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The Journal of Neuroscience, February 15, 2001, 21(4):1302-1312
Late Oligodendrocyte Progenitors Coincide with the Developmental
Window of Vulnerability for Human Perinatal White Matter Injury
Stephen A.
Back1, 3,
Ning Ling
Luo1,
Natalya S.
Borenstein3,
Joel M.
Levine2,
Joseph J.
Volpe3, and
Hannah C.
Kinney3, 4
1 Department of Pediatrics, Oregon Health Sciences
University, Portland, Oregon 97201, 2 Department of
Neurobiology and Behavior, State University of New York, Stony Brook,
New York 11794, and the Departments of 3 Neurology and
4 Pathology, Children's Hospital and Harvard Medical
School, Boston, Massachusetts 02115
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ABSTRACT |
Hypoxic-ischemic injury to the periventricular cerebral white
matter [periventricular leukomalacia (PVL)] results in cerebral palsy
and is the leading cause of brain injury in premature infants. The
principal feature of PVL is a chronic disturbance of myelination and
suggests that oligodendrocyte (OL) lineage progression is disrupted by
ischemic injury. We determined the OL lineage stages at risk for injury
during the developmental window of vulnerability for PVL (23-32 weeks,
postconceptional age). In 26 normal control autopsy human brains, OL
lineage progression was defined in parietal white matter, a region of
predilection for PVL. Three successive OL stages, the late OL
progenitor, the immature OL, and the mature OL, were characterized
between 18 and 41 weeks with anti-NG2 proteoglycan, O4, O1, and
anti-myelin basic protein (anti-MBP) antibodies. NG2+O4+ late OL
progenitors were the predominant stage throughout the latter half of
gestation. Between 18 and 27 weeks, O4+O1+ immature OLs were a minor
population (9.9 ± 2.1% of total OLs; n = 9). Between 28 and 41 weeks, an increase in immature OLs to 30.9 ± 2.1% of total OLs (n = 9) was accompanied by a
progressive increase in MBP+ myelin sheaths that were restricted to the
periventricular white matter. The developmental window of high risk for
PVL thus precedes the onset of myelination and identifies the late OL
progenitor as the major potential target. Moreover, the decline in
incidence of PVL at ~32 weeks coincides with the onset of myelination
in the periventricular white matter and suggests that the risk for PVL
is related to the presence of late OL progenitors in the
periventricular white matter.
Key words:
development; cell lineage; cerebral white matter; cerebral cortex; myelination; O4 antibody; O1 antibody; NG2; myelin
basic protein; immunohistochemistry; neurofilament protein; microglia; periventricular leukomalacia; prematurity
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INTRODUCTION |
Periventricular leukomalacia (PVL)
is a common lesion of the periventricular cerebral white matter that
has its peak incidence in the premature infant and underlies the
subsequent development of cerebral palsy and cognitive impairment in
survivors of the newborn critical care nursery (Volpe, 2000 ). The risk
for PVL is high during a well defined period in human brain development (23-32 weeks, postconceptional age). Two major factors related to cerebrovascular immaturity predispose the developing periventricular white matter to injury from hypoxia-ischemia (Volpe, 1998). These are
the presence in periventricular white matter of arterial end and border
zones (Takashima and Tanaka, 1978; Nakamura et al., 1994) and a
propensity for the sick premature neonate to exhibit a pressure-passive
circulation related to a disturbance of cerebral autoregulation (Pyrds,
1991; Menke et al., 1997).
The major pathological feature of PVL is a chronic disturbance of
myelination and suggests that oligodendrocyte (OL) progenitors are a
target of ischemic injury in PVL (Back and Volpe, 1997). The
susceptibility of the rat OL lineage to oxidative stress, a well
established sequela of ischemia and reperfusion (Traystman et al.,
1991), is a maturation-dependent phenomenon, and late OL progenitors
in vitro are markedly more susceptible than mature OLs to
free radical-mediated injury (Back et al., 1998; Fern and Moller,
2000). However, the susceptibility of human OL progenitors to
hypoxia-ischemia has been difficult to evaluate directly, and there is
no animal model that reproduces the myelination disturbances of PVL.
The objectives of the present study were to determine the human OL
lineage stages at risk for injury during the developmental window of
vulnerability for PVL and to determine whether the risk for PVL might
correlate with the presence of more immature stages in the cerebral
white matter.
The human OL lineage has been characterized mainly in first trimester
spinal cord (for review, see Hardy, 1997), and there are no
studies from the human cerebrum that are relevant to the critical
period of vulnerability for PVL. During rodent white matter development
in vivo, OL progenitors express the platelet-derived growth
factor- receptor and the NG2 proteoglycan
(Levine et al., 1993; Nishiyama et al., 1996, 1999; Reynolds and
Hardy, 1997). Late OL progenitors (pre-OLs) are identified by labeling
with the O4 antibody and for NG2 but not with the O1 monoclonal
antibody (Warrington and Pfeiffer, 1992; Reynolds and Hardy, 1997).
Next in succession are immature OLs that label with both the O4 and O1
monoclonal antibodies but not with myelin basic protein (Warrington and
Pfeiffer, 1992). The mature OL is characterized by expression of
myelin-associated markers that include myelin basic protein (MBP)
(Sternberger et al., 1978).
We report here that the developmental window of high risk for PVL
coincides with a period in human cerebral white matter development during which one major population of late OL progenitors predominates and identifies them as a potential target for injury in PVL. The decline in incidence of PVL at ~32 weeks coincides with the onset of
myelination in the periventricular white matter and suggests a
developmental explanation for PVL that is related to the timing of
appearance of late OL progenitors in the periventricular white matter.
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MATERIALS AND METHODS |
Study population. Cases without significant brain
pathology were prospectively collected from the pediatric autopsy
populations of the Departments of Pathology at Oregon Health Sciences
University (Portland, OR), Brigham and Women's Hospital (Boston, MA),
Children's Hospital (Boston, MA), and the National Institutes of
Health Developmental Brain and Tissue Bank at the University of
Miami (Miami, FL; provided under the direction of Dr. Carol Petito).
