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J Neuropathol Exp Neurol
Copyright Ó 2012 by the American Association of Neuropathologists, Inc.
Vol. 71, No. 3
March 2012
pp. 00Y00
ORIGINAL ARTICLE
Microglial Reaction in Axonal Crossroads Is a Hallmark of
Noncystic Periventricular White Matter Injury in Very
Preterm Infants
Catherine Verney, PharmD, PhD, Ivana Pogledic, MD, Vale´rie Biran, MD,
Homa Adle-Biassette, MD, PhD, Catherine Fallet-Bianco, MD, and Pierre Gressens, MD, PhD
Abstract
Disabilities after brain injury in very preterm infants have mainly
been attributed to noncystic periventricular white matter injury
(PWMI). We analyzed spatiotemporal patterns of PWMI in the
brains of 18 very preterm infants (25Y29 postconceptional weeks
[pcw]), 7 preterm infants (30Y34 pcw), and 10 preterm controls
without PWMI. In very preterm infants, we examined PWMI in
detail in 2 axonal crossroad areas in the frontal lobe: C1 (lateral to the
lateral angle of the anterior horn of the lateral ventricle, at the exit of
the internal capsule radiations) and C2 (above the corpus callosum
and dorsal angle of the anterior horn). These brains had greater
microglia-macrophage densities and activation but lesser astroglial
reaction (glial fibrillary acidic protein and monocarboxylate transporter 1 expression) than in preterm cases with PWMI. In preterm
infants, scattered necrotic foci were rimmed by axonal spheroids and
ionized calcium binding adaptor molecule 1Ypositive macrophages.
Diffuse lesions near these foci consisted primarily of hypertrophic and
reactive astrocytes associated with fewer microglia. No differences in
Olig2-positive preoligodendrocytes between noncystic PWMI and
control cases were found. These data show that the growing axonal
crossroad areas are highly vulnerable to PWMI in very preterm
infants and highlight differences in glial activation patterns between
very preterm and preterm infants.
From the INSERM, U676 (CV, IP, VB, HA-B, PG); Universite´ Paris 7,
Faculte´ de Me´decine Denis Diderot (CV, VB, HA-B, PG); PremUP (CV,
VB, PG); Reanimation et Pe´diatrie Ne´onatales, Hoˆpital Robert Debre´
(VB); and APHP, Neuropathologie, Hoˆpital Saint-Anne (CF-B), Paris,
France; Croatian Institute for Brain Research, Medical School (IP), University of Zagreb, Zagreb, Croatia; and Institute for Reproductive and
Developmental Biology (PG), Imperial College, Hammersmith Campus,
London, United Kingdom.
Send correspondence and reprint requests to: Catherine Verney, PharmD,
PhD, INSERM U676, Hoˆpital Robert Debre´, 48 Blvd Se´rurier, 75019
Paris, France; E-mail: [email protected]
This study was supported by grants from Inserm, Universite´ Paris Diderot,
PremUP, Seventh Framework Program of the European Union (Grant
HEALTH-F2-2009-241778/NEUROBID), Fondation Leducq, Fondation
Grace de Monaco, Fondation Roger de Spoelberch, ELA Foundation,
Fondation Planiol, and Assistance Publique Hoˆpitaux de Paris (APHPContrat Hospitalier de Recherche Translationnelle to Dr Pierre Gressens).
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions
of this article on the journal’s Web site (www.jneuropath.com).
J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Key Words: Gliosis, Immunohistochemistry, Ionized calcium binding adaptor molecule 1, Monocarboxylate transporter 1, Periventricular crossroads, Periventricular leukomalacia, Preoligodendrocyte,
Prematurity.
INTRODUCTION
Infants born before 33 gestational weeks or 31 postconceptional weeks (pcw) (1) are at high risk for brain damage and subsequent cognitive, behavioral, and/or motor
deficits (2Y4). Periventricular leukomalacia (PVL) is classically described as the main pattern of white matter (WM)
damage associated with cerebral palsy in premature infants
(5, 6). Magnetic resonance imaging (MRI) and sonogram
studies in the intensive care unit have shown not only cystic
lesions but also more subtle noncystic abnormalities indicating periventricular WM injury (PWMI) (7Y13). Currently,
noncystic PWMI is thought to account for more than 95%
of brain lesions in preterm infants (2, 14, 15). Magnetic resonance imaging is widely used to predict long-term outcomes
of preterm infants (4, 7, 10, 16Y19). However, MRI studies of
noncystic PWMI suggest that the pattern of injury may differ
between very preterm infants born before 31 pcw and preterm
infants born between 31 and 34 pcw (1, 9, 20, 21).
