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The Journal of Neuroscience, November 1, 1998, 18(21):8780-8793
Myelin Gene Expression after Experimental Contusive Spinal
Cord Injury
Jean R.
Wrathall1,
Wen
Li2, and
Lynn D.
Hudson2
1 Neurobiology Division, Department of Cell Biology,
Georgetown University, Washington, DC 20007, and
2 Laboratory of Developmental Neurogenetics, National
Institute for Neurological Disorders and Stroke, Bethesda, Maryland
20892
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ABSTRACT |
After incomplete traumatic spinal cord injury (SCI), the spared
tissue exhibits abnormal myelination that is associated with reduced or
blocked axonal conductance. To examine the molecular basis of the
abnormal myelination, we used a standardized rat model of incomplete
SCI and compared normal uninjured tissue with that after contusion
injury. We evaluated expression of mRNA for myelin proteins using
in situ hybridization with oligonucleotide probes to
proteolipid protein (PLP), the major protein in central myelin; myelin
basic protein (MBP), a major component of central myelin and a minor
component of peripheral myelin; and protein zero (P0), the major
structural protein of peripheral myelin, as well as myelin
transcription factor 1 (MYT1). We found reduced expression of PLP and
MBP chronically after SCI in the dorsal, lateral, and ventral white
matter both rostral and caudal to the injury epicenter. Detailed
studies of PLP at 2 months after injury indicated that the density of
expressing cells was normal but mRNA per cell was reduced. In addition,
P0, normally restricted to the peripheral nervous system, was expressed
both at the epicenter and in lesioned areas at least 4 mm rostral and
caudal to it. Thus, after SCI, abnormal myelination of residual axons
may be caused, at least in part, by changes in the transcriptional
regulation of genes for myelin proteins and by altered distribution of
myelin-producing cells. In addition, the expression of MYT1 mRNA and
protein seemed to be upregulated after SCI in a pattern suggesting the
presence of undifferentiated progenitor cells in the chronically
injured cord.
Key words:
myelin; spinal cord injury; mRNA; proteolipid
protein; myelin basic protein; protein zero; myelin transcription
factor 1; in situ hybridization; Western analysis; immunocytochemistry
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INTRODUCTION |
Traumatic spinal cord injury (SCI)
results in loss of tissue including important myelinated fiber tracts
carrying descending motor and ascending sensory information. However,
in ~50% of patients, the injury is incomplete because some residual
function remains (Bracken et al., 1990 ). In animal models of incomplete
SCI, the residual white matter in the chronic injury site has been
found to be hypomyelinated, and this has been associated with abnormal impulse conduction properties of surviving axons (Blight, 1983a,b). There is also evidence of abnormal myelination after human SCI (Bunge
et al., 1993). That such abnormalities contribute to functional impairment in human SCI is indicated by the beneficial effect of
treatment with 4-aminopyridine (Hansebout et al., 1993; Hayes et al.,
1994), a potassium channel blocker that restores conduction in
deymyelinated internodes.
A demyelination-remyelination sequence of events has been postulated
to account for chronic hypomyelination after SCI (for review, see
Blight, 1993). Demyelinated axons have been observed in the cord after
experimental SCI (Gledhill et al., 1973; Griffiths and McCulloch, 1983;
Bunge et al., 1994). Loss of oligodendrocytes by apoptotic mechanisms
has recently been demonstrated (Li et al., 1996; Crowe et al., 1997;
Liu et al., 1997; Shuman et al., 1997), which could contribute to
aberrant remyelination of residual axons. However, the regulation of
genes for myelin proteins in surviving oligodendrocytes may also be
affected by SCI. To explore this hypothesis, we used in situ
hybridization to examine the expression of genes involved in
myelination after a standardized incomplete experimental spinal
contusion injury.
Myelin in the CNS is characterized by two major structural
proteins (for review, see Hudson, 1990). Proteolipid protein (PLP) accounts for ~50% of CNS myelin protein. Myelin basic protein (MBP)
constitutes 30% of CNS myelin protein and is also a minor component
(5-15%) of peripheral nervous system (PNS) myelin. The major protein
of PNS myelin, protein zero (P0), is not normally detected in the
spinal cord. However, P0 might be expected in pathological situations
such as SCI characterized by Schwann cell invasion of the CNS
(Griffiths and McCulloch, 1983). In recent years transcription factors
that seem to be involved in the coordinate regulation of expression of
the program of genes for myelin synthesis have been identified. The
first of these, myelin transcription factor 1 (MYT1), is a zinc finger
protein (Kim and Hudson, 1992) that has been shown to be expressed not
only in developing oligodendrocytes (Armstrong et al., 1995) but also
in many glial tumors and in areas of the CNS where progenitor cells are
found (Armstrong et al., 1997). To the extent that remyelination after
SCI may involve the differentiation of progenitor cells into new
oligodendrocytes to replace those lost by injury, we might expect
increased expression of MYT1. We therefore used oligonucleotide probes
to PLP, MBP, P0, and MYT1 to examine expression of these myelin-related
genes after a standardized incomplete spinal cord contusion injury in the rat.
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MATERIALS AND METHODS |
Spinal cord injury. A standardized contusive
experimental SCI was produced in adult Sprague Dawley rats as described
previously (Wrathall et al., 1985). Briefly, a 10 gm weight was allowed
to drop 2.5 cm onto a impounder (2.4-mm-diameter tip) resting on exposed dura at the T8 vertebral level to produce a contusion injury.
This results in an incomplete chronic lesion characterized at the
epicenter by a central cavity devoid of normal CNS tissue and a
peripheral rim of residual white matter (Noble and Wrathall, 1985,
1989).