"Normalcy" was defined as cases without gross brain pathology and
was confirmed by histological data with reference to agonal-metabolic
derangements, pertinent clinicopathologic data, and the cause of death.
Postmortem intervals of <24 hr were accepted for 24 of the 26 cases
(one case each at 26 and 30 hr). Cases were processed for standard
histological stains [hematoxylin and eosin (H+E) and H+E plus Luxol
fast blue] to permit evaluation of tissue sections by standard
histopathological criteria.
Tissue preparation and sectioning. Tissue was immersed at
the time of collection in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer and stored at 2-4°C.
Immunocytochemical labeling with the O4 and O1 mAbs was unaffected by
fixation in excess of 18 months. However, fixation for 6-18 hr yielded
optimal immunocytochemical labeling for NG2. Tissue blocks were adhered
to a metal stage, chilled on ice, and embedded in 1% low-gelling
temperature agarose (A-4018; Sigma, St. Louis, MO). Free-floating
sections (50 µm) were cut on a Leica VTS-1000 vibrating microtome in
ice-cold PBS.
Primary antibodies. The O4 and O1 mouse monoclonal
antibodies were isolated and purified by ammonium sulfate fractionation from the media of cultured hybridoma cells (Bansal et al., 1989) that
were the generous gift of Dr. Steven Pfeiffer (University of
Connecticut Health Center, Farmington, CT). Before dilution, the
concentration of purified O4 was 11.3 mg/ml, and that of O1 was 3.8 mg/ml. The panaxonal neurofilament marker SMI 312 and the anti-myelin
basic protein antibody SMI 99 were mouse monoclonal antibodies from
Sternberger Monoclonals (Lutherville, MD). The Ricinus
communis lectin microglial marker (biotinylated agglutinin RCA120, L-2641) and the mouse monoclonal antibody
neuronal marker anti- -tubulin isotype III (T-8660) were from Sigma.
A rabbit anti-bovine glial fibrillary acidic protein (GFAP) antibody
(Z-0334) was from Dako (Carpinteria, CA). Unless otherwise noted, all
immunohistochemical procedures used a 1 hr incubation in blocking
buffer (5% NGS in PBS), overnight incubation at 2-4°C with primary
antibodies, and a 2 hr incubation at room temperature (RT; 20°C) with
secondary antibodies, and all washes were 10 min in a minimum of 5 ml
of PBS at RT. Antibodies were diluted in PBS with 3% NGS.
Immunofluorescence histochemistry for O4 or O1 antibodies.
For single immunofluorescent labeling, the O4 or O1 mAb (1:1000) was
visualized with a µ-chain-specific fluorescein-conjugated IgM
secondary antibody (1:100; FI-2020; Vector Laboratories, Burlingame, CA). For double immunofluorescent labeling with O4 and O1, a
biotinylated O4 (bO4) antibody was synthesized (Research
Genetics, Huntsville, AL), because both primary antibodies are of the
same class (IgM). Tissue sections were first labeled overnight with O1
(1:250), followed by incubation for 2 hr at RT in
fluorescein-conjugated IgM secondary antibody (1:100). Sections were
next incubated overnight with bO4 antibody (1:100), followed by
incubation with rhodamine red X-conjugated streptavidin (1:400 in PBS;
016-290-084; Jackson ImmunoResearch, West Grove, PA). For double
immunofluorescent labeling with O4 or O1 (1:1000) and SMI 312 (1:1000),
anti--tubulin isotype III (1:100), anti-GFAP (1:1000), or
biotinylated agglutinin RCA120 (1:100), both
primary antibodies were coincubated with tissue sections. SMI 312 and
anti--tubulin isotype III were visualized with Fc fragment-specific
rhodamine red X-conjugated anti-mouse IgG (1:200; 115-295-071; Jackson
ImmunoResearch). Anti-GFAP was visualized with anti-rabbit IgG
conjugated to Texas Red (TI-1000; Vector Laboratories). Biotinylated
agglutinin RCA120 was visualized with rhodamine
red X-conjugated streptavidin (1:400 in PBS). Nonspecific labeling with
O1 mAb, when noted, was related to a prolonged postmortem interval or a
high titer of O1. It was markedly decreased both in magnitude and
distribution as the concentration of O1 was reduced from 800 to 4 µg/ml, whereas OL-specific labeling with O1 was unchanged. Specific
labeling with O1 was achieved at a concentration of 1-2 µg/ml.
Immunofluorescence double labeling of O4 or O1 antibodies and
NG2. After tissue sections were labeled with O4 or O1 (1:1000), they were washed, incubated for 5 min in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and immediately washed
three times in PBS at RT. After a 2 hr incubation with
µ-chain-specific fluorescein-conjugated IgM (1:100), all subsequent
incubations were done in the dark. Tissue sections were next incubated
overnight at 2-4°C with a rabbit polyclonal antibody raised against
NG2 (Ong and Levine, 1999) (1:1000 in PBS with 3% NGS and 0.1% Triton X-100). After incubation with biotinylated goat anti-rabbit IgG (1:200;
111-065-046; Jackson ImmunoResearch), sections were incubated for 2 hr
with rhodamine red X-conjugated streptavidin (1:400). Immunocytochemical controls, processed under conditions identical to
those described above, revealed no cross-reactivity between O4 or O1
and the Fc fragment-specific IgG secondary antibody. With omission of
both primary antibodies, there was no detectable nonspecific labeling
with either secondary antibody. It should be noted that the 5 min
incubation in 4% paraformaldehyde was required to prevent the
solubilization of the O4-antigen complex by 0.1% Triton X-100 that
was necessary for optimal visualization of NG2.
Quantitative morphometric analysis of O4- and O1-labeled
cells. Cell counts were obtained from a minimum of three sets of 50-µm-thick adjacent sections that were alternately stained with O4
or O1. Absolute counts were not determined because of the uneven thickness inherent in vibrating microtome-cut sections (Guillery and
Herrup, 1997). The nucleus was selected as the smallest countable object and was visualized by immunofluorescent counterstain with Hoechst 33324. Cells were systematically counted in similar sectors of
adjacent sections that included superficial, mid and deep cerebral white matter, and the germinal matrix. Cell profiles that contained a
nucleus were counted with a 20× objective equipped with a counting grid that was mounted on a Nikon Eclipse TE 300 inverted fluorescent microscope. The total O4-labeled cells counted varied with the gestational age of the case and ranged from 433 to 2867. In 14 cases,
at least 1000 total cells were counted. Immature OLs were calculated as
the percentage of total (i.e., O4-labeled) cells that labeled with O1.
