Acute Lipopolysaccharide-Mediated Injury in Neonatal White
Matter Glia: Role of TNF-?, IL-1?, and Calcium1
Catherine Sherwin and Robert Fern2
Bacterial infection is implicated in the selective CNS white matter injury associated with cerebral palsy, a common birth disorder.
Exposure to the bacterial endotoxin LPS produced death of white matter glial cells in isolated neonatal rat optic nerve (RON) (a
model white matter tract), over a 180-min time course. A delayed intracellular Ca2?concentration ([Ca2?]i) rise preceded cell
death and both events were prevented by removing extracellular Ca2?. The cytokines TNF-? or IL-1?, but not IL-6, mimicked
the cytotoxic effect of LPS, whereas blocking either TNF-? with a neutralizing Ab or IL-1 with recombinant antagonist prevented
LPS cytotoxicity. Ultrastructural examination showed wide-scale oligodendroglial cell death in LPS-treated rat optic nerves, with
preservation of astrocytes and axons. Fluorescently conjugated LPS revealed LPS binding on microglia and astrocytes in neonatal
white and gray matter. Astrocyte binding predominated, and was particularly intense around blood vessels. LPS can therefore
bind directly to developing white matter astrocytes and microglia to evoke rapid cell death in neighboring oligodendroglia via a
calcium- and cytokine-mediated pathway. In addition to direct toxicity, LPS increased the degree of acute cell death evoked by
ischemia in a calcium-dependent manner. The Journal of Immunology, 2005, 175: 155–161.
no effective remedy or prophylactic strategy (1–3). In the majority
of cases, cerebral palsy involves a lesion centered upon the devel-
oping white matter adjacent to the ventricles (periventricular leu-
komalacia (PVL)3). PVL arises in mid-gestation at a point when
myelination is initiating (4). Injury of white matter glial cells is
prevalent and may involve selective loss of oligodendroglia with
subsequent hypomyelination (5–7).
An increasing number of studies have implicated intrauterine
infection and chorioamnionitis in the genesis of PVL. Early reports
demonstrated that i.v. injection of the bacterial endotoxin LPS can
produce selective white matter injury in neonatal CNS (8, 9). Re-
cent studies have shown that direct LPS injection into the devel-
oping brain can evoke white matter damage (10, 11), whereas in-
duction of intrauterine infection can produce diffuse glial cell death
and cavitation in fetal white matter (12, 13). The mechanism of
LPS cytotoxicity in developing white matter appears to involve
cell-cell interactions. For example, cultured oligodendroglia die
when exposed to LPS provided that other glial types are present
(14–17), whereas media collected from LPS-treated microglia or
astrocytes is toxic to oligodendroglia in culture (15, 18). A recent
report suggests that microglia are the only neural cell type to ex-
erebral palsy is a common human birth disorder manifest
in the first years of life, which continues unabated
throughout the lifetime of the patient. There is currently
press the LPS receptor TLR-4, although this has not been exam-
ined in vivo (17).
LPS can promote the release of cytokines from glia, whereas
cytokine production is implicated in the association between in-
trauterine infection, preterm birth and neonatal brain damage (see
Discussion). However, because i.v. injection of LPS produces sys-
temic hypotension (19), whereas chorioamnionitis is associated
with hypotension in the fetus (20), it is uncertain whether bacterial
infection produces white matter injury as a direct effect, or subse-
quent to ischemia associated with hypotension. Experimental stud-
ies have repeatedly demonstrated that immature white matter is
predisposed to ischemia during hypotension, and is highly sensi-
tive to ischemic injury (1). The immature oligodendroglia that pop-
ulate white matter at this age are exquisitely sensitive to ischemic
injury (21, 22). The current study examines LPS-mediated cyto-
toxicity in glial cells in isolated white matter, to establish the po-
tential for direct LPS-mediated white matter injury independent of
hemodynamic factors. The mechanism and characteristics of direct
LPS cytotoxicity are investigated and the pattern of glial cell bind-
ing of LPS examined in situ.