Our previous studies with this model have shown hindlimb paralysis and
areflexia at 1 d followed by partial recovery of function that
stabilizes to reveal the chronic functional deficits by 3 weeks after
injury (Gale et al., 1985; Wrathall et al., 1985). Functional deficits
correlate with loss of spinal cord tissue at the epicenter, the region
of maximal damage at the injury site (Noble and Wrathall, 1985, 1989).
By 2 months after injury, a typical lesion extends from the epicenter
longitudinally for 10-12 mm, with the lesion area tapering
rostrocaudally to a small region in the dorsal white matter.
Electron microscopy. To characterize chronic histopathology
further, three rats at 2 months after SCI and four normal controls were
anesthetized and perfused with saline followed by fixative solution
(2% glutaraldehyde, 2% paraformaldehyde, and 2 mM
CaCl2 in 0.1 M cacodylate buffer, pH 7.3). A
tissue chopper was used to prepare 200 µm cross-sections that were
post-fixed (1% osmium and 1.5% potassium ferricyanide in cacodylate
buffer for 2 hr), stained en bloc with uranyl acetate, dehydrated, and
flat embedded in Araldite resin (Ted Pella, Redding, CA). One
micrometer sections through the lesion epicenter were cut with glass
knives on an ultramicrotome, stained with toluidine blue, and examined
by light microscopy. Smaller blocks were prepared that represented
specified regions of the epicenter, and 50-70 nm thin sections
were cut with a diamond knife and mounted on copper grids. After
they were stained with lead citrate, the sections were viewed in a JOEL (Tokyo, Japan) 1200 EX electron microscope.
For quantitative analyses of myelinated axons, electron micrographs of
ventromedial white matter were prepared at an original magnification of
2000× and printed all with the same enlargement. For each rat,
micrographs representing different grid squares of a single section
were randomly selected for quantitative analysis. Each myelinated axon
whose cross-section could be seen in its entirety was measured in terms
of the diameter of the axoplasm (A) and the diameter of the axon plus
myelin (AM). Myelin index (MI) was calculated as MI = A/AM.
In situ hybridization. At 1 week, 2 months, and 6 months after SCI, rats were killed by decapitation, and spinal cords
from injured rats (n = 3 at each time point) and normal
controls were rapidly removed. The tissue was frozen in blocks that
contained both one uninjured control and one or more injured spinal
cords (e.g., cords at different times after injury). Serial 12 µm
cross-sections were prepared on a cryostat, thaw mounted on slides
coated with 3-aminopropyltriethoxysilane (Sigma, St. Louis, MO), and
stored frozen until they were used for in situ
hybridization. Slides representing each millimeter length of cord were
stained with luxol blue and counterstained with hematoxylin and eosin
and used to reconstruct the injury site, as described previously (Noble and Wrathall, 1989; Teng and Wrathall, 1997). Based on this, a series
of slides representing the lesion epicenter and specified distances
rostral and caudal to it was selected for hybridization with the
various probes.
Oligonucleotide antisense sequences with a length of 48 bases were used
as probes for the following genes associated with myelination (Hudson,
1990): P0, the major structural protein of PNS myelin (50%); PLP, a
major protein of CNS myelin (50%); and MBP, a major component of CNS
(30%) and a minor component of PNS (5-15%) myelin. In addition we
probed for myelin transcription factor 1 (MYT1) that is expressed in
developing oligodendrocytes and seems to be involved in the coordinate
expression of genes for myelin proteins (Hudson et al., 1997). Figure
1 shows the probe sequences and the
location of these sequences in their respective genes. The
oligonucleotides were labeled with S35-dATP
homopolymer tails using terminal deoxynucleotidyl transferase (Stratagene, La Jolla, CA). For in situ hybridizations,
slides were post-fixed with 4% formaldehyde in PBS, pH 7.4, for
10 min, acetylated (0.25% acetic anhydride in 0.1 M
triethanolamine HCl, pH 8, for 10 min), and dehydrated with graded
alcohols and chloroform. They were then incubated overnight at 37°C
with hybridization buffer (50% formamide, 600 mM NaCl, 80 mM Tris-HCl, pH 7.5, 4 mM EDTA, 0.1%
sodium pyrophosphate, 0.2% SDS, 0.2% sodium heparin, 10%
dextran sulfate, and 100 mM dithiothreitol) containing
0.5-1 × 105 cpm per µl of
S35-labeled probe. The next day the slides were
washed sequentially with 1× SSC (0.15 M NaCl, and 15 mM sodium citrate, pH 7.0), 2× SSC with 50% formamide
(four times for 15 min each at 40°C), and 1× SCC (60 min ambient)
and then dehydrated and allowed to air dry. The slides were dipped in
Kodak (Rochester, NY) NTB2 liquid photographic emulsion, exposed
in the dark at 4°C for 6-14 d, and then developed and counterstained
lightly with hematoxylin or hematoxylin and eosin.
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Figure 1.
Oligonucleotide probe sequences used and the
location of these sequences in their respective genes.
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Quantification was performed with slides exposed for a short period
(e.g., 6 d) to limit the number of grains and thus errors of
coincidence. Grains were counted with the aid of a Zeiss IBAS image analysis system, in which control and injured tissue sections were compared on the same slide and background counts from slide areas
devoid of tissue were subtracted. For area counts, a rectangular template encompassing 7912 µm2 was placed
at specified positions in the ventral, lateral, or dorsal funicular
white matter on both left and right sides of the tissue section, and
counts were taken from the template areas in at least three section
pairs per slide. For evaluating PLP grains per cell, a circular
template delimiting an area of 227 µm2 was
centered on each sequential hematoxylin-stained nucleus in the
specified white matter areas, and counts were recorded from these as
well as nearby background areas devoid of tissue. All cell areas with
counts less than the average background were considered unlabeled
cells. Counts of net grains and the number of labeled or unlabeled
cells were compared for the sampled areas (n = 6-8) of
injured and control tissue sections on the slide by the use of
Student's t test (p < 0.05).
