; Sullivan et al., 1999). In
a recent study TNF-/ mice subjected
to an extradural compression of the thoracic spinal cord showed no
better recovery of hind limb function after SCI than the wild-type
controls (Farooque et al., 2001). Recombinant murine interleukin-1
(IL-1), IL-6, and TNF- administered to the lesioned spinal cords
of adult mice after SCI reduced the amount of tissue loss (Klusman and
Schwab, 1997). Our data demonstrated more severe tissue damage and
worse functional outcome in mice that were deficient in TNFR.
NF-B was activated during the acute stage of SCI but was less
pronounced in TNFR-deficient mice than in wild-type mice. The NF-B
heterodimer p50/RelA is known to upregulate various inflammatory mediator genes such as inducible nitric oxide synthase (iNOS), cytokines, cellular adhesive molecules, and proteases, which may be
detrimental by perpetuating and intensifying an inflammatory reaction
(Shimizu et al., 1990; Ledebur and Parks, 1995; Baldwin, 1996; Barnes,
1997; Barnes and Karin, 1997; O'Neill and Kaltschmidt, 1997; Schmedtje
et al., 1997; Ghosh, 1999; Xu et al., 2001b). However, NF-B also may
exert a cytoprotective role (Beg and Baltimore, 1996; Grilli et al.,
1996; Van Antwerp et al., 1996). Lack of the p50 NF-B subunit
increases the vulnerability of hippocampal neurons to excitotoxic
injury (Yu et al., 1999). A cytoprotective action of NF-B could be
mediated via the induction of endogenous IAP genes. c-IAP2 expression
is induced via NF-B activation (Chu et al., 1997; Wang et al.,
1998). c-IAP2, originally identified as a molecule recruited to the
TNFR complex, contains two NF-B-binding DNA consensus sequences
within its promoter region (Hong et al., 2000).
IAP overexpression has been shown to suppress apoptosis induced by a
variety of stimuli (Duckett et al., 1996; Liston et al., 1996; Deveraux
et al., 1997). Overexpression of an X chromosome-linked IAP attenuated
ischemia-induced caspase-3 activation and CA1 neuron degeneration in
the rat hippocampus (Xu et al., 1999). Targeted expression of
baculovirus p35 caspase inhibitor, an IAP analog, in oligodendrocytes
protected mice against autoimmune-mediated demyelination (Hisahara et
al., 2000). In the present study a decrease in NF-B binding activity
was accompanied by reduced expression of c-IAP2 in
TNFR1/ mice after SCI. This reduction
in c-IAP2 expression in both TNFR1/
and TNFR2/ mice initially may cause
more cell death after SCI. However, the delayed increase in c-IAP2
expression observed in TNFR2/ mice may
limit cell death in the injured cord, thus resulting in an intermediate
level of functional outcome.
Consistent with a reduced expression of c-IAP2, an increase in the
expression of the active form of caspase-3, together with an increase
in the number of TUNEL-positive cells, was observed in the injured cord
in TNFR1/ mice after SCI. The
molecular machinery necessary for activation and execution of the
caspase-3 apoptotic pathway is operational after traumatic SCI in rats
associated with an intense expression of DNA fragmentation
factor/caspase-activated DNase in oligodendrocytes (Springer et al.,
1999). An increase in caspase-3 expression and the numbers of dead
cells in TNFR1/ mice followed a
blunted NF-B response and reduced c-IAP2 expression after SCI. These
results are consistent with the contention that an endogenous
inhibitory mechanism on cell death may be activated via the
TNFR-NF-B cascade. In the TNFR1/
and TNFR2/ mice, to a lesser extent,
this anti-apoptotic mechanism may be deficient.
The extent of demyelination and axonal disruption in
TNFR1/ mice was increased after SCI.
The loss of locomotor function from traumatic SCI is known to depend on
the extent of demyelination and axonal destruction (Blight and
Decrescito, 1986; Wrathall et al., 1996; Beattie et al., 1997). Lesion
expansion through secondary demyelination of white matter tract has
been attributed to delayed oligodendrocyte apoptosis distant from the
lesion center (Crowe et al., 1997). Cycloheximide, known to inhibit
apoptosis in the nervous system, produced substantial preservation of
tissue, reduced central cavitation, and improved recovery of function after SCI (Liu et al., 1997). In addition, the inhibition of caspase-1 and -3 activity reduced post-traumatic lesion size and improved motor
performance in a mouse SCI model (Li et al., 2000). Therefore, limiting
apoptotic cell death in the spinal cord might reduce tissue damage and
enhance functional recovery after SCI.
The individual contribution of TNFR1 or TNFR2 has not been elucidated
clearly. Compared with the wild-type mice,
TNFR2/ and especially
TNFR1/ mice showed lower intensity of
NF-B activation as well as poorer functional recovery after SCI.
Shared signaling pathways may account for the close functional
relationship between TNFR1 and TNFR2. These two receptors have the same
proximal adapter domain, TNFR-associated factor 2, which activates
NF-B-inducing kinase for NF-B nuclear translocation (Hsu et al.,
1996). The cooperative signaling by TNFR1 and TNFR2 may be explained by
the "ligand-passing effect" of TNFR2. TNFR2 is known to regulate
the rate of TNF- association with TNFR1, possibly by increasing the
local concentration of TNF- at the cell surface via rapid ligand
association and dissociation (Tartaglia et al., 1993; Riches et al.,
1998). Results from the present study raise the possibility that TNFR1
and TNFR2 interact via either a cooperative cross-linking effect of
individual TNFR or a shared anti-apoptosis signaling pathway.
In summary, results from the present study are the first to demonstrate
an increase in c-IAP2 expression after SCI and support the contention
that the TNFR-NF-B-c-IAP2 cascade may mediate an important
endogenous protective mechanism against apoptosis after SCI. Our
findings suggest that TNFR1, and probably to a lesser extent TNFR2,
plays a role in limiting cell death, tissue damage, and functional
recovery after SCI. Results from our study infer that therapeutic
strategies aimed at suppressing TNF- production and/or NF-B
activation after SCI should be reevaluated. Moreover, a regimen
directed at limiting apoptosis in the acute post-traumatic period also
may be beneficial in improving functional outcome after SCI.
| |
FOOTNOTES |
Received March 1, 2001; revised June 11, 2001; accepted June 13, 2001.
This study is supported by National Institutes of Health (NIH) Grants
NS37230, NS36350, and NS40162, by the Paralyzed Veterans of America, by
the International Spinal Research Trust, and by NIH spinal cord injury
PPG Grant NS39577. We thank Dr. Y. Y. He for providing expert
technical assistance in tissue preparation.
Correspondence should be addressed to Dr. Chung Y. Hsu, Department of
Neurology, Washington University School of Medicine, Box 8111, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: hsuc{at}neuro.wustl.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