Because the nucleus was selected as the smallest countable object, a
difference in the size of the nucleus for an O4- or an O1-labeled cell
would introduce a bias into the relative cell counts. To estimate
whether the nucleus differed in size for cells labeled with O4 versus
O1, the average nuclear area was determined for O4- or O1-labeled cell
profiles from three adjacent sections from a case at 18 and at 35 weeks. We predicted that if nuclear size varied with the state of OL
differentiation, that this difference would be amplified by comparing a
case at midgestation (i.e., 18 weeks) with one close to term. Nuclei,
labeled with Hoechst 33324, were randomly but systematically sampled
from a sector that included superficial, mid, and deep cerebral white
matter. Double-labeled profiles were visualized with a 40× oil
immersion objective (Nikon Plan Fluor; numerical aperture, 1.30) on a
laser-scanning confocal microscope (Noran Instruments) equipped with
argon ion and UV lasers coupled to an inverted microscope (Nikon
Diaphot 200). Nuclei were visualized by excitation with 360 nm ( )
light using a UV bandpass filter, and fluorescence emission was
detected at >470 nm (). The beam was attenuated using a 25 nm
dichroic confocal aperture. For all measurements, the laser settings
that included brightness, contrast, and exposure time (100 nsec) were held constant. Frame-averaged confocal images (128 frames/image) were
digitalized at 512 × 480 pixels using microcomputer-based imaging
software (Noran OZ with Intervision). For analysis of nuclear area,
regions of interest were selected by image threshold analysis. Mean
nuclear area was calculated from a minimum of 40 cellular profiles for
all four groups sampled.
At 18 weeks, there was no significant difference (paired Student's
t test) between the size of the nucleus of an O4+ cell (39.0 ± 8.3 µm2; n = 112 profiles) and that of an O1+ cell (37.5 ± 8.3 µm2; n = 40 profiles).
At 35 weeks, there was also no difference between the size of the
nucleus of an O4+ cell (54.5 ± 10.9 µm2; n = 164 profiles)
and that of an O1+ cell (54.8 ± 10.6 µm2; n = 93 profiles).
The size of the nucleus was significantly less (unpaired Student's
t test) at 18 weeks than at 35 weeks for an O4+ cell
(p < 0.0001) or for an O1+ cell
(p < 0.0001). We concluded that at any given
gestational age, there was no difference in the size of the nucleus of
a pre-OL or an immature OL.
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RESULTS |
Clinicopathologic information
The parietal white matter at the level of the trigone was analyzed
in 26 human brains, ranging in age from 18 to 41 postconceptional weeks. The mean postmortem interval of autopsy cases
was 19 ± 7 hr. This study involved analysis of cases at the
following postconceptional ages in weeks: 18 (n = 1),
20 (n = 3), 21 (n = 1), 22 (n = 3), 24 (n = 2), 26 (n = 1), 27 (n = 2), 28 (n = 1), 30 (n = 2), 31 (n = 1), 34 (n = 1), 35 (n = 2), 36 (n = 1), 37 (n = 1), 40 (n = 3), and 41 (n = 1). The brains were free of PVL or other major malformative or acquired lesions. The causes of death were as follows:
therapeutic abortion (n = 7), stillborn
(n = 1), extreme prematurity (n = 1),
congenital heart disease (n = 3), major noncerebral congenital malformations (n = 5), respiratory distress
(n = 5), fetal distress at delivery (n = 2), sepsis (n = 1), and undetermined (n = 1).
Numerous O4- and NG2-labeled cells populate the human cerebrum
by midgestation
In a series of 13 cases between 18 and 27 weeks, we first examined
the distribution of cells that labeled with the O4 antibody. At 18 weeks, numerous O4-labeled somata were present in both the parietal
white matter and the cortical mantle. The distribution of O4-labeled
somata in the white matter was nonuniform (Fig. 1A). They were more
numerous in the superficial (Fig. 1B) and mid (Fig.
1C) cerebral white matter, and fewer somata were present in
the deep white matter (Fig. 1D) or the germinal
matrix (Fig. 1E). Many O4-labeled somata were also
present in the cortical mantle as early as 18 weeks (Fig.
2A). These cells
appeared less differentiated than those in the white matter and were
associated with a fine meshwork of narrow processes.
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Figure 1.
Distribution of O4 mAb-labeled cells in the
cerebral white matter at 18 weeks. A, Low-power
photomontage showing the regional distribution of cells in the
superficial (SWM), mid
(MWM), and deep (DWM)
cerebral white matter and the germinal matrix
(GM). B-E, Detail of the
morphology and distribution of cells shown in A in the
SWM (B), MWM
(C), DWM
(D), and GM
(E). Scale bar: A, 110 µm.
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Figure 2.
A, Low-power photomontage shows the
regional distribution of O4 mAb-labeled cells in the cortical mantle
from a case at 18 weeks. B-E, Confocal laser digital
images demonstrate that OL precursors in the cortical mantle (B,
C) and in the cerebral white matter (D, E)
labeled for both the O4 antibody (B, D) and NG2
(C, E). Note that the cells in the cortical mantle
appear morphologically less mature compared with those in the white
matter. Scale bars: B, C, 20 µm; D,
E, 25 µm.
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NG2-labeled cells were visualized in four cases between 18 and 26 weeks
of gestation. In all cases, numerous NG2-labeled cells were present in
the white matter and cortical mantle. The morphology and distribution
of these cells were very similar to that visualized with the O4
antibody. Immunocytochemical double-labeling studies with the O4 and
anti-NG2 antibodies demonstrated that the entire population of
NG2-labeled cells in the cortical mantle (Fig. 2C) and in
the white matter (Fig. 2E) also labeled with O4 (Fig.
2B,D).
The O4+NG2+ cells were morphologically diverse (Fig.