Materials and Methods
Optic nerves were dissected from P8-P12 (called “P10” throughout) Lister
hooded rats and placed in artificial cerebrospinal fluid (aCSF) composed
(in mM) 153 Na?, 3 K?, 2 Mg2?, 2 Ca2?, 131 Cl?, 26 HCO3
10 dextrose, bubbled with 95% O2/5% CO2. All procedures involving the
use of animals were approved by local ethical review. At this age, the rat
optic nerve (RON) is initiating the process of myelination, with the first
wraps of myelin appearing (23, 24). This is the same developmental point
at which periventricular white matter is subject to PVL in the fetus (4, 25).
Bacterial LPS (from Escherichia coli; Sigma-Aldrich) was dissolved in
aCSF immediately before the experiment to give a final concentration of 1
?g/ml. Ischemia was induced by changing from aCSF to perfusion with
zero-glucose aCSF that had been bubbled with 95% N2/5% CO2for at least
60 min. The atmosphere in the recording chamber was switched simulta-
neously to 95% N2/5% CO2(see Ref. 26 for further details). Ischemia was
maintained for 60 min, at which point normal conditions were re-estab-
lished for a further 15 min. Zero-calcium solutions were made up by ex-
cluding Ca2?from the composition of aCSF and adding the calcium che-
lator EGTA at a concentration of 50 ?M. IL-1?, TNF-?, IL-6, anti-TNF-?
(used at 10 ?g/ml), and IL-1 receptor antagonist (IL-1ra; used at 5 ?g/ml)
were purchased from R&D Systems. Catalase (250 U/ml) and 4-hydroxy-
2,2,6,6-tetramethylpiperidinyloxy (TEMPO; 500 ?M) were purchased
?, 2 H2PO4
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester,
Received for publication December 17, 2004. Accepted for publication April
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institute of Neurological Disorders and Stroke
Grant NS44875 (to R.F.).
2Address correspondence and reprint requests to Dr. Robert Fern, Department of Cell
Physiology and Pharmacology, University of Leicester, P.O. Box 138, University
Road, Leicester, U.K. LE1 9HN. E-mail address: RF34@le.ac.uk
3Abbreviations used in this paper: PVL, periventricular leukomalacia; aCSF, artificial
cerebrospinal fluid; [Ca2?]i, intracellular Ca2?concentration; RON, rat optic nerve;
IL-1ra, IL-1 receptor antagonist; ROS, reactive organ species.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
from Calbiochem. Unless otherwise stated, all other chemicals were pur-
chased from Sigma-Aldrich. Data are presented as the mean ? SEM, with
statistical significance determined by ANOVA with the Tukey posttest
Intracellular Ca2?concentration ([Ca2?]i) imaging
The low affinity, Ca2?-sensitive dye Fura-FF was used for Ca2?imaging
to limit the extent of Ca2?-buffering. 1 mM Fura-FF AM (Molecular
Probes) stock solution was made in dry DMSO and 10% pluronic acid.
RONs were incubated for 50 min at room temperature in aCSF containing
10 ?M FURA-FF AM. RONs were maintained in hydrated 95% O2/5%
CO2atmosphere during the incubation period and were washed in aCSF
before being mounted in the perfusion chamber. The ends of the optic
nerves were fixed to a 22 ? 40 mm glass coverslip with small amounts of
cyanoacrylate glue, leaving the majority of the nerve completely free of
glue (26). The coverslip was sealed onto a Plexiglas perfusion chamber
(atmosphere chamber; Warner Instruments) with silicone grease. The aCSF
was perfused through the chamber at a rate of 2–3 ml/min, with a fluid level
of ?2 mm completely covering the RON. 95% O2/5% CO2was blown
over the aCSF at a rate of 0.3 L/min. The chamber was mounted on the
stage of an Nikon Eclipse TE200 inverted epifluorescence microscope.