Periventricular leukomalacia has been the focus of
many histopathologic studies (22Y27). Typically, PVL is described as a combination of focal abnormalities (which may
be acute, subacute, or chronic) and diffuse abnormalities
characterized by reactive gliosis and microglia activation in
the deep WM surrounding the necrotic foci. Studies done in
the past decade have suggested loss of preoligodendrocytes
followed by defective myelination as critical to the onset of
PVL (28Y30). However, a 2008 study challenged this hypothesis by showing qualitative abnormalities in myelin
maturation (assessed based on myelin basic protein immunostaining) but no decrease in the density of Olig2-positive
preoligodendrocytes within PWMI lesions (31). A new hypothesis was developed regarding the potential deleterious
and/or reparative role of microglial-macrophage activation
in the onset of PWMI (32Y34). Clinical epidemiological data
suggest the involvement of inflammatory processes and responses (35, 36). Furthermore, microglia-macrophages from
newborns with PWMI have been shown to express interleukin
1
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Verney et al
(IL)-1A, IL-2, IL-6, and tumor necrosis factor (37, 38). Similarly, in animal models of PWMI involving excitotoxicity,
inflammation, and hypoxia-ischemia-asphyxia, microgliamacrophage activation has been the first cellular event detected in or around lesions after the injury (32, 39Y41).
In a previous study of normal telencephalic development in human fetuses, we found transient patches of microglia located in junctional regions of the WM anlage, most
notably at junctions connecting the anterior and posterior
limbs of the internal capsule to the external capsule between
14Y17 pcw (42) and in the rostral portion of the centrum
semiovale in the corona radiata extending caudally to the
occipital lobe between 19 and 30 pcw (corresponding to very
preterm birth) (43, 44). The periventricular distribution of
microglial accumulations corresponds to 3 of the periventricular crossroads of growing axonal pathways in the WM
during the very preterm period, which are designated C1, C2,
and C5 (8, 45, 46). The presence of activated microglia in
these crossroads during early development may be related to
phagocytic events in developing axonal bundles, as observed
TABLE 1. Clinical Data
(A) Cases With Noncystic Periventricular White Matter Injury
Case No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Age at Birth*
24 + 2
24
25 + 5
25 + 6
26
26
27 + 3
27 + 6
27 + 4
28 + 5
28 + 6
25 + 6
30
30 + 2
28
33
33
34
Age at Death*
d
24 + 5
25
26
26
26 + 1
26 + 2
27 + 6
27 + 6
28 + 1
28 + 6
28 + 6
29 + 3
30 + 2
31
32
33
33 + 3
34 + 4
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
Sex
Clinical Diagnoses
F
F
M
M
F
M
M
F
M
M
M
M
M
F
M
M
M
F
RDS
RDS
RDS
Twin-to-twin transfusion syndrome
RDS
RDS
RDS
RDS
Septic shock
RDS
Intrapartum death
RDS
RDS, pneumothorax
RDS, septic shock
Necrotizing enterocolitis
Septic shock
RDS
Oligohydramnios, renal failure
(B) Cases With Cystic Periventricular White Matter Injury
Case No.
1
2
3
4
5
6
7
Age at Death
Sex
Clinical Diagnoses
25
25 + 2 d
26 + 4 d
27
28 + 4 d
28,5
30 + 1 d
F
M
M
M
M
F
M
Twin-to-twin transfusion syndrome
RDS
Stillbirth, thoracic tumor
Stillbirth
RDS, maternofetal infection
RDS, maternofetal infection
RDS, maternofetal infection
(C) Controls Without Periventricular White Matter Injury
Case No.
1
2
3
4
5
6
7
8
9
10
Age at Death*
Sex
Clinical Diagnoses
24
25 + 2 d
25 + 2 d
25 + 5 d
27
29 + 2 d
30 + 4 d
32
33
33
F
F
M
M
M
M
F
F
M
F
Twin
Therapeutic abortion (cleft lip and palate)
Spontaneous abortion
Therapeutic abortion (growth retardation)
Spontaneous abortion
Therapeutic abortion (posterior urethral valve)
Spontaneous abortion
Uropathy
Spontaneous abortion
Spontaneous abortion
*Ages are in postconceptional weeks + days (d).
F, female; M, male; RDS, respiratory distress syndrome.
2
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
for exuberant transcallosal projections eliminated by microglia during postnatal development in kittens (47Y49). Periventricular crossroads are composed of numerous intersecting
callosal, associative, and thalamocortical axons involved in
motor, sensory, and associative functions (45). During the very
preterm period of normal development, these axonal crossroads
are rich in extracellular matrix molecules such as chondroitin
sulfate and in guidance molecules such as semaphorin3A and
Eph3A receptor (45). Microglial interactions in these areas
with these molecules may supply directional cues to axonal
projections on their way to their targets (45).
Here, we investigated the responses of glial cells and
axonal fascicles at sites of PWMI in the frontal- and occipitallobe axonal crossroads and compared abnormalities in these
areas in very preterm infants (24Y30 pcw), preterm infants
(31Y34 pcw), and age-matched controls.