Western analysis. Protein levels for MBP in spinal cord
tissue at 2 months after SCI were compared with those in laminectomy controls. For Western analysis, rats (n = 5 per group)
were decapitated, and 2 cm lengths of spinal cord centered on T8 (the
injury site) were rapidly removed, weighed, and frozen at  70°C
until used. Tissue homogenates were prepared in 0.32 M
sucrose using a Kontes Dounce homogenizer (Fisher Scientific, Houston,
TX), a myelin fraction was prepared by sucrose density centrifugation
(Norton and Poduslo, 1973), and protein concentrations were determined (Lowry et al., 1951). Myelin fraction samples (4.5 µg of total protein per well) and MBP (M 1891; Sigma) standards were subjected to
SDS-acrylamide electrophoresis (Laemmli and Favre, 1973) and transferred onto nitrocellulose filters. MBP was detected with a
polyclonal rabbit anti-bovine MBP (1:1000) generously provided by Dr.
John Whittaker (Birmingham, AL), using a peroxidase-conjugated goat
anti-rabbit IgG (Amersham, Arlington Heights, IL) as the secondary
antibody. LumaGLO (Kierkegaard & Perry, Gaithersburg, MD)
chemiluminescent substrate was used, and the reaction was detected with
Hyperfilm-MP autoradiography film (Amersham) and quantified by
densitometry using PDI Discovery System (PDI, Huntington Station, NY)
scanner and software, based on curves calculated from the MBP standards
run with each gel. Values for injured and control samples
(n = 5 each group) were compared by the use of Student's t test. A p value of <0.05 was
considered significant.
Immunocytochemistry. For MBP immunocytochemistry, rats at 2 months after injury (n = 4) and uninjured controls
(n = 2) were anesthetized and perfused intracardially
with saline followed by 4% phosphate-buffered paraformaldehyde, pH
7.4. After fixation for an additional hour, 1.5 cm lengths of spinal
cord tissue centered at T8 (the injury site) were cryoprotected by
transfer through 10-30% sucrose solutions and frozen in blocks that
contained both normal and injured tissue; serial cross-sections (20 µm) were prepared and stored frozen until used. The lesion was
reconstructed by staining slides representing each millimeter of tissue
with luxol blue, hematoxylin, and eosin; additional slides representing the epicenter and levels 2 and 4 mm rostral and caudal to it were used
for MBP immunocytochemistry. These slides were air dried and then fixed
for 10 min with 10% buffered formalin. After washes with PBS
containing 1% Triton X-100 (TPBS), they were dehydrated through graded
alcohols and xylene, washed with TPBS, and air dried. After being
blocked with hydrogen peroxide and normal goat serum, they were
incubated overnight at 4°C with rabbit anti-MBP (catalog # 08-0038;
Zymed, San Francisco, CA), and the antigen-antibody complex was
detected using the Zymed Histostain-SP kit.
Immunohistochemistry was also performed on normal tissue and tissue at
6 weeks after SCI (n = 3) using  MYT1-his polyclonal rabbit antibody (Armstrong et al., 1995) and MYT1L rabbit
polyclonal antibody (Kim et al., 1997) essentially according to the
avidin-biotin peroxidase complex (ABC) method described by Hsu et al.
(1981). To block endogenous peroxidase activity and nonspecific
immunostaining, we immersed sections in Tris-buffered saline (TBS)
containing 3% hydrogen peroxide before treating them with 10% normal
goat serum containing 1% bovine serum albumin and 0.1% sodium azide for 40 min at room temperature. Sections were incubated with primary antibody (1:50 dilution for MYT1; 1:400 dilution for MYT1L) overnight at 4°C. After incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 40 min at room temperature, the avidin-biotin peroxidase complex reagent in a Vectastain Elite kit
(Vector Laboratories) was applied according to the manufacturer's instructions, using a freshly prepared 0.05% diaminobenzidine solution containing 0.01% H2O2. All TBS
buffers contained 0.1% Triton X-100. Double immunostaining for the
glial markers GFAP (astrocytes), CC1 [oligodendrocytes (Bhat et
al., 1996; Shuman et al., 1997)], or OX42 (microglia) and MYT1
was also performed with enzymatic methods. The monoclonal anti-GFAP
(1:50 dilution; Boehringer Mannheim, Indianapolis, IN), anti-CC1
(1:1000 dilution; APC-7; Oncogene Research Products, Cambridge, MA), or
OX42 (Serotec, Harlan, Westbury, NY: 1:1000 dilution) antibodies
were detected by alkaline phosphatase anti-mouse IgG (Promega, Madison,
WI), and polyclonal MYT1 was detected by the horseradish peroxidase system (Vector Laboratories).
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RESULTS |
Hypomyelination in the chronic injury site at 2 months
after SCI
The injury epicenter (Fig.
2A) typically contained
a central cavity devoid of normal spinal tissue but containing variable numbers of non-neuronal cells (Fig. 2B). These
included large numbers of debris-laden macrophages as well as vascular
elements and glia (see also Fig. 10). The central lesion was surrounded by a peripheral rim of residual white matter. By electron microscopy, the residual white matter appeared abnormal (Fig.
2D). Hypertrophied astrocytic processes produced a
pale matrix of interaxonal material. Compared with uninjured controls
(Fig. 2C), there was an obviously reduced number of axons,
especially large axons. Although the residual axons were myelinated by
oligodendrocytes, as expected, many axons appeared to be hypomyelinated
(Fig. 2D); occasionally, some also seemed to have
myelin sheaths that were thicker than normal (data not shown).