Chamber temperature was maintained closely at 37°C with a flow-through
feedback tubing heater (Warner Instruments) positioned immediately be-
fore the aCSF entered the chamber, and a feedback objective heater
(Bioptechs) that warmed the objective to 37°C. This combination of heat-
ing systems regulated the temperature of the bath and cover slip to 37°C,
as established periodically with a temperature probe.
Cells within the RON were illuminated at 340, 360, and 380 nm by
Monochromator (Optoscan; Cairn Research), and images were collected at
520 nm using an appropriate filter set (Chroma Technology). Images were
taken with a cooled CCD camera (CoolSNAP HQ; Roper Scientific) every
60 s. This low recording frequency limited any damaging effect of illumi-
nation on the cells and reduced dye bleaching over the long recording times
that were used. Changes in 340:380 ratio were taken to indicate changes in
[Ca2?]i(27). The 360 intensity (isosbestic point) was monitored to assess
the capacity of cells to retain dye. The sudden loss of 360 signal correlated
with loss of cell membrane integrity and the release of dye into the extra-
cellular space (see Ref. 26). Data were collected and stored with the image
acquisition program Metafluor (Universal Imaging) running on Windows
XP. Because of slow shifting of the preparation, cells were occasionally
refocused during experiments. A small proportion of cells slowly drifted
out of the focal plane and were discarded. Many of the experiments re-
quired long recording periods (190 min), which had a relatively low suc-
cess rate due to dye fading and/or shifting of the preparation.
Dead cell counts
Ethidium bromide, a lipophobic nuclear dye that does not stain intact living
cells, was used to stain nuclei of dead cells. RONs were dissected and
placed in aCSF. The nerves were incubated for 180 min at 37°C in 1 ml of
an experiment solution. RONs were maintained in hydrated 95% O2/5%
CO2atmosphere during the incubation period and were subsequently
washed with fresh aCSF. RONs were then incubated for 10 min at room
temperature with 0.5 nM ethidium bromide. RONs were then washed in
aCSF and live-mounted on a Nikon LABOPHOT-2 epifluorescence mi-
croscope equipped with a 20? fluorescence objective and appropriate fil-
ter. Whole RON images (excluding the cut ends) were captured with a
CCD camera, averaging 16 frames. Cell counting was performed blind
using ImageJ (NIH). The mean number of dead cells found following ex-
posure to any given condition was compared with the number seen in
control RONs (180 min aCSF only), and are expressed as a percentage
change relative to this level of control cell death.
Whole P10 rat brains were dissected free, and RONs were dissected free
and left on the optic chiasm, before fixation in 3% paraformaldehyde for 30
min at room temperature and mounting in Tissue Tec (Sakura). Sections
(20 ?m) were cut with a cryostat before mounting on slides. Fluorescently
conjugated LPS, (LPS-Alexa Fluor 488, E. coli; Molecular Probes), was
used to detect LPS binding (1 ?g/ml in PBS). Sections were blocked for 60
min at room temperature with PBS containing 10% goat serum and 0.5%
Triton X-100 before incubation in conjugated LPS for 120 min at room
temperature. Sections were either simultaneously costained with monoclo-
nal anti-glial fibrillary acidic protein (GFAP) conjugated to Cy3 (Sigma-
Aldrich; 1:500 in PBS), or stained overnight at 5°C with isolectin IB4
conjugated to Alexa-Fluor 594 (Molecular Probes; 1:100 in PBS). Sections
were washed briefly after incubation in LPS-conjugate before fixation with
4% formaldehyde for 30 min in PBS. Slices were then washed in PBS (2 ?