MATERIALS AND METHODS
Cases
T1
We studied 35 preterm fetuses obtained from fetal
pathology units, including 18 with noncystic PWMI, 7 with
cystic PWMI, and 10 controls (Table 1). Of the 25 fetuses
with PWMI, 18 were very preterm (24Y29 pcw or 26Y31
gestational weeks) and 7 were preterm (30Y34 pcw or 32Y36
gestational weeks). Postmortem delays were less than 48 hours.
Written consent was obtained from all parents. The study was
approved by the institutional review board of the Paris North
Hospitals, Paris 7 University, AP-HP (No. 09-062), France, and
by the ethics committee of the Zagreb University, School of
Medicine (No. 108-1081870-1876), Croatia.
In each fetus, the brain was removed from the skull and
fixed in a 10% formaldehyde solution containing NaCl (9 g/L)
and ZnSO4 (3 g/L) for 3 to 6 weeks according to brain size.
The brain was then cut in the coronal plane, and sections of 1
hemisphere or both were embedded in paraffin. The selected
sections included the frontal lobe at different levels of the
basal ganglia and anterior thalamic nuclei. In some cases,
sections were selected in the parieto-occipital lobe. The sections were stained with hemalun-phloxine and cresyl violet
Microgliosis in Very Preterm White Matter Injury
or cresyl violetYLuxol fast blue (Klu¨ver-Barrera), according
to standard methods. Each brain was examined by 2 neuropathologists blinded to group assignment. Controls were defined as brains free of abnormalities, such as microvacuolar
changes (spongiosis), necrosis, or marked microglial reaction
(Table 1C); a minor astroglial reaction, as typically detected
in human postmortem brains, was not considered an abnormality. No histologic differences were noted between control
specimens from fetuses with versus those without survival
after delivery.
Immunocytochemistry
Ten-micrometer-thick sections were deparaffinized in
a series of xylene/alcohol solutions. After citrate buffer treatment (0.01 mol/L, pH 6, for 40 minutes at 94-C), the sections were placed in phosphate-buffered saline (PBS 1,
pH 7.6/7.4)/H2O2 (0.25%) at room temperature for 15 minutes
to block endogenous peroxidase and then rinsed in PBS with
2% gelatin and 0.5% Triton (42, 43). Primary antibodies were
incubated at the dilutions indicated in Table 2 in the abovedescribed solution with 8% human serum albumin and 0.02%
sodium azide at 4-C for 48 hours.
The primary antibodies were used to identify astrocytes
(glial fibrillary acidic protein [GFAP] and monocarboxylate transporter 1 [MCT1]) (50), microglia-macrophages (ionized calcium binding adaptor molecule 1 [Iba1], CD68,
CD45, and the antiYclass II major histocompatibility complex
class II [MHC-II] molecule antibody LN3) (42, 43), preoligodendrocytes (Olig2), vessel walls (CD34 and MCT1)
(50), and axons (monoclonal mouse SMI31 antibody to axonal phosphorylated epitopes on 168- and 220-kD neurofilament proteins). Immunolabeling was achieved using the
streptavidin-biotin-peroxidase method (42, 43) with a mixture of 3,3¶-diaminobenzidine tetrahydrochloride and nickel
ammonium sulfate (6%), which produced a black reaction
product. For double immunostaining, the second primary antibody raised in another species was used with the peroxidaseantiperoxidase method (42, 43); the chromogen was 0.05%
3,3¶-diaminobenzidine tetrahydrochloride in 0.1 mol/L PBS,
which yielded a brown reaction product. Sections were counterstained with neutral red, dehydrated, and mounted. For both
TABLE 2. Primary Antibodies
Antibody
Structures Labeled
Company
Species
Dilution
GFAP
Vimentin
Iba1
CD68
CD45
LN3
Olig2
MCT1
CD34
SMI31
Astrocytic cells
Radial glia
Macrophages/microglia
Macrophage lysosomes
Hematopoietic nucleated cells
Class II major histocompatibility complex
Preoligodendrocyte
Astrocytes and endothelial cells
Endothelial cells
Axonal neurofilament
Sigma, St Louis, MO
Amersham, Buckinghamshire, UK
Wako, Osaka, Japan
Dako, Grostrup, Denmark
Dako
Neomarkers, Fremont, CA
Immuno-biological Lab, Fujioka, Japan
Chemicon-Millipore, Billerica, MA
Serotec, Raleigh, NC
Affinity, Exeter, UK
Mouse
Mouse
Rabbit
Mouse
Mouse
Mouse
Rabbit
Chicken
Mouse
Mouse
1:500
1:200
1:1000
1:800
1:50
1:1500
1:200
1:1000
1:200
1:1000
GFAP, glial fibrillary acidic protein; Iba1, ionized calcium binding adaptor molecule 1; MCT1, monocarboxylate transporter 1.