3). Some cells were characterized by an
asymmetric bipolar morphology (Fig. 3A). The asymmetry
derived from an initial short process that branched into two major
processes. Other bipolar cells were typically elongated or fusiform in
shape with a similar degree of branching from each pole (Fig.
3B). Another population of asymmetric simple multipolar
cells had several processes that were elaborated from one pole of the
soma (Fig. 3C). Other simple multipolar cells had more
uniformly distributed processes (Fig. 3D). Both types of
bipolar cells were most numerous in the superficial white matter and
were less common in the mid and deep white matter.
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Figure 3.
Representative confocal laser microscopic digital
three-dimensional reconstructions of NG2-positive cells in the cerebral
white matter. Representative examples of the variety of morphological
subtypes of pre-OLs identified in the white matter are shown.
A, An asymmetric bipolar cell. B, A
symmetric bipolar cell. C, An asymmetric multipolar
cell. D, Simple and complex multipolar cells. Scale
bars: A-D, 25 µm.
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To confirm that the NG2+O4+ cells were distinct from other neural cells
in fetal human brain, we stained sections with antibodies specific for
neurons, microglia, and astrocytes. Neuronal somata and processes
identified by -tubulin isotype III differed in morphology and
distribution from that of O4+ somata in the cortical mantle (Fig.
4A) and white matter
(Fig. 4B). Astrocytes identified by glial fibrillary
acidic protein also did not overlap with O4+ somata (data not shown).
We examined two markers that label microglia in the developing human
brain (Streit et al., 1999). The LN-3 antibody, reactive against
HLA-DR antigen, detected reactive microglia at sites of cerebral
necrosis in a case at term gestation but failed to detect most of the
resident population of ramified and/or resting microglia (data not
shown). However, the R. communis lectin RCA120 detected numerous ramified microglia in
the cerebral white matter and cortical mantle in this same case and
five additional cases at 18, 20, 31, 35, and 40 weeks of gestation.
Although the RCA120-labeled microglia had a
morphology and distribution similar to that of the bipolar and simple
multipolar cells visualized with the O4 antibody, we found no overlap
in the distribution of cells labeled by these two markers (Fig.
4C).
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Figure 4.
Representative confocal laser microscopic digital
images demonstrate the specificity of the O4 antibody for OL
precursors. A, B, O4-labeled cells
(green) differ in morphology and distribution
from neuronal somata (red) in the cortical mantle
(A) and from neuronal processes
(red) in the superficial white matter
(B) that were visualized with anti--tubulin
isotype III. C, Microglia labeled with the R.
communis lectin (red) have a similar morphology
but a distinctly different distribution from that of O4-labeled cells
(green) in the superficial white matter. Scale
bars: A, 20 µm; B, C, 25 µm.
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Few O1-labeled cells are present in the human cerebrum between 18 and 27 weeks
In all 13 cases examined between 18 and 27 weeks, numerous
cells were visualized with the O4 antibody (Fig.
5A), but few O1-labeled cells
were visualized in the white matter (Fig. 5B), and none was
visualized in the cerebral cortex. All of the O1-labeled cells displayed a complex multipolar morphology characterized by a round soma
that elaborated numerous extensively branched processes.
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Figure 5.
Pre-OLs are more abundant than immature OLs in
human parietal white matter at midgestation. Representative examples of
the morphology and distribution of pre-OLs and immature OLs from cases
between 18 and 22 weeks of gestation in human parietal white matter are
shown. A, B, Cells labeled with O4
(A) were more numerous than those labeled with O1
(B). C, D, The morphology and
distribution of cells labeled with the native O4 antibody
(C) and a bO4 antibody (D)
were quite similar. E, F, Immunofluorescent double
labeling with the bO4 (E) and O1
(F) antibodies in the mid cerebral white matter
demonstrates that bO4 and O1 labeled a minor population of immature OLs
with a complex multipolar morphology. G, H, Confocal
laser microscopic digital images are shown of the morphology of complex
multipolar immature OLs that labeled with both the bO4
(G) and the O1 (H)
antibodies. Note the less differentiated-appearing pre-OLs that labeled
with bO4 but not O1 (arrows). Scale bars: A,
B, 100 µm; C-F, 60 µm; G, H,
25 µm.
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We next compared the distribution of cells visualized with the O1
antibody with that of cells labeled with a bO4 antibody (see
Materials and Methods). We first determined the effect of biotinylation
of O4 on the specificity of the antibody. Double-labeling studies with
the bO4 (Fig. 5D) and native O4 (Fig. 5C)
antibodies demonstrated a complete overlap in the staining obtained
with the two antibodies. The O1-labeled cells (Fig.
5F) were a small subset of the cells that labeled
with bO4 antibody (Fig. 5E). All of the O1-labeled cells
also labeled with the bO4 antibody. The double-labeled cells displayed
a complex multipolar morphology (Fig. 5G,H), whereas
the cells that labeled with only the bO4 antibody had a more simple
appearance (Fig. 5G, arrows). Double-labeling studies with O1 and for NG2 revealed an occasional NG2-positive cell
that stained weakly for O1, but most of these cells did not stain for
O1 (data not shown; see also Fig. 7C,D).
O4+O1+ immature OLs increase at ~30 weeks
Approximately 30 weeks marked the onset of an increase
in the number of O4+ cells that displayed a complex multipolar
morphology (Fig. 6A,B).
These cells were most extensive in the deep and mid cerebral white
matter, were sparsely localized to the superficial white matter, and
were not detected in the cerebral cortex. In 12 cases between 30 and 41 weeks, the O4 and O1 antibodies identified a similar population of
complex multipolar cells. Fluorescent double-labeling studies with the
bO4 and O1 antibodies confirmed that these cells were a single
population of O4+O1+ immature OLs (Fig. 6C,D). Interspersed
among the immature OLs were numerous O4+O1 pre-OLs (Fig.
6C,D, arrows). The pre-OLs appeared
morphologically less mature than did the immature OLs and were most
numerous in the superficial white matter.
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Figure 6.