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Figure 2.
The injury epicenter at 2 months after incomplete
contusive SCI. A, One micrometer plastic section showing
the partial peripheral rim of residual white matter surrounding a
central lesion cavity containing a loose accumulation of cells. 22×.
B, Higher magnification showing residual white matter
(wm), central cavity, and cells in the lesioned area
(lc). 80×. C, D, Electron
micrographs of ventral white matter from a normal uninjured spinal cord
(C) and a lesion epicenter
(D). 5000×. Compared with normal, the residual
white matter of the chronic epicenter demonstrates reduced axon density
interspersed with hypertrophic astrocytic processes resulting in a pale
interaxonal matrix. Large axons are conspicuously absent, and the
myelination of the remaining axons is frequently thinner than
normal.
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The myelin index (ratio of axon diameter to axon diameter plus its
myelin sheath) showed an approximately normal distribution with a peak
at 0.7 or 0.8 for axons in the ventromedial white matter of four normal
rats (Fig. 3A). The
distribution curves appeared wider and peaked at a higher ratio
(thinner myelin) ranging from 0.8 to 0.9 in the preserved ventromedial
white matter of three rats at 2 months after SCI (Fig. 3B).
The average myelin index was not significantly different with the
sample size studied. However, the proportion of axons with very thin
myelin sheaths (myelin index  0.9) was significantly greater
(Student's t test, p = 0.026) in the
injured group (31.9 ± 10.4%) than in uninjured controls
(3.7 ± 2.1%).
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Figure 3.
The myelin index (ratio of axon diameter to the
diameter of the axon plus its myelin sheath) of axons in normal and
chronic SCI ventromedial white matter. A, The index
showed an approximately normal distribution with a peak at 0.7 or 0.8 for axons in the ventromedial white matter of four normal rats
(n = 92-124 axons per rat). B, The
distribution curves appeared wider and peaked at a higher ratio
(thinner myelin), ranging from 0.8 to 0.9, in the preserved
ventromedial white matter in three rats at 2 months after SCI
(n = 100-167 axons per rat).
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PLP and MBP gene expression after SCI
In normal adult rat spinal cord, there was a stereotypic pattern
of myelin gene expression, as shown in Figure
4. PLP mRNA was heavily expressed as
clumps of grains in the white matter and to a lesser extent in the gray
matter but was not detected in peripheral nerve roots. MBP mRNA was
strongly expressed as evenly distributed grains in the white matter.
Its expression in the spinal cord showed a regional distribution that
was similar to PLP with reduced expression in the gray matter. It was
also expressed in peripheral nerve roots. P0 mRNA was seen only in peripheral nerve roots in which a clumped distribution was the characteristic pattern.
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Figure 4.
Pattern of myelin gene expression in normal adult
rat spinal cord. Diagram of the dorsal horn region of a spinal cord
cross-section indicates the location of gray matter
(GM) of the dorsal horn with the superficial
substantia gelatinosa (Gel) that is devoid of
myelin, lateral-myelinated white matter (WM),
and, external to the cord, a portion of a peripheral dorsal nerve root.
Sequential serial sections hybridized with probes for myelin structural
protein genes show PLP expression absent in the nerve root and
substantia gelatinosa but heavily expressed in the white matter with a
clumped pattern. MBP expression has a similar distribution in the
spinal cord, but grains are more dispersed; MBP also shows some
expression in the dorsal root. P0 is not seen in the spinal cord but is
heavily expressed in a clumped pattern in the dorsal root.
44×.
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All tissue examined after SCI at 7 d, 2 months, or 6 months after
injury showed reduced PLP and MBP expression compared with that in
uninjured control tissue on the same slide (Fig.
5). The reduced expression was seen in
sections representing the injury epicenter (see Fig.
10B) and up to 6 mm rostral and caudal to the epicenter, the farthest distance at which tissue was examined. Quantification of the effect of SCI on PLP and MBP expression is shown
in Figure 6 for a representative pair of
normal and injured spinal cords with tissue sections at ± 4 mm
from the lesion epicenter at 2 months after SCI. White matter in the
dorsal, lateral, and ventral funiculi both rostral (Fig.
6A,B) and caudal (Fig.
6C,D) to the epicenter showed
significantly reduced grain counts per area for MBP and PLP in injured
compared with normal tissue. To determine whether this was likely
caused by a reduction in the number of myelin gene-expressing cells
chronically after SCI, we compared the number of cell nuclei per area
positive for grains after hybridization with the PLP probe in normal
and injured white matter (Fig.
7A). We found no difference in
the number of cells expressing PLP mRNA but a shift in the grain
density (grains per cell) as compared with normal for cells in both the
dorsal and ventral funicular white matter (Fig.
7B,C). However, the number of
unlabeled cells and the total number of cells per area were significantly increased. For example there were 41 ± 2.7 nuclei per white matter area in control versus 67 ± 6.2 nuclei per white matter area in chronically injured ventral white matter (t
test, p < 0.05). Similar increases were seen in dorsal
and lateral white matter (data not shown), and counts of the ventral
gray matter revealed an even greater increase in unlabeled cell density
(65 ± 2.5 vs 167 ± 15.7).
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Figure 5.
Reduced expression of PLP and MBP mRNA chronically
after SCI. A, PLP expression in normal uninjured
ventromedial white matter is primarily seen as grains associated with a
subset of nuclei in the white matter. B, MBP expression
is more evenly distributed over nuclei and cell processes.
C, PLP expression shows the same distribution but
appears reduced in amount in a section that is 4 mm caudal to the
epicenter from a cord at 2 months after SCI and that was mounted on the
same slide shown in A. D, MBP expression
that is 4 mm caudal to the epicenter is also reduced as seen in this
section of injured tissue from the same slide shown in
B. A-D, 158×.
Arrowheads, PLP-positive cells.