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T2
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Fig 1 4/C
Verney et al
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Microgliosis in Very Preterm White Matter Injury
TABLE 3. Summary of Histologic and Immunohistochemical Data
Very Preterm PWMI (24Y29 pcw)
Crossroad
Area C1
Crossroad
Area C2
Preterm PWMI (30Y34 pcw)
White Matter
Around C1-C2
Dispersed
Necrotic Foci
Diffuse
Lesions
Parenchymal and
cellular loss
Macrophages on the rim
of the necrotic foci
Core of the necrotic
foci (GFAP-negative,
MCT1-negative,
Vim-negative)
Parenchymal cell
loss
Slight microglial
activation
Hypertrophic astroglia,
astrogliosis
Parenchymal lesions
Axonal swelling
Parenchymal cell loss
No axonal lesions
Microglia-macrophages
(Iba1*)
Astrocytes (GFAP,
Vim, MCT1*)
Intense microglial
activation
No clear astrogliosis
Intense microglial
activation
No clear astrogliosis
Slight microglial
activation
No astrogliosis
MCT1-negative
astrocytes
MCT1-negative
hypertrophic vessels
No loss
MCT1-negative
astrocytes
MCT1-positive
hypertrophic vessels
No significant loss
MCT1-variable
astrocytes
MCT1-positive
hypertrophic vessels
No loss
In the core: vessel wall
loss, MCT1 negative
Slight loss
Hypertrophic vessels,
MCT1 variable
No significant loss
Axonal spheroids
No axonal spheroids
No axonal spheroids
Axonal spheroids
No axonal spheroids
Vessel walls
(CD34, MCT1*)
Preoligodendrocytes
(Olig2*)
Axonal bundles
(SMI31*)
MCT1-variable
*Antibodies used for immunohistochemical assessment.
GFAP, glial fibrillary acidic protein; MCT1, monocarboxylate transporter 1; PWMI, noncystic periventricular white matter injury; vim, vimentin.
methods, controls without the primary antibody were performed to confirm absence of cross-reactivity.
Quantification of Immunoreactive Cells
The densities of cells labeled by anti-GFAP, Iba1, and
Olig2 antibodies in C2 (centrum semiovale) were assessed in
each brain. For each brain, labeled cells were counted at 400
magnification in 4 fields of 0.065 mm2 each. Results were
compared using ANOVA with Bonferroni multiple comparison of means test (GraphPad Prism; GraphPad Software, La
Jolla, CA). p G 0.05 was considered significant.
Regional Analyses
F1
Frontal sections of the precentral gyrus and central
sulcus corresponding to plates 154 to 158 in the Bayer and
Altman atlas were examined (51) (Fig. 1A), as well as sections at the level of the parieto-occipital junction (plates
168Y170 (51)) (Figure, Supplemental Digital Content 1, Part
A, http://links.lww.com/NEN/A316). From 24 to 29 pcw on,
the cortical wall was composed of the cortical plate, extended
subplate layer, ‘‘white matter,’’ subventricular zone, and
ventricular zone (Figure, Supplemental Digital Content 1,
Parts A and B, http://links.lww.com/NEN/A316). We focused
on several crossroad areas of the periventricular WM, mainly
C1 and C2, but also C5. Crossroad C1 is lateral to the lateral
angle of the anterior horn of the lateral ventricle at the exit
of the internal capsule radiations; crossroad C2 is above the
corpus callosum and dorsal angle of the anterior horn (45, 51).
Crossroad C1 is the peduncular part of the prospective corona radiata extending in the main body of the WM between
the external capsule and anterior limb of the internal capsule;
crossroad C2 extends into the centrum semiovale (Figs. 1A,
B). Crossroad C5 is dorsolateral to the posterior horn of
the lateral ventricle, close to the parieto-occipital junction
(45, 51) (Figure, Supplemental Digital Content 1, Part A,
http://links.lww.com/NEN/A316). We compared the phenotypic expressions of several markers in the crossroads areas
versus the surrounding WM (Figure, Supplemental Digital
Content 2, http://links.lww.com/NEN/A317).
RESULTS
Spatial Distribution of Noncystic White
Matter Injury in Crossroads in Very
Preterm Brains (24Y29 pcw)
In very preterm control brains, a crescent of GFAPpositive astrocytes was seen in the deep dorsolateral WM
encompassing the periventricular crossroads C1 and C2
(Fig. 1B). In the surrounding WM, GFAP-positive astrocytes were sparser and exhibited small proximal processes
FIGURE 1. Frontal sections of brains from very preterm (VPT) cases. (A, B) Control section (26 postconceptional weeks [pcw])
stained with hemalun-phloxine (HPS) (A) and double immunolabeled for glial fibrillary acidic protein (GFAP, brown) and ionized
calcium binding adaptor molecule 1 (Iba1) (black) (B). The boxes in B are the crossroad areas C1 and C2 in the periventricular
white matter (WM); CC, corpus callosum; CP, cortical plate; SP, subplate layer; SVZ, subventricular zone; VZ, ventricular zone; V,
ventricle, IC, internal capsule. (C, D) Microcystic periventricular white matter injury (PWMI) (29 pcw) in C1 and C2 indicated in a
diagram (C) and on an Iba1-immunolabeled section (D). (E, F ) Noncystic PWMI (26 pcw) with subtle early-stage lesions in C1
and C2 (long arrows) stained with HPS (E) and immunolabeled with the axonal marker SMI 311 (F ). Magnifications: (AYD) 2;
(E, F) 4.