Pre-OLs persist as the expansion in the immature
OL population occurs at ~30 weeks. Representative confocal laser
microscopic digital images from a case at 31 weeks demonstrate the
striking change in the morphology of OL precursors that occurs with the
expansion of the immature OL population in the parietal white matter.
A, B, A low-power image (A) and
detail (B) demonstrate the marked
increase in complex multipolar cells visualized here in the mid
cerebral white matter with the O4 antibody. C, D, Double
immunofluorescent-labeling studies with a biotinylated O4 antibody
(C) and the O1 antibody (D)
revealed that many of the O4-labeled cells were O4+O1 pre-OLs
(arrows) that were interspersed among the O4+O1+
immature OLs. Scale bars: A, B, 100 µm; C,
D, 20 µm.
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In two cases at 32 and 37 weeks, numerous cells were identified that
labeled with O4 and for NG2. In general, cells that appeared morphologically less mature labeled with similar intensity for both
antibodies (Fig. 7A,B).
However, complex multipolar cells that labeled with O1 (Fig.
7D, arrow) or with O4 (data not shown) labeled
weakly for NG2. The less mature-appearing cells labeled strongly for
NG2 but did not label with O1 (Fig. 7C, arrowheads).
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Figure 7.
Double immunofluorescent-labeling studies from a
case at 32 weeks. Pre-OLs are a morphologically and
immunohistochemically distinct population of OL precursors that persist
in human cerebral white matter during the expansion in the immature OL
population. Pre-OLs were distinguished by diverse morphologies similar
to those observed earlier in development. A, B,
Representative examples of pre-OLs in the superficial white matter that
strongly labeled with O4 (A) and for NG2
(B) are shown. C, Note in
C that the NG2-positive pre-OLs
(arrowheads) appear morphologically less mature compared
with an O1-positive immature OL (D) with a complex
multipolar morphology (arrow). Scale bars:
A-D, 20 µm.
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Distribution of MBP between 18 and 40 weeks
The distribution of MBP, visualized by SMI 99 immunoreactivity,
was determined in the parietal white matter in 15 cases between 18 and
40 weeks. In seven cases examined between 18 and 27 weeks, no MBP
labeling was detected (Fig.
8A). In eight cases
examined between 30 and 40 weeks, MBP labeling was first detected at
~30 weeks and was restricted to the periventricular white matter, which included the optic radiation (Fig. 8B). By 40 weeks, MBP labeling was much more extensively visualized in the
periventricular white matter (Fig. 8C) and was often present
in more superficial regions of the white matter (data not shown).
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Figure 8.
The temporal progression of myelinogenesis in
human parietal periventricular white matter between 18 and 40 weeks was
assessed by immunohistochemical localization of MBP. Representative
examples of the distribution of MBP are shown at 20 weeks
(A), 30 weeks (B), and 40 weeks (C). The asterisk is
adjacent to the ependymal surface of the lateral ventricle. Scale bar:
A-C, 100 µm.
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Pre-OLs predominate throughout the high-risk period for PVL
To identify the major OL lineage stage in the parietal white
matter when the risk of PVL is high (i.e., 23-32 weeks), we
determined the relative percentage of pre-OLs and immature OLs present
in 18 individual cases that ranged in age from 18 to 41 weeks. To quantify these two OL stages, the nucleus was selected as the smallest
countable object. We confirmed that at any given gestational age there
was no difference in the size of the nucleus of a pre-OL or an immature
OL (see Materials and Methods). Figure 9
shows the temporal progression of the percentage of immature OLs
between 18 and 41 weeks. Between 18 and 27 weeks (n = 9), the O1-labeled cells comprised 9.9 ± 2.1% of total cells
(mean ± SE; one-way ANOVA). At ~28-30 weeks, a progressive
increase in the number of O1-labeled cells was observed. Between 28 and
41 weeks (n = 9), the O1-labeled cells comprised
30.9 ± 2.1% of total cells (mean ± SE; one-way ANOVA).
There was thus approximately a threefold increase in the percentage of
O1-positive cells observed at 28-41 weeks when compared with 18-27
weeks (p < 0.0001).
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Figure 9.
Quantitative analysis of the timing of appearance
of pre-OLs and immature OLs in human cerebral white matter between 18 and 41 weeks. Each time point represents an individual
case in which the percentage of immature OLs was determined from the
total OL somata that labeled with the O1 antibody. Because the O4
antibody was found to label the entire population of OL precursors at
all ages examined, total OL precursors were determined from the number
of cells that labeled with O4 (see Materials and Methods). Note that
O1+ immature OLs are a minor population between 18 and 28 weeks.
Approximately 28-30 weeks mark the onset of an expansion in the
immature OL population.
|
|
| |
DISCUSSION |
Definition of the cellular and molecular events that regulate OL
lineage progression in the human CNS is important for the understanding
of developmental disorders of cerebral white matter (Back and Volpe,
1999). This study identified three successive stages of the human OL
lineage between midgestation and term birth in human cerebral white
matter (Fig. 10): the pre-OL,
the immature OL, and the mature OL. The major features of the lineage
progression were the following: (1) Pre-OLs were present as early as 18 weeks in both the cerebral white matter and cortex and were the major OL stage throughout the latter half of gestation. Pre-OLs labeled for
NG2 and with the O4 antibody but not with the O1 antibody. (2) Between
18 and 27 weeks, immature OLs were a minor population, present only in
the white matter, that labeled strongly with the O4 and O1 antibodies
but very weakly for NG2. Immature OLs were further distinguished by a
complex multipolar morphology, whereas pre-OLs were morphologically
more diverse and appeared less differentiated. (3) At ~30 weeks,
immature OLs increased in number only in the white matter. The increase
in immature OLs coincided with the restricted localization of
MBP-positive myelin sheaths to the periventricular white matter. Thus,
during the high-risk period for PVL (Fig. 10), pre-OLs comprised
~90% of the total cells derived from the OL lineage.
View larger version (8K):
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|
Figure 10.