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Figure 6.
Quantification of the relative expression of PLP
and MBP mRNA in normal white matter and residual white matter at 2 months after SCI. White matter in the dorsal, lateral, and ventral
funiculi both rostral (A, B) and caudal
(C, D) showed significantly reduced grain
counts per area for PLP (A, C) and MBP
(B, D) in injured compared with normal
tissue mounted on the same slide. Bars represent the mean ± SEM
of grains counted in six sampled areas (7912 µm2
each); * indicates values significantly different from uninjured
control tissue on the same slide (t test,
p  0.05). P.I., Postinjury.
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Figure 7.
PLP mRNA-expressing cells and
the relative expression per cell at 2 months after SCI.
A, Comparison of the number of cell nuclei per area
positive for grains greater than background after hybridization with
the PLP probe in normal and injured white matter. Bars represent mean
number ± SEM based on six to eight areas analyzed for each white
matter region from sections on the same slide representing normal and
chronic injured tissue at 4 mm rostral to the epicenter.
B, C, Frequency distributions of net
grains per cell in labeled cells in the ventral white matter at 4 mm
rostral (B) and 4 mm caudal
(C) to the epicenter of in-jured compared
with normal tissue on the same slides. Net grains (minus
background) were counted in 227 µm2 circles
centered on each nucleus in the areas sampled for a total of 35-40
cells analyzed per group. P.I., Postinjury.
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MBP protein chronically after SCI
Myelin preparations from normal and injured spinal cords contained
three major bands of MBP-immunoreactive protein (Fig.
8A). These corresponded
to the 14, 17 and 18.5 (not fully resolved in our gels), and 21.5 kDa
isoforms characteristic of rodent MBP (Carson et al., 1983; de Ferra et
al., 1985). Quantitative analysis (Fig. 8B) indicated
a small reduction in MBP protein per microgram of myelin preparation
protein that was statistically significant only with respect to band 1 (21.5 kDa). However, the myelin preparation yield was lower for the
injured tissues. Thus, there was a significant reduction in each of the
bands and in total MBP protein per gram of spinal cord tissue compared
with that in uninjured controls (Fig. 8C).
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Figure 8.
Expression of MBP protein after SCI.
A, Western blots showed three major bands of
MBP-immunoreactive protein in myelin preparations from normal thoracic
spinal cord (N) and in tissue from the
injury sites at 2 months after SCI (I).
S, MBP standard. Molecular weights in kilodaltons (kDa)
of the major bands are shown on the right.
B, Quantification of MBP bands as micrograms per
microgram of total protein applied in normal and injured samples
(n = 5) shows a significant (*) reduction in band 1 (t test, p 0.05).
C, MBP expressed as micrograms per gram wet weight of
spinal cord tissue demonstrates significant (*) reductions in each
isoform as well as in total MBP. P.I., Postinjury.
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Immunocytochemistry demonstrated preferential and relatively even MBP
staining in the white matter of normal spinal cord tissue (data not
shown). At 2 months after SCI there was also considerable staining with
antibody to MBP, but in this case it was primarily associated with
spherical microcyst-like structures (Fig.
9A) seen in the residual white
matter. These MBP-positive structures appeared similar in shape and
size to the numerous myelin microcysts seen by electron microscopy at 2 months after injury (Fig. 9B).
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Figure 9.
Myelin microcysts and debris at 2 months after
SCI. A, Immunocytochemistry with antibody to MBP
demonstrates staining primarily associated with spherical
microcyst-like structures (arrow) seen in the residual
white matter of the chronic injury epicenter. 79×. B,
Myelin microcysts (arrow) and debris seen by electron
microscopy at 2 months after injury are shown. 4250×.
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P0 expression and Schwann cell myelination after SCI
P0 expression was not seen in the normal spinal cord or at 7 d after injury (data not shown) but was characteristically present at 2 and 6 months after SCI both at the epicenter and in the lesioned area
of the cord at least 4 mm distal from the epicenter (Fig. 10C,G). At the
epicenter the P0 expression was seen to be associated both with the
dorsal and ventral root entry zones as well as with the central
"cavity" that contains loosely arranged cells. Electron microscopy
demonstrated that this lesion area contained many axons myelinated by
Schwann cells (Fig. 10D,E), as well
as nests of ependymal cells and macrophages containing myelin debris.
Schwann cell-myelinated axons were distinguished by the presence of
cytoplasm, and sometimes the nucleus, of the myelinating cell located
externally to the myelin sheath and surrounded by a basal lamina
(Peters et al., 1991). The thickness of the myelin sheaths appeared
normal, although a formal analysis of myelin index was not performed.
In some cases, astrocytic processes were seen to separate groups of
axons myelinated by Schwann cells (Fig. 10D).
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Figure 10.
P0 expression in the chronic injured spinal cord.
A, SCI epicenter at 2 months after injury with a
peripheral rim of residual white matter (wm) and cells
in the lesion area (lc) within the central cavity is
shown. 35×. B, Dark-field microscopy of in
situ hybridization demonstrating PLP expression in an adjacent
section is reduced and restricted to the peripheral rim of residual
white matter. C, P0 expression is strong both in a
circular profile of dorsal root near the
top of the field as well as in the lesion zone of the
epicenter and also shows an expanded distribution near the dorsal and
ventral root entry zones. D, Electron microscopy of the
junction between residual spinal cord tissue and lesion zone reveals
many Schwann cell (sc)-ensheathed axons near an
astrocytic process (as). 10,000×. E, In
the lesion zone at the center of the epicenter, axons myelinated by
Schwann cells (sc) are frequently seen. 6000×.