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
T3
and radially or longitudinally oriented longer processes,
as well as a meager network of GFAP-positive processes
(Figure, Supplemental Digital Content 2, Parts C and D,
http://links.lww.com/NEN/A317). These fibrous astrocytes
appeared slightly activated, which was probably due to perimortem events. In addition, as previously described (42Y44),
a patch of activated microglia (Iba1-positive, CD68-positive,
CD45-positive) was restricted to crossroad C2 during this
very preterm period (Fig. 1B). Cystic PWMI in very preterm cases was predominantly located in crossroads C1 and
C2 (Table 3 and Figs. 1C, D; Figure, Supplemental Digital
Content 3, http://links.lww.com/NEN/A320).
In 8 very preterm cases, there was a subtle but specific
lesion pattern in the WM in C1 and C2. These lesions consisted of parenchymal loss with axonal swellings in C1 and
spongiosis in C2 (Figs. 1E, F). In both areas, there was a
specific glial reaction different from that in the surrounding
WM (Table 3; Figure, Supplemental Digital Content 2,
http://links.lww.com/NEN/A317); this may constitute the
earliest stage of PWMI in very preterm infants. In 4 very
preterm cases, subtle PWMI seen as spongiosis was detected
in C5 (Figure, Supplemental Digital Content 1, Parts A and
B, http://links.lww.com/NEN/A316).
Crossroad C1
F2
Hemalun-phloxine staining of C1 showed parenchymal
loss with axonal lesions and no necrosis (Figs. 2A, B). Within
C1, GFAP-positive astrocytes had smaller cell bodies than in
the controls (Figs. 2C, D) and expressed less MCT1 immunoreactivity versus controls (Figs. 2E, F). MCT1 immunolabeling demonstrated more prominent vessel walls than in
the controls (Figs. 2E, F). These lesions contained numerous
Iba1-positive cells with a macrophage-like morphology and
immunophenotype (Figs. 2G, F); they were also CD68 positive, slightly CD45 positive, and MHC-II negative (not
shown). Axonal injury was confirmed by the presence of
SMI31-positive spheroids that were not present in the controls (Figs. 2I, J). No loss of Olig2-positive preoligodendrocytes was observed in PWMI cases compared to controls
(Figs. 2K, L).
Crossroad C2
F3
The centrum semiovale exhibited a loose parenchyma
with microvacuolar changes and increased cellularity (Figs. 3A,
B; Figure, Supplemental Digital Content 2, Parts A and B,
http://links.lww.com/NEN/A317). The most severe cases displayed parenchymal loss with microcysts and perivascular
edema or petechial hemorrhages. At the level of the centrum
Microgliosis in Very Preterm White Matter Injury
semiovale in C2, GFAP-positive astrocytes had small cell
bodies with only 1 or 2 long processes included in a GFAPpositive network of thin processes (Fig. 3C). They were
generally less numerous than in controls (Figs. 3C and 4A).
The distribution of GFAP-positive astrocytes in the surrounding WM was similar to that in the same zone in controls (Figure, Supplemental Digital Content 2, Parts C and D,
http://links.lww.com/NEN/A317). Astrocytes in the injured
crossroad C2 had less MCT1 expression compared to controls, whereas most of the vessels expressed comparable
MCT1 levels to those in controls (Fig. 3E compared to F). Iba1positive cells had a macrophage immunophenotype (CD68
positive and slightly CD45 positive, MHC-II negative) that
were present in high density in controls and noncystic cases
(Figs. 3G, H). They tended to be more numerous in cases with
PWMI than in controls (Fig. 4B). Axonal injury demonstrated
by SMI 31 immunostaining was less prominent than in C1
(Fig. 3I vs Fig. 2I). No significant difference in Olig2-positive
preoligodendrocytes was observed among very preterm and
preterm cases with and without PWMI (Figs. 3K, L and 4C).
Crossroad C5
Increased cellularity and spongiosis similar to the findings in C2 were detected in crossroad C5 of cases with PWMI.
Glial fibrillary acidic proteinYpositive astrocytes were smaller
than in controls, and numerous Iba1-positive microglia of
intermediate sizes were seen (Figure, Supplemental Digital
Content 1, http://links.lww.com/NEN/A316). No clearly visible SMI31-positive spheroids were detected in the PWMI, and
no early glial reaction or axonal lesions were detected in the
WM surrounding focal lesions in crossroads C1, C2, and C5 in
very preterm infants (Table 3; Figure, Supplemental Digital
Content 2, http://links.lww.com/NEN/A317).