15 min) and mounted on an Olympus IX70 inverted confocal microscope
for image collection. Conjugated probes were used to eliminate cross-re-
actions and the specificity of LPS-conjugate binding was tested by dis-
placement with unconjugated LPS (see Results). No bleed through of flu-
orescence was detected between channels.
natal white matter glia. A, Changes in the [Ca2?]i-dependent 340/380 ratio
and [Ca2?]i-independent 360 intensity in a representative FURA-FF loaded
glial cell within a RON perfused in aCSF ? LPS (1 ?g/ml) for 180 min.
Following a period of control recording, exposure to LPS induced a de-
layed Ca2?rise associated with cell death (dashed line). B, Degree of cell
death during 180 min of control perfusion with aCSF and during 180 min
of perfusion with LPS (n ? cells/RONs). C, Time-course of cell death
among the 64 cells that died following exposure to LPS, determined by loss
of cytoplasmic FURA-FF and expressed as a percentage. Note the gradual
increase in cell death with the period of LPS exposure, with death first
becoming apparent after ?60–90 min.
LPS induces [Ca2?]irises and subsequent cell death in neo-
156ACUTE LPS-MEDIATED INJURY OF NEONATAL GLIA
RONs were exposed to 1 ?g/ml LPS in aCSF or control aCSF for 180 min
before washing in Sorenson’s buffer and postfixation in 3% glutaraldehyde/
Sorenson’s for 90 min at room temperature. The RONs were subsequently
postfixed with 2% osmium tetroxide and dehydrated in ethanol and pro-
pylene oxide. The nerves were infiltrated in epoxy resin and the ends cut
back before taking ultrathin sections. Sections were counterstained with
uranyl acetate and lead citrate and examined with a JEOL 100CX electron
microscope. To avoid bias in the data, electron micrographs were collected
blind (by R. Fern) to the experimental procedure used to produce each
The 340/380 ([Ca2?]i-sensitive) ratio of FURA-FF loaded P10
RON glial cells was stable during control perfusion with aCSF for
190 min, with little cell death observed (1 of 53 cells, in 3 RONs).
Following 10 min of control perfusion, glial cells in RONs ex-
posed to LPS (1 ?g/ml) generally had a stable [Ca2?]iuntil a rapid
rise preceded cell death in 59.4 ?10.6% of cells (n ? 63 cells, 4
RONs, p ? 0.001: Fig. 1, A and B). The incidence of cell death
tended to increase with the period of LPS exposure (Fig. 1C).
The P10 RON is at a similar developmental point to the human
mid-term periventricular white matter that is subject to PVL (24,
25, 28). Ultrastructural examination of control RONs showed the
typical components previously reported at this age: premyelinated ax-
ons and some early myelinated axons; astrocytes; and cells of the
oligodendroglial lineage. Immature oligodendroglial cells could be
in the P10 RON. A and B, Control RON perfused with
aCSF for 180 min postdissection. Typical immature oli-
godendrocytes are shown, characterized by the pres-
ence of myelinating and premyelinating axons embed-
ded within the somata and processes (“*”), numerous
mitochondria (“m”), a relatively narrow bore endoplas-
mic reticulum (“er”), and evenly dispersed chromatin
within the nucleus that generally contained at least one
prominent nucleolus (29). Golgi apparatus (“g”) was
often apparent. C–F, RON after 180 min LPS treat-
ment. C and D, Typical LPS treated immature oligo-
dendrocytes. The nuclear morphology is largely unaf-
fected but mitochondria are either severely swollen or
completely disrupted (D) and the cell membrane may
be breached (note the free-floating distended mitochon-
dria in C, arrow). Endoplasmic reticulum may also be
distended, but gross cell swelling and wide-scale ne-
crosis, such as that seen in postischemic neonatal RON
glia (29), is not apparent. E, Mitochondrial damage is
also evident in immature oligodendroglial processes
(arrow). Note the healthy appearance of neighboring
axons in C–E. F, Astrocyte in LPS treated RON. The
endoplasmic reticulum is swollen but mitochondria are
healthy and cell membrane is intact. Scale bar ? 1 ?m
in all cases.