F, At 4 mm caudal to the epicenter, the lesion
(arrowheads) is restricted to the dorsal funicular white
matter. 28×. G, In situ
hybridization shows strong expression of P0 mRNA in this lesion area
(arrowheads).
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Increased expression of MYT1 after SCI
MYT1 mRNA expression in the normal spinal cord was barely
detected in the white matter and was low in gray matter (Fig.
11A). The expression
of MYT1 mRNA appeared to be increased in injured SCI. At 2 months after
injury, increased MYT1 expression was seen at the epicenter (Fig.
11B) and in tissue rostral and caudal to the
epicenter (Fig. 11C,D). The increased MYT1
expression was seen primarily in the central gray matter of the cord
and was less but not completely absent in the lesioned zone where P0
was highly expressed (compare Figs. 11B,
10C).
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Figure 11.
MYT1 mRNA expression in the normal spinal cord
and 2 months after SCI. A, In the uninjured cord, MYT1
mRNA was barely detected in the white matter and was low in gray matter
(gm). B, MYT1 mRNA expression at
the lesion epicenter in a section adjacent to those shown in Figure
10A-C is shown. Expression is seen in the rim of
residual white matter, in the central lesion zone, and even in some
foci within an expanded dorsal root that appears in Figure
10C to be intensely positive for P0. C,
At 4 mm rostral to the epicenter, MYT1 is increased both in white
matter and especially in gray matter. D, At 4 mm caudal
to the injury site, in a section adjacent to those shown in Figure 10,
F and G, MYT1 mRNA appears primarily but
not completely absent in the lesion zone where PO expression is intense
(Fig. 10G) and seems especially prominent immediately
adjacent to the lesion zone (arrowheads).
A, C: Dark field, 26×. B,
D: Bright field, 38×.
|
|
Because the oligonucleotide probe for MYT1 is highly homologous to a
recently cloned family member of MYT1 named MYT1L (Kim et al., 1997),
the elevated hybridization signals found with MYT1 could reflect in
part an overexpression of MYT1L, a gene whose expression is restricted
to the neuronal lineage during development and is found in neurons at
the time of their terminal mitosis. To assess the relative expression
of MYT1 and MYT1L after SCI, we used antibodies generated against
portions of MYT1 (Armstrong et al., 1995) or MYT1L (Kim et al., 1997).
Both MYT1 and MYT1L were overexpressed in distinct cell populations of
the injured spinal cord. In normal rat, immunostaining for MYT1 (Fig.
12A) was detectable
primarily in cells forming the central canal and some neurons in the
gray matter. Although there was also some staining of glia in white
matter, it was mostly in the lateral rather than the dorsal or ventral
funiculi. Six weeks after SCI, the immunostaining for MYT1 was
elevated, both at the lesion epicenter (Fig. 12C) and at
least 4 mm distally (Fig.
12E,G,J).
MYT1 at the epicenter (Fig. 12C) was high in the peripheral
rim of preserved white matter but was also seen in the central lesioned
zone. Distal to the epicenter there was increased expression in neurons
of the gray matter as well as in cells in the white matter (Fig. 12E), especially the ventral medial white matter
(Fig. 12G) adjacent to the ventral median fissure. Two types
of MYT1 staining were observed. There was staining of virtually all of
the hypertrophic astrocytic processes (Fig. 12G) as
confirmed by GFAP and MYT1 double immunostaining (Fig.
12J); no nuclear staining for MYT1 was detected in
astrocytes. In addition, many nuclei of small round GFAP-negative cells
expressed MYT1 (Fig. 12G,J). As
shown in Figure 13, these cells with
MYT1-positive nuclei were distinct from mature oligodendrocytes labeled
with antibody to CC1 and from microglia stained with the OX42
antibody.
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Figure 12.
Immunostaining for MYT1(A,
C, E, G,
J) and MYT1L (B, D,
F, H, I,
K) proteins in normal and injured spinal cord.
A, In the normal spinal cord, MYT1 immunoreactivity is
present in cells of the central canal, in large neurons in the gray
matter, and to a lesser extent in cells in the white matter.
B, MYTL1 immunoreactivity in the uninjured cord is very
faint. C, At the lesion epicenter 6 weeks after SCI, the
intensity of MYT1 immunostaining is increased both in the peripheral
rim of preserved white matter and in the central lesion zone.
D, MYT1L staining at the epicenter in an adjacent
section is also increased but limited to portions of the peripheral rim
of white matter. E, Four millimeters distal to the
epicenter, MYT1 expression is still greater than that in normal cord
both in large neurons in the gray matter and in the white matter.
F, An adjacent section shows increased MYT1L
immunoreactivity. G, The most intense MYT1 expression is
in the ventromedial white matter (boxed region in
E) where many nuclei of small round cells are positive
as well as in the cytoplasm of cells with the morphology of astrocytes.
H, Enlargement of the boxed region in
B shows MYT1L immunoreactivity only in the most
superficial layers of the dorsal horn of normal spinal cord.
I, Similar enlargement of the dorsal horn
(boxed region in F) indicates that
after SCI the proportion of MYT1L-labeled cells is much increased.
J, Double immunocytochemistry for MYT1
(brown) and GFAP (blue) shows that
reactive astrocytes in white matter express both antigens
(arrow) in their hypertrophic processes. MYT1-positive
nuclei (arrowhead) are also present in many
GFAP-negative cells. K, Double immunocytochemistry for
MYT1L (brown) and GFAP (blue) shows that
some reactive astrocytes in white matter express both antigens
(arrow); other astrocytic processes are MYT1L-negative.
MYT1L staining is seen in the nuclei of GFAP-negative cells in
the white matter (small arrowhead) and in both the
nucleus and cytoplasm of large ventral horn neurons (large
arrowhead) in the adjacent gray matter. cc,
Central canal; dfu, dorsal funiculus; GM,
gray matter; lfu, lateral funiculus; vfu,
ventral funiculus. A-F: 35×. G-I:
135×. J, K: 320×.