Dispersed Noncystic White Matter Injury in
Preterm Cases
In the preterm cases, noncystic PWMI included crossroads C1, C2, and C5 but involved the WM more extensively
than in the very preterm cases (4) (Table 3 and Fig. 5). The
lesions consisted of paucicellular necrotic foci of various sizes
surrounded by diffuse tissue damage (Fig. 5A).
Necrotic Foci in Preterm Cases
Necrotic foci were characterized by hypocellularity and
axonal swellings (Figs. 5B, D, G). In larger foci without tissue loss, GFAP-positive, MCT1-positive, and Vim-positive
FIGURE 2. High magnification of crossroad area C1 in very preterm (VPT) cases. (AYL) Noncystic periventricular white matter injury
(PWMI) (25 postconceptional weeks [pcw]) (A, C, E, G, I, K ) and control (26 pcw) (B, D, F, H, J, L ). (A, B ) Hemalun-phloxine
staining (HPS). (C, D ) In a focus of PWMI, glial fibrillary acidic protein (GFAP)Ypositive astrocytes (brown) have small cell bodies
and are less numerous than ionized calcium binding adaptor molecule 1 (Iba1)Ypositive microglial cells (black); the latter are more
densely stained than those compared in the control. (E, F ) CD34-positive vessel walls in brown and monocarboxylate transporter
1Ypositive (MCT1-positive) astrocytes in black. The astrocytes appear to express less MCT1 immunoreactivity in the PWMI case
than in the control. Note the absence of MCT1 immunostaining in the vessels in both specimens. (G, H ) Iba1 immunostaining
showing increased numbers of reactive microglia-macrophages. (I, J ) SMI31 immunostaining confirms the presence of axonal
lesions such as enlarged axon bundles and spheroids. (K, L ) Olig2-positive preoligodendrocytes. Scale bar = 50 Km.
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Microgliosis in Very Preterm White Matter Injury
served around the foci. There were swollen axons and spheroids at the borders of the foci that exhibited SMI31 positivity
(Fig. 5H).
Diffuse Lesions in Preterm Cases
Part of the WM around the necrotic foci displayed diffuse lesions with gliosis (Fig. 5A). These areas contained
hypertrophic/reactive astrocytes with large cell bodies and numerous processes (Fig. 6C vs D; Figure, Supplemental Digital
Content 4, Parts C and D, http://links.lww.com/NEN/A321).
Their density was slightly lower than that in controls and most
of them were MCT1-negative (Figs. 4 and 6E, F). The evenly
distributed Iba1-positive microglial cells were significantly less
dense compared to very preterm PWMI (Fig. 4B) and only
slightly activated (Fig. 5G compared to H). Some cases displayed an intermediate phenotype of microglia with granules
located along processes (Fig. 5H; Figure, Supplemental Digital
Content 4, Part E, http://links.lww.com/NEN/A321). Double
labeling showed close contact between GFAP-positive astrocytes and Iba1-positive macrophages, suggesting interactions
between these cell types (Figure, Supplemental Digital Content 4, Parts C and D, http://links.lww.com/NEN/A321). Axonal
bundles were disrupted, but no axonal spheroids were detected
(Figs. 6J, K).
Preoligodendrocytes
FIGURE 4. Noncystic periventricular white matter injury (PWMI)
in crossroad area C2. (AYC) Assessments of glial fibrillary acidic
protein (GFAP)Ypositive astrocytes (A), ionized calcium binding
adaptor molecule 1 (Iba1)Ypositive microglia-macrophages (B),
and Olig2-positive preoligodendrocytes (C) in very preterm
(VPT), preterm (PT) cases with and without (control) noncystic
PWMI. *, p G 0.001, ANOVA with Bonferroni multiple comparison tests.
The density of Olig2-positive preoligodendrocytes varied across cases in very preterm and preterm fetuses compared
to controls (Figs. 2K, L, and 3K, L). Assessment of these
markers in the centrum semiovale (C2) showed no significant
differences between controls and very preterm or preterm
PWMI cases (Fig. 4C).