Summary diagram of OL lineage progression in
human cerebral white matter during the latter half of gestation. Note
that the high-risk period for PVL coincides with the developmental
epoch when the white matter is mostly populated by O4+O1 pre-OLs that
also label with NG2. O4+O1+ immature OLs are a minor population until
~30 weeks (dotted line) when they undergo a marked
expansion. This expansion in the immature OL population is accompanied
by the appearance in the periventricular white matter of MBP+ mature
OLs. Hence the decline in incidence of PVL at ~32 weeks coincides
with OL maturation in the periventricular white matter.
wks, Weeks.
|
|
The human OL lineage was defined with several OL-specific markers and
by morphological criteria (Pfeiffer et al., 1993; Nishiyama et al.,
1999). Despite the morphological diversity of human pre-OLs, their
immunohistochemical phenotype identified them as oligodendroglial in
origin and distinct from astroglia, resting or reactive microglia, and
neuronal precursors. Restricted foci of O4-positive progenitors were
detected in the ventral ventricular zone of the human spinal cord as
early as 6 weeks (Hajihosseini et al., 1996). In agreement with most
(Levine et al., 1993; Nishiyama et al., 1997; Reynolds and Hardy, 1997;
Chang et al., 2000) but not all (Pouly et al., 1999) studies, our
results in developing human brain also indicate that NG2 is specific to
the OL lineage in vivo.
The timing of appearance of early human OL progenitors cannot be
established from our study. Although early OL progenitors were cultured
from fetal (Rivkin et al., 1995) and adult (Scolding et al., 1995; Roy
et al., 1999) human brain, we did not detect NG2+O4 OL progenitors at
midgestation or later in parietal white matter or at the foramen of
Monro, a region with a well developed ventricular zone (data not
shown). These previous studies may have isolated pre-OLs that reverted
to an early progenitor phenotype in culture, as do rat pre-OLs in
culture (Gard and Pfeiffer, 1993; Back et al., 1998). We also did not
observe migratory streams of OL progenitors, but rather pre-OLs were
diffusely distributed throughout the white matter by 18 weeks. In
contrast to the rodent spinal cord (for review, see Hardy, 1997), in
which the migration of OL progenitors occurs at midgestation, O4+
progenitors migrate from the human ventral ventricular zone of the cord
considerably earlier between 6 and 8 weeks (Hajihosseini et al., 1996).
NG2+ OL progenitors are abundant in the adult rat and human brain
(Levine et al., 1993; Nishiyama et al., 1996, 1997; Reynolds and Hardy, 1997; Ong and Levine, 1999; Chang et al., 2000). In adult human brain,
NG2+ progenitors appear to be oligodendroglial in origin and as
abundant as mature OLs (Chang et al., 2000). The relationship of these
progenitors to pre-OLs in fetal or adult (Armstrong et al., 1992;
Wolswijk, 1998) human brain is presently unclear. Because adult
NG2-positive progenitors would appear to be more abundant than we
observed in developing human brain, it is possible that adult human
progenitors may subserve more diverse functions than during development.
The role of the minor population of human immature OLs between 18 and
28 weeks is unclear. Although O1-positive cells at 18 weeks had no
significant difference in nuclear area compared with that of
O4-positive cells (see Materials and Methods), they were morphologically more complex. In contrast to the rat, in which O1
positivity correlates with a rapid commitment to myelination (Warrington and Pfeiffer, 1992), human immature OLs were detected at
least 3 months before the onset of myelinogenesis. Myelinogenesis began
at ~30 weeks and was restricted to the periventricular white matter
despite the presence of numerous immature OLs in more superficial white
matter. Hence, factors extrinsic to the immature OL seem to influence
the onset and progression of myelinogenesis.
The restricted localization of early myelinogenesis to the
periventricular white matter appears to be developmentally specific. Although MBP-positive myelin sheaths were detected
immunohistochemically by 30 weeks, sulfatide by thin layer
chromatography (TLC) and MBP by SDS-PAGE were not detected in parietal
white matter until after birth (Kinney et al., 1994). The appearance of
microscopic myelin does not occur until at least after the first
postnatal month, and myelin tubes were not detected until 11-13
postconceptional months (Brody et al., 1987; Kinney et al., 1989).
Hence, immunohistochemistry appears to be more sensitive than TLC or
SDS-PAGE to detect myelin constituents. Interestingly, recent studies
of infants between 24 and 40 weeks after conception with diffusion
tensor magnetic resonance imaging showed changes in water diffusion
that correlate with the time course for parietal myelinogenesis
determined here by immunohistochemistry. Thus, in central white matter,
relative anisotropy, a measure of preferred directionality of water
parallel to fiber tracts, increased markedly from 28 to 40 weeks and
occurred in parallel with a decline in overall water diffusion, as
measured by the apparent diffusion coefficient (Huppi et al., 1998).
This combination of findings implies restriction of diffusion
perpendicular to fiber tracts and could relate to the progressive
increase in myelinogenesis that we observed between 30 and 40 weeks.
We found substantial differences in the temporal progression of
myelinogenesis between rodents and man. Whereas numerous human immature
OLs are present before birth, they do not increase in the rat until the
first postnatal week (Reynolds and Hardy, 1997; Back et al., 1998).
Human myelinogenesis begins early in the third trimester, but
myelinogenesis does not begin in the rat forebrain until approximately
the second postnatal week. The marked difference in timing of OL
lineage progression between humans and rats may partly account for the
failure to develop a suitable perinatal rat model of PVL. Our results
underscore the importance of human developmental studies to identify
temporal mismatches between lineage progression in humans and
experimental animal models.
Clinical correlations
A central unresolved question regarding the pathogenesis of PVL is
the cellular basis for the increased susceptibility of fetal cerebral
white matter to injury as gestational age decreases (Back and Volpe,
1997). Rorke (1982) reported an apparent depletion of myelination glia,
putative OL precursors, in cases with PVL, but this observation has not
been reevaluated with OL-specific markers. One aim of our study was,
thus, to identify the stages in the OL lineage that may be susceptible
to injury at sites of PVL. Because impaired myelination is the
principal pathological sequela of PVL, OL precursors are hypothesized
to be a target cell of ischemia-reperfusion injury to the white matter
(Kinney and Back, 1998). That OL precursors may be susceptible to
injury in PVL is supported by recent in vitro studies that
demonstrated that OL progenitors are significantly more susceptible
than are mature OLs to death triggered by oxidative stress (Back et
al., 1998; Fern and Moller, 2000). We demonstrated here that the
parietal periventricular white matter, a region of high predilection
for PVL, is populated mostly by pre-OLs between 24 and 32 weeks of gestation, the period when the incidence of PVL is highest (Fig. 10).