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Figure 13.
Double immunostaining for MYT1 and the
oligodendrocyte marker CC1 (A, B) or the
microglial marker OX42 (C, D). At 6 weeks
after injury, sections 4 mm from the epicenter demonstrate ventromedial
immunostaining for MYT1 (brown) as well as CC1
(blue; A, B) and OX42
(blue; C, D). However, at
high magnification, no colocalization of MYT1 (arrow)
staining was seen with CC1 (B, arrowhead)
or OX42 (D, arrowhead). A,
C: 280×. B, D:
630×.
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|
The staining with MYT1L antibody in normal spinal cord (Fig.
12B) was limited to the nuclei of cells in the first
two layers of dorsal horn gray matter, where staining was barely
detectable (Fig. 12H). Six weeks after injury, MYT1L
expression was increased, perhaps to a relatively
greater extent than was MYT1. Elevated nuclear staining for MYT1L was
seen throughout the gray matter (Fig.
12F,I), and some neurons
also demonstrated cytoplasmic immunoreactivity (Fig.
12K). MYT1L immunostaining appeared increased
at the epicenter after SCI (Fig. 12D) where it was
restricted to the peripheral rim of surviving white matter. White
matter distal to the epicenter (Fig. 12F) also
demonstrated increased staining; some reactive astrocytes were positive
as well as the nuclei of a number of GFAP-negative cells (Fig.
12K).
| |
DISCUSSION |
Our results demonstrate chronic alterations in the myelination of
axons after experimental contusion injury of the spinal cord associated
with chronic alterations in myelin gene expression. These include
apparent downregulation of mRNA for PLP and MBP, major structural
proteins in CNS myelin, expression of P0, usually only found in the
PNS, and increased expression of MYT1, a gene associated with the
transcriptional regulation of myelin proteins and normally expressed
during development and in progenitor cells of the adult CNS.
We found that the residual white matter in the peripheral rim of
preserved tissue that we evaluated previously by light microscopy (e.g., Noble and Wrathall, 1985; Teng and Wrathall, 1997) exhibits profound ultrastructural abnormalities. These include reduced numbers
of axons compared with that in normal white matter, particularly large
myelinated axons. This is consistent with reports on residual white
matter in chronically contused ferret (Eidelberg et al., 1977), cat
(Blight, 1983a), and rat (Salgado-Ceballos et al., 1998) spinal cords.
It is also consistent with our finding significant axonal loss,
especially of large axons, 4 hr after contusion in the rat SCI model
used in the current study (Rosenberg and Wrathall, 1997). We also found
abnormal myelination, particularly hypomyelination, reflected in a
shift of the myelin index of axons in chronic SCI tissue, consistent
with previous data from the rat (Salgado-Ceballos et al., 1998) and
from cat, where hypomyelination was associated with abnormalities of
impulse conduction (Blight, 1983b).
Reduced myelination could result from loss of myelinating cells and/or
reduced myelin synthesis by surviving oligodendrocytes. There is a
prolonged loss of oligodendrocytes via apoptosis after SCI (e.g., Crowe
et al., 1997). This seems to be associated with Wallerian degeneration
of axons, involves the loss of only a small portion of cells at any
particular time, and thus might not significantly affect the ratio of
oligodendrocytes to axons. We found that the concentration of
myelinating cells (cells per area) that express PLP mRNA is not
significantly reduced in the chronically injured spinal cord. However,
the amount of PLP mRNA per cell is reduced, and more cells than normal
appear to express low levels. MBP mRNA cannot be similarly studied at
the cellular level because it is not restricted to a perinuclear
distribution (Zeller et al., 1985). However, based on area counts, its
reduction is at least equal to that of PLP. Because myelin synthesis
seems to be regulated at the transcriptional level (Cook et al., 1992),
our results suggest that the hypomyelination of surviving axons after
SCI may be caused, at least in part, by transcriptional downregulation in oligodendrocytes over a distance of many millimeters in the chronically injured rat spinal cord.
This conclusion must be considered tentative. We do not know that the
rate of myelin synthesis is actually reduced. That there is reduced
myelin in the injured cord is clearly established by the combination of
morphological and biochemical techniques used in this study. Western
analysis showed reduced MBP bands, and immunocytochemistry showed that
much of what remains is in inclusion bodies. In combination with the
evidence of reduced mRNA levels for both MBP and PLP, our data suggest,
but do not prove, that there is reduced myelin synthesis in the injured
spinal cord. This may reflect the reduced numbers of axons after SCI
and/or may contribute to the hypomyelination that characterizes many of
these axons.
In either case, the abnormal myelination seen in the chronically
injured spinal cord raises important and interesting questions. Do
surviving axons provide an inadequate signal for normal myelination by
oligodendrocytes? Are many of the myelinating cells damaged by SCI so
that they can no longer respond normally to the axonal signal(s) for
myelination? Are a significant proportion of the myelinating cells
after SCI derived from precursors present in adult white matter
(Armstrong et al., 1992) but incapable of myelinating axons in a normal
manner? In this respect, the recent report (Shihabuddin et al., 1997)
that a progenitor cell population can be isolated from adult rat spinal
cord and stimulated to divide with basic fibroblast growth factor
(FGF2) is of considerable interest. Cells in these cultures
differentiated into both neuronal and glial cell types, including
oligodendrocytes. Because FGF2 mRNA and protein are upregulated in the
spinal cord after SCI (Follesa et al., 1994; Mocchetti et al., 1996), a
similar stimulation of proliferation of progenitor cells may occur
in vivo. Our data suggest that the increased number of cells
in the white matter that are PLP-negative may include representatives
of such a progenitor cell population that express MYT1 and/or
MYT1L.