DISCUSSION
astrocytes were virtually undetectable in the core of the foci;
however, large activated astrocytes were present around the
foci (Figs. 5C, E, F, I; Figure, Supplemental Digital Content
1, Part F, http://links.lww.com/NEN/A316). These reactive
astrocytes displayed large cell bodies and long processes
(Figs. 5C, E, F, I; Figure, Supplemental Digital Content 4,
Part B, http://links.lww.com/NEN/A321). The inner rim
around the foci was composed of activated Iba1-positive,
CD68-positive microglia-macrophages intermingling with the
outer rim containing GFAP-positive astrocytes. A few MCT1negative vessel walls were seen in the core of the necrotic
foci, and prominent MCT1-positive vessel walls were ob-
In this study, we demonstrate differences in cellular
reactions in PWMI between very preterm and preterm infants
and selective vulnerability of the periventricular crossroads
of axonal pathways containing callosal associative and thalamocortical axons. Early microglial investment has also been
reported during normal development (43, 44) and shown to
be developmentally related to retraction of transiently projecting axons (45, 47Y49). We observed that in very preterm
brains, necrosis was restricted to periventricular crossroad
C1, whereas spongiosis was more common in periventricular
crossroads C2 and C5. The significance of the periventricular
crossroad areas as a source of extracellular matrix substrate
and axonal guidance molecules was first proposed by Judax
FIGURE 3. High magnification of crossroad area C2 in very preterm (VPT) cases. (AYL) Noncystic periventricular white matter injury
(PWMI) (25 postconceptional weeks [pcw]) (A, C, E, G, I, K) and control (26 pcw) (B, D, F, H, J, L ). (A, B ) Hemalun-phloxine
stain (HPS). (C, D) Glial fibrillary acidic protein (GFAP)Ypositive astrocytes (brown) have small cell bodies and are less numerous in a
focus of PWMI, whereas ionized calcium binding adaptor molecule 1 (Iba1)Ypositive microglial cells (black) appear to be reactive
compared to those in the control. (E, F) Astrocytes do not express MCT1 in a focus of PWMI in contrast to those in the control.
Note the MCT1 expression in most of the vessels in both. (G, H) Iba1-positive microglial cells have more of a macrophage
phenotype in the PWMI lesion than in the control. (I, J ) SMI31-positive axon labeling demonstrates slight axonal swelling in the
PWMI lesion. (K, L) Olig2-positive preoligodendrocytes are similarly immunostained in lesion and control. Scale bar = 50 Km.
Ó 2012 American Association of Neuropathologists, Inc.
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F6
J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Fig 5 4/C
Verney et al
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
et al (45). The mixture of callosal associative and projection
pathways in these areas might underlie the motor, sensory,
and cognitive deficits seen after periventricular injury (8, 10,
11, 14, 15, 17, 19). The developmental changes in extracellular matrix are reflected in MRI signal intensities and could
lead to differences in pathogenetic responses between very
preterm and preterm brains. Indeed, the developmental differences in glial reactions in foci of PWMI were not limited to
microglia as massive astrogliosis was found in the preterm
brains in contrast to the early microglial activation characteristic of very preterm brains. Importantly, the age-related differences in the periventricular crossroad areas did not include
a decrease in the density of Olig2-positive cells.
The different PWMI patterns between very preterm
and preterm infants may reflect different pathophysiological
mechanisms linked to differences in brain maturation but
we cannot exclude the possibility that the PWMI patterns in
very preterm and preterm infants correspond to 2 successive
stages of the same pathophysiological processes. Indeed, if
we assume that the insult starts in utero, PWMI restricted to
crossroads C1 and C2 in very preterm infants may correspond
to the early postinsult stage and a more extensive pattern of
PWMI distribution including C1 and C2 in preterm infants to
a later postinsult stage. Serial MRI studies of very preterm and
preterm infants would be useful to assess this hypothesis.
Evidence suggests that ischemic-hypoxic and/or inflammatory processes may not only damage growing axons but
also permanently impair axon navigation (41). Therefore, we
conducted a detailed investigation of 2 axonal crossroads
where a complex axon pathfinding process occurs during development. The C1 crossroad is part of the junction of the
external capsule to the anterior limb of the internal capsule (8)
and contains several corticosubcortical bundles that include
the pyramidal tract and thalamocortical reciprocal connections.
The C2 crossroad consists mainly of corticocortical connections (callosal and intrahemispheric fibers), as opposed to corticosubcortical connections. Crossroad C5 contains subcortical
pulvinar and basal forebrain fibers intermingling with cortical
external capsule and callosal radiations. We detected axonopathy in all 3 crossroads. Conceivably, axonopathy may further
disrupt axon pathfinding resulting in motor and cognitive
function impairments for C1 and somatosensory and cognitive
function impairments for C2 and C5. Thus, in very preterm
Microgliosis in Very Preterm White Matter Injury
infants, whose axons are still growing out toward their targets,
functional deficits may result not only from direct lesions to
specific cortical areas but also from fiber-bundle lesions that
prevent normal target finding by spared processes (8).
Microglia-macrophage activation in axonal crossroads
is a well-documented characteristic of the very preterm brain.
During normal human brain development, the junction of the
external and internal capsules (C1) contains more microgliamacrophages at approximately 14 to 17 pcw (42). The
centrum semiovale exhibits a similar increase in microgliamacrophage density from 19 to 30 pcw (43, 44). The developmentally regulated accumulation of microglia-macrophages
in these areas may explain the selective distribution of PWMI
in crossroad areas of very preterm infants. Indeed, a pathophysiological role for activated microglia-macrophages has
been described in several animal models of PWMI caused
by excitotoxic, inflammatory, hypoxic, or hypoxic-ischemic
insults (32, 39Y41). Microglia-macrophage activation may be
either deleterious or neuroprotective depending on the time
since the insult, type of lesion, and location within the lesion.