Ultrastructural changes evoked by LPS
dent. Histogram showing the number of dead cells in RONs exposed to
various experimental conditions, relative to control. LPS produced a sig-
nificant increase in the number of dead cells in RONs, an effect that was
blocked by removing extracellular Ca2?or by the ROS scavengers
TEMPO ? catalase. RONs were incubated for 180 min in the various
conditions, before ethidium bromide staining. Error bars ? SEM, n ?
number of RONs; ?, p ? 0.001 vs control.
LPS-mediated acute cell death is Ca2?- and ROS-depen-
157The Journal of Immunology
reliably identified by their characteristic nuclear morphology, narrow-
bore endoplasmic reticulum and the presence of myelinated axons
within the cell body or processes (29) (Fig. 1, A and B).
RONs subjected to 180 min of LPS treatment showed wide scale
injury of immature oligodendrocytes (Fig. 2, C and D). Nuclear
morphology appeared normal in these cells but mitochondria were
swollen or ruptured and areas where the cell membrane had been
lost could be identified in many cases. Similar changes were ap-
parent in immature oligodendrocyte processes, although the degree
of injury was less marked and cell membrane breakdown was not
observed (Fig. 2E). No pathology was evident in axons, even those
that were being ensheathed by immature oligodendrocytes that had
died (Fig. 2C). Astrocytes were also largely unaffected in post-LPS
treated RON (Fig. 2F).
The cytotoxic effect of LPS was confirmed by counting dead
cells in P10 RONs following a standard 180 min exposure to 1
?g/ml LPS. RONs maintained in the presence of LPS had a 68.0 ?
10.2% increase in the number of dead cells compared with control
(p ? 0.001; Fig. 3). The cytotoxic effect of LPS was blocked by
removing extracellular Ca2?(?50 ?M EGTA), or by addition of
the reactive oxygen species (ROS) scavengers TEMPO ? catalase
(30) (Fig. 3). Neither zero Ca2?nor ROS scavengers significantly
affected the number of dead cells in the absence of LPS.
Cytokines are implicated as intermediaries in various bacterial
infection models of white matter injury. Exposure for 180 min to
the cytokines TNF-? (1,000 U/ml) or IL-1? (1,000 U/ml), but not
IL-6 (1,000 U/ml), mimicked the cytotoxic effect of LPS (1 ?g/ml)
(Fig. 4). Exposure to all three cytokines (with IL-1? increased to
25,000 U/ml) had no additive cytotoxic effect (Fig. 4). Removing
the biological activity of TNF-? by incubation in an inactivating
Ab (10 ?g/ml) abolished the cytotoxic action of LPS, as did in-
cubation with an antagonist of IL-1, IL-1ra (5 ?g/ml). Although
showing the number of dead cells in neonatal RON after 180 min of several
experimental conditions, relative to control. A similar increase in the num-
ber of dead cells was apparent following 180 min exposure to LPS, TNF-?,
IL-1? or TNF-? ? IL-1? ? IL-6. Although 120-min exposure to TNF-?
was toxic, 120-min exposure IL-1? was not. IL-6 alone did not signifi-
cantly increase the number of dead cells compared with control. Anti-
TNF-? or IL-1ra blocked the cytotoxic effect of LPS. n ? number of
RONs, error bars ? SEM; ?, p ? 0.001 vs control.