The presence of Schwann cells and axons ensheathed with PNS myelin in
the injured spinal cord at chronic time points has been noted in
previous studies (e.g., Griffiths and McCulloch, 1983). There is also
electrophysiological evidence that Schwann cell-myelinated axons in the
chronically injured spinal cord conduct impulses (Blight and Young,
1989). Our results confirm and extend these earlier studies. It is
intriguing that P0 expression is high in the tapered distal extensions
of the lesion characteristically present in the dorsal funiculus just
above the gray matter, an area that normally includes corticospinal
axons in the rat. Thus, an interesting question is whether any
surviving corticospinal axons become myelinated by Schwann cells or
whether the area becomes repopulated with other types of axons from the
CNS, or with sensory axons from the dorsal roots.
The abnormally thin myelin seen in residual white matter chronically
after SCI is thought to arise from a demyelination-remyelination sequence of events (Blight, 1993). Evidence of demyelination after SCI
has been reported by a number of investigators (Balentine, 1978;
Bresnahan, 1978; Griffiths and McCulloch, 1983; Bunge et al., 1994;
Rosenberg and Wrathall, 1997). Remyelination after SCI may be performed
by the surviving mature oligodendrocytes and/or oligodendrocyte
progenitors that could be stimulated to differentiate and myelinate
after injury. We used two approaches to examine the role of progenitor
cells in remyelination. One was to characterize the isoforms of myelin
proteins present after injury, because progenitor cells induced to
myelinate follow a typical developmental pattern in which exon
2-containing MBP isoforms (21 and 18 kDa) are initially highly
expressed. The other approach was to assess alterations in the
distribution and size of the progenitor pool after SCI using
transcription factor markers of oligodendrocyte (MYT1) and neuronal
(MYT1L) progenitors (Armstrong et al., 1995, 1997; Kim et al.,
1997).
We found MYT1 mRNA and protein were elevated after SCI. The most
intense concentration of MYT1-positive cells was in ventromedial white
matter (Fig. 12G), reminiscent of the ventral origin of
oligodendrocyte progenitor cells in the developing spinal cord (for
review, see Richardson et al., 1997). These cells with nuclear MYT1
staining were not positive for astrocyte, microglial, or mature
oligodendrocyte markers. They seem to represent a population of glial
progenitor cells present in the chronically injured spinal cord that
are distinct from reactive astrocytes expressing cytoplasmic MYT1.
Apart from this putative glial progenitor cell population marked by
MYT1, another cell population was revealed with an antibody to a
closely related family member, MYT1L (Kim et al., 1997). Restricted to
the neuronal lineage in normal development, MYT1L-positive cells
were conspicuous throughout the gray matter of the injured spinal cord.
Thus, both neuronal (MYT1L-positive) and glial (MYT1-positive) progenitor cells may have been stimulated to divide and/or been recruited to the site of injury.
The presence of cells expressing MYT1 or MYT1L hints that a block in
the developmental progression of these cells exists chronically after
SCI injury. Our finding a significantly increased cell density in white
matter of the chronically injured cord, cells not expressing detectable
levels of PLP mRNA, is consistent with this hypothesis. In injured
tissue, the relative depletion of the 21.5 kDa isoform of MBP (Fig. 8),
which is normally one of the first MBP proteins expressed in
oligodendrocyte development (Carson et al., 1983), also suggests that
oligodendrocyte precursors present in the lesion have not been induced
to fully differentiate and myelinate axons. We postulate that the
remyelination after SCI, marked by thin myelin sheaths, was likely
performed by mature oligodendrocytes that survived the injury and
continue to express myelin protein isoforms characteristic of mature
cells. Future studies examining the survival of oligodendrocytes after
SCI and the origin and fate of cells expressing MYT1 and MYT1L will be
needed to test these hypotheses.
In summary, we found that chronically after incomplete contusive SCI
there is abnormal myelination of residual axons that may be caused, at
least in part, by changes in transcriptional regulation of genes for
myelin proteins. There is also a dramatically altered distribution of
myelin-producing cells with substantial Schwann cell myelination in the
lesion zone. Additionally, an increased cell density in residual white
matter is associated with increased expression of MYT1, a transcription
factor present in oligodendrocyte precursors. These data suggest the
presence of undifferentiated progenitor cells in the chronically
injured cord. Thus, the abnormal myelination of spared axons after SCI may be less a problem of oligodendrocyte cell death than a problem in
the signaling pathways that normally lead to differentiation of
progenitor cells and their participation in axonal (re)myelination.
| |
FOOTNOTES |
Received April 27, 1998; revised July 20, 1998; accepted Aug. 14, 1998.
This work was supported by National Institutes of Health Grants NS28130
and NS35647 to J.W. and by intramural National Institute for
Neurological Disorders and Stroke funds. We thank Dr. Arnulf Koeppen
(VA Medical Center, Albany, NY) for the MBP antibody used in, and Dr.
Richard Quarles for teaching us his protocol for, Western blot analysis
of MBP. The densitometric analysis was performed in the Lombardi Cancer
Center's Macromolecular Synthesis and Sequencing Shared Resource,
supported in part by United States Public Health Service Grant
P30-CA-51008. Excellent technical assistance during the course of these
studies was provided by Lily Verma, David Choiniere, and Robert
Marriott. We also thank Carolyn Feltes for the MBP immunocytochemistry
that she performed during a research rotation in the laboratory.
Correspondence should be addressed to Dr. Jean Wrathall, Department of
Cell Biology, Georgetown University, 3900 Reservoir Road, Northwest,
Washington, DC 20007.
| |
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Top
Abstract
Introduction