In agreement with an earlier study (52), we found no MHC-IIY
positive cells in noncystic PWMI, suggesting that this reaction
may be unrelated to an immunologic stimulus.
Foci of PWMI contained only a few reactive GFAPpositive astrocytes in very preterm brains, whereas marked
astrogliosis was seen in preterm brains. The low level of
MCT1 expression by astrocytes in very preterm PWMI suggests astrocyte immaturity, which may explain the absence of
astrogliosis in these brains (50, 53). A recent study showed
GFAP-positive astrocytes undergoing apoptosis in preterm
PWMI (54), and in an animal model, astrocytic death was
described as a primary response of the developing WM to
excitotoxic injury (32).
Preoligodendrocyte cell death has been considered a
key factor in the pathogenesis of PWMI (4, 28Y30). In keeping with a recent study by Billiards et al (31), we found no
decrease in Olig2-positive cell density in PWMI compared
to controls. This finding indicates that preoligodendrocyte
cell death is probably not a major pathogenic factor in PWMI
in very preterm or preterm neonates, but our data do not
exclude impairment of oligodendroglial cell maturation with
subsequent myelination defects in very preterm and preterm
survivors.
FIGURE 5. Coronal sections of a noncystic lesion in a preterm case (33 postconceptional weeks [pcw]). (A) Diagram of necrotic foci
(outlined in red) in white matter in and around the C1 and C2 crossroad areas; area of diffuse damage is outlined in green. CP,
cortical plate: WM, white matter: Ca, caudate (head), CC, corpus callosum, IC, internal capsule, V, ventricle. The pound (#) symbol
indicates the level enlarged (B, D, E, F, B, D, G ): different magnification of the necrotic foci of sections stained with hemalunphloxine (HPS). (B) Arrowheads indicate axonal swellings delimiting the necrotic focus including C2; the arrow indicates the level
enlarged in C. (C) Core of the necrotic focus (asterisk) containing very few glial fibrillary acidic protein (GFAP)Ypositive astrocytes
(brown), delineated by a band of ionized calcium binding adaptor molecule 1 (Iba1)Ypositive microglia-macrophages (black), and
surrounded by reactive astrocytes. (DYF) Serial sections (vessel indicated by an arrow) stained with HPS (D), GFAP-positive
astrocytes (brown) and Iba1-positive microglia-macrophages (black) (E), and vimentin labeling (vim) (F ) showing numerous
dispersed prenecrotic and necrotic foci indicated by black asterisks. The box in E shows the level enlarged in serial sections (GYI).
(GYI ) High magnification at the edge of the necrotic focus. Axonal swellings are labeled by HPS (G); SMI31-positive axonal
spheroids (arrows) are indicated in H; the core of the necrotic focus (asterisk) with very few GFAP-positive astrocytes (brown),
delineated by a band of Iba1-positive microglia-macrophages (black), and surrounded by reactive GFAP-positive astrocytes (I).
Scale bars = 2 mm (B); 200 Km (C); 500 Km (DYF); 50 Km (GYI ).
Ó 2012 American Association of Neuropathologists, Inc.
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
Fig 6 4/C
Verney et al
FIGURE 6. High magnification of a white matter area at a distance from the necrotic foci in a preterm (PT) case (33 postconceptional weeks [pcw]) with noncystic periventricular white matter injury (PWMI) (A, C, E, G, H, J ) and a control case (32 pcw) (B, D,
F, I, K). (A, B ) Hemalun-phloxine staining (HPS). (C, D) Double immunolabeling for glial fibrillary acidic protein (GFAP) (brown)
and ionized calcium binding adaptor molecule 1 (Iba1) (black). (E, F) Double immunolabeling for CD34 (vessel walls, brown) and
monocarboxylate transporter 1 (MCT1, black). (GYI) Iba1-positive intermediate microglia in PWMI (G) compared to more ramified
microglia in the control (I ). Note the large intermediate microglia exhibiting enlargements on processes (arrows) (arrowhead: cell
body) in an abnormal case. ( J, K) Immunolabeling for the axonal filament SMI31. Scale bar = 50 Km (AYK).
12
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J Neuropathol Exp Neurol Volume 71, Number 3, March 2012
In conclusion, our results show that the growing axonal
crossroad areas are cellular compartments that are highly
vulnerable to PWMI in very preterm brains. Research focusing on these axonal crossroad areas within the WM will likely
shed new light on the imaging characteristics and pathophysiology of PWMI.
ACKNOWLEDGMENT
The authors thank Prof. I. Kostovi( for his help with the
anatomic brain studies.
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