Interestingly, the developmental window for PVL thus coincides with a
period before the onset of myelination of periventricular white matter.
This period is dominated by one major population of pre-OLs and
identifies this OL stage as a potential target for hypoxic-ischemic
injury in PVL. The decline in the incidence of PVL coincides with the
onset of maturation of the periventricular white matter at ~30 weeks.
Hence, the differentiation of OL precursors to mature OLs may underlie
the increased resistance of the periventricular white matter to injury
and could account for the decline in the incidence of PVL as term
gestation approaches.
Future studies
Further studies are needed to define better the biology of human
pre-OLs in vivo. Although rodent pre-OLs are mitotically active, it remains unresolved, for example, whether human pre-OLs actively divide in vivo during development or adulthood and
whether their proliferation is affected by brain injury (Wolswijk,
1998). Also unclear is whether human pre-OLs are directly killed by
hypoxia-ischemia or whether cell death might be triggered by disruption
of trophic support during a critical period in white matter
development. To understand the cellular pathogenesis of PVL, studies
are needed to determine whether late OL progenitors are depleted at
sites of PVL and, if depleted, what mechanisms may regulate their
potential to repopulate the white matter after injury.
| |
FOOTNOTES |
Received Sept. 1, 2000; revised Nov. 17, 2000; accepted Nov. 20, 2000.
This work was supported by National Institutes of Health (NIH) Grant NS
01855 to S.A.B., NIH Grant P30 HD 33703, Oregon Child Health Research
Center, Training Program Award for Pediatric Physician-Scientists to
S.A.B., NIH-National Institute of Child Health and Human Development Grant P30 HD 18655 to J.J.V., a Grass Foundation Morison
Fellowship and a Hearst Foundation Award to S.A.B., and an award from
the Sudden Infant Death Syndrome Alliance to H.C.K. We gratefully acknowledge the support of Drs. Linda Wallen, Randall Nixon, and Geoffrey Murdoch at Oregon Health Sciences University and the NIH
Developmental Brain and Tissue Bank at the University of Miami under
the direction of Dr. Carol Petito (NO1-HD-8-3284) for their support in
the acquisition of cases. We also thank Drs. Edward G. Jones, Gregory
Popken, and Clifford Saper for guidance on the morphometric studies,
Dr. Peter Rotwein for the generous use of his microscope, and Dr. Gary
Sexton for generous assistance with statistical analysis. We are
grateful to Drs. Julie Ellison, John Fiala, Jonathan Flax, Montey
Gates, John Kornhauser, Jeffrey Macklis, Akiko Nishiyama, Steven
Pfeiffer, Scott Pomeroy, and Wolfgang Streit for their helpful advice
and discussions. We thank Luciana Rava and Richard Belliveau for their
excellent technical assistance.
Correspondence should be addressed to Dr. Stephen A. Back, Department
of Pediatrics, Hatfield Research Center, Room 516, Oregon Health
Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR
97201-3098. E-mail: Backs{at}ohsu.edu.
| |
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February 1, 2007;
14(2):
182 - 191.
[Abstract]
[PDF]
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S. A. Back, A. Riddle, and M. M. McClure
Maturation-Dependent Vulnerability of Perinatal White Matter in Premature Birth
Stroke,
February 1, 2007;
38(2):
724 - 730.
[Abstract]
[Full Text]
[PDF]
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N. G. Anderson, I. Laurent, L. J. Woodward, and T. E. Inder
Detection of Impaired Growth of the Corpus Callosum in Premature Infants
Pediatrics,
September 1, 2006;
118(3):
951 - 960.
[Abstract]
[Full Text]
[PDF]
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S. A. Back, A. Riddle, and A. R. Hohimer
Topical Review: Role of Instrumented Fetal Sheep Preparations in Defining the Pathogenesis of Human Periventricular White-Matter Injury
J Child Neurol,
July 1, 2006;
21(7):
582 - 589.
[Abstract]
[PDF]
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M Allin, M Rooney, T Griffiths, M Cuddy, J Wyatt, L Rifkin, and R Murray
Neurological abnormalities in young adults born preterm
J. Neurol. Neurosurg. Psychiatry,
April 1, 2006;
77(4):
495 - 499.
[Abstract]
[Full Text]
[PDF]
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A. Kanaan, R. Farahani, R. M. Douglas, J. C. LaManna, and G. G. Haddad
Effect of chronic continuous or intermittent hypoxia and reoxygenation on cerebral capillary density and myelination
Am J Physiol Regulatory Integrative Comp Physiol,
April 1, 2006;
290(4):
R1105 - R1114.
[Abstract]
[Full Text]
[PDF]
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A. Riddle, N. Ling Luo, M. Manese, D. J. Beardsley, L. Green, D. A. Rorvik, K. A. Kelly, C. H. Barlow, J. J. Kelly, A. R. Hohimer, et al.
Spatial heterogeneity in oligodendrocyte lineage maturation and not cerebral blood flow predicts fetal ovine periventricular white matter injury.
J. Neurosci.,
March 15, 2006;
26(11):
3045 - 3055.
[Abstract]
[Full Text]
[PDF]
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R. D. Folkerth
Neuropathologic Substrate of Cerebral Palsy
J Child Neurol,
December 1, 2005;
20(12):
940 - 949.
[Abstract]
[PDF]
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F. E. Jensen
Role of Glutamate Receptors in Periventricular Leukomalacia
J Child Neurol,
December 1, 2005;
20(12):
950 - 959.
[Abstract]
[PDF]
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P. Svedin, I. Kjellmer, A.-K. Welin, S. Blad, and C. Mallard
Maturational Effects of Lipopolysaccharide on White-Matter Injury in Fetal Sheep
J Child Neurol,
December 1, 2005;
20(12):
960 - 964.
[Abstract]
[PDF]
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I. Jakovcevski and N. Zecevic
Olig Transcription Factors Are Expressed in Oligodendrocyte and Neuronal Cells in Human Fetal CNS
J. Neurosci.,
November 2, 2005;
25(44):
10064 - 10073.
[Abstract]
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[PDF]
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A. S. Paintlia, M. K. Paintlia, M. Khan, T. Vollmer, A. K. Singh, and I. Singh
HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis
FASEB J,
September 1, 2005;
19(11):
1407 - 1421.
[Abstract]
[Full Text]
[PDF]
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C. Sherwin and R. Fern
Acute Lipopolysaccharide-Mediated Injury in Neonatal White Matter Glia: Role of TNF-{alpha}, IL-1{beta}, and Calcium
J. Immunol.,
July 1, 2005;
175(1):
155 - 161.
[Abstract]
[Full Text]
[PDF]
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D. M. Ferriero
Neonatal Brain Injury
N. Engl. J. Med.,
November 4, 2004;
351(19):
1985 - 1995.
[Full Text]
[PDF]
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C. Nosarti, T. M. Rushe, P. W. R. Woodruff, A. L. Stewart, L. Rifkin, and R. M. Murray
Corpus callosum size and very preterm birth: relationship to neuropsychological outcome
Brain,
September 1, 2004;
127(9):
2080 - 2089.
[Abstract]
[Full Text]
[PDF]
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W. Deng, H. Wang, P. A. Rosenberg, J. J. Volpe, and F. E. Jensen
Role of metabotropic glutamate receptors in oligodendrocyte excitotoxicity and oxidative stress
PNAS,
May 18, 2004;
101(20):
7751 - 7756.
[Abstract]
[Full Text]
[PDF]
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P. L. Follett, W. Deng, W. Dai, D. M. Talos, L. J. Massillon, P. A. Rosenberg, J. J. Volpe, and F. E. Jensen
Glutamate Receptor-Mediated Oligodendrocyte Toxicity in Periventricular Leukomalacia: A Protective Role for Topiramate
J. Neurosci.,
May 5, 2004;
24(18):
4412 - 4420.
[Abstract]
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[PDF]
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O. Baud, A. E. Greene, J. Li, H. Wang, J. J. Volpe, and P. A. Rosenberg
Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes
J. Neurosci.,
February 18, 2004;
24(7):
1531 - 1540.
[Abstract]
[Full Text]
[PDF]
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M. Derrick, N. L. Luo, J. C. Bregman, T. Jilling, X. Ji, K. Fisher, C. L. Gladson, D. J. Beardsley, G. Murdoch, S. A. Back, et al.
Preterm Fetal Hypoxia-Ischemia Causes Hypertonia and Motor Deficits in the Neonatal Rabbit: A Model for Human Cerebral Palsy?
J. Neurosci.,
January 7, 2004;
24(1):
24 - 34.
[Abstract]
[Full Text]
[PDF]
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B. Vollmer, S. Roth, J. Baudin, A. L. Stewart, B. G. R. Neville, and J. S. Wyatt
Predictors of Long-Term Outcome in Very Preterm Infants: Gestational Age Versus Neonatal Cranial Ultrasound
Pediatrics,
November 1, 2003;
112(5):
1108 - 1114.
[Abstract]
[Full Text]
[PDF]
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S. Rakic and N. Zecevic
Emerging Complexity of Layer I in Human Cerebral Cortex
Cereb Cortex,
October 1, 2003;
13(10):
1072 - 1083.
[Abstract]
[Full Text]
[PDF]
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J. Li, J. C. Lin, H. Wang, J. W. Peterson, B. C. Furie, B. Furie, S. L. Booth, J. J. Volpe, and P. A. Rosenberg
Novel Role of Vitamin K in Preventing Oxidative Injury to Developing Oligodendrocytes and Neurons
J. Neurosci.,
July 2, 2003;
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[Abstract]
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J. J. Volpe
Cerebral White Matter Injury of the Premature Infant--More Common Than You Think
Pediatrics,
July 1, 2003;
112(1):
176 - 180.
[Full Text]
[PDF]
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W. Deng, P. A. Rosenberg, J. J. Volpe, and F. E. Jensen
Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors
PNAS,
May 27, 2003;
100(11):
6801 - 6806.
[Abstract]
[Full Text]
[PDF]
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P. S. McQuillen, R. A. Sheldon, C. J. Shatz, and D. M. Ferriero
Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia
J. Neurosci.,
April 15, 2003;
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[Abstract]
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S. M. Curristin, A. Cao, W. B. Stewart, H. Zhang, J. A. Madri, J. S. Morrow, and L. R. Ment
Disrupted synaptic development in the hypoxic newborn brain
PNAS,
November 26, 2002;
99(24):
15729 - 15734.
[Abstract]
[Full Text]
[PDF]
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E. R. Sowell, P. M. Thompson, S. N. Mattson, K. D. Tessner, T. L. Jernigan, E. P. Riley, and A. W. Toga
Regional Brain Shape Abnormalities Persist into Adolescence after Heavy Prenatal Alcohol Exposure
Cereb Cortex,
August 1, 2002;
12(8):
856 - 865.
[Abstract]
[Full Text]
[PDF]
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S. Lehnardt, C. Lachance, S. Patrizi, S. Lefebvre, P. L. Follett, F. E. Jensen, P. A. Rosenberg, J. J. Volpe, and T. Vartanian
The Toll-Like Receptor TLR4 Is Necessary for Lipopolysaccharide-Induced Oligodendrocyte Injury in the CNS
J. Neurosci.,
April 1, 2002;
22(7):
2478 - 2486.
[Abstract]
[Full Text]
[PDF]
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G. Wolswijk
Oligodendrocyte precursor cells in the demyelinated multiple sclerosis spinal cord
Brain,
February 1, 2002;
125(2):
338 - 349.
[Abstract]
[Full Text]
[PDF]
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S. A. Back, B. H. Han, N. L. Luo, C. A. Chricton, S. Xanthoudakis, J. Tam, K. L. Arvin, and D. M. Holtzman
Selective Vulnerability of Late Oligodendrocyte Progenitors to Hypoxia-Ischemia
J. Neurosci.,
January 15, 2002;
22(2):
455 - 463.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