Cytokines mediate the cytotoxic effects of LPS. Histogram
present on both microglia and astro-
cytes in neonatal RON. A, GFAP
staining of astrocytes in P10 RON. B,
Conjugated LPS binding of the same
section. C, Merge, showing LPS bind-
ing to astrocyte somata and processes
(arrows). D, GFAP staining of astro-
cytes in P10 RON. E, Conjugated
LPS binding of the same section, per-
formed in the presence of a 10 times
excess of unconjugated LPS. Note the
absence of LPS binding. F, Conju-
gated LPS binding of P10 RON. G,
Same section showing conjugated-
isolectin B4 (IB4) binding on a micro-
glial cell. H, Merge, showing coex-
pression of LPS and IB4 binding sites
on the microglial cell (arrow). I, Con-
jugated LPS binding in a P10 brain
slice, showing intense binding around
a blood vessel (vessel lumen indicated
by “*”). J, GFAP binding of the same
section showing GFAP?astrocytes
surrounding the blood vessel. F,
Merge, showing intense LPS binding
to the perivascular astrocytes. Bar ?
10 ?m in all cases.
LPS-binding sites are
158ACUTE LPS-MEDIATED INJURY OF NEONATAL GLIA
120 min exposure to IL-1? was toxic, 120 min of IL-1? was not
To probe the site of action of LPS, conjugated LPS binding was
examined in P10 RON and brain. LPS binding was widespread
throughout P10 RON, colocalizing with GFAP?astrocyte somata
and processes (Fig. 5, A–C). Conjugated LPS binding was dis-
placed by coincubation with nonconjugated LPS (10 ?g/ml) (Fig.
5, D and E), proving the selectivity of the binding. Microglial
labeling with I-B4 revealed a sparse population of I-B4?cells that
bound conjugated LPS (Fig. 5, F–H). These cells were rare, and
the great majority of LPS binding cells were I-B4?. Widespread
binding of conjugated LPS was also found in P10 brain slices,
again largely colocalized with GFAP?astrocytes. Binding in brain
slices was particularly intense on astrocytes surrounding blood
vessels (Fig. 5, I–K).
Several reports suggest that bacterial and ischemic injury may
be additive in developing white matter (31). Consistent with our
previous results, ischemic conditions evoked [Ca2?]irises associ-
ated with cell death in FURA-FF loaded P10 RON glia (Fig. 6, A
and B), (29, 32). Qualitatively similar events were seen during
ischemia in the presence of LPS (1 ?g/ml) (Fig. 6, C and D), and
the degree of cell death produced by ischemia was significantly
higher in the presence of LPS (1 ?g/ml) (Fig. 7).
We have recently reported that removing extracellular Ca2?is
protective against oligodendroglial death, and increases astrocyte
death during acute ischemia in the P10 RON (29, 32). The overall
level of glial death during ischemia was significantly reduced by
removing extracellular Ca2?(p ? 0.01), and the increase in cell
death during ischemia produced by LPS was absent in zero-Ca2?
(Fig. 7). Unlike for the LPS-mediated cell death produced by 180
min of exposure to LPS, the additive effect of LPS upon ischemic
cell death was not reduced by the ROS scavengers TEMPO ?
catalase (Fig. 7).
Induction of maternal bacterial infection in rabbits can reproduce
a focal and diffuse white matter injury in fetal brain that has some
similarities to PVL (12, 13, 33). Intracerebral or ventricular injec-
tion of LPS in neonatal rat can also produce white matter damage
(10, 11). The current study demonstrates LPS toxicity in immature
white matter oligodendrocytes in a whole mount preparation. LPS
is therefore directly and acutely toxic to developing white matter
glia, irrespective of vascular factors that may come into play dur-
ing infection (20). The rapid death of immature oligodendrocytes
found in this preparation was not reported following exposure to
LPS in cell culture (15, 16), suggesting that interactions between
cell types in situ underlies the acute toxicity.
during ischemia: effect of LPS. A, [Ca2?]i-sensitive 340/380 ratio changes
in a typical FURA-FF-loaded cell evoked by 60 min of ischemia (oxygen
and glucose withdrawal). B, The accompanying 360 intensity changes,
showing cell death (dashed line), which is associated with an elevation in
[Ca2?]i. C, 340/380 ratio changes in a typical cell during 60 min of isch-
emia in the presence of LPS. D, Accompanying 360 intensity changes,
showing cell death (dashed line).
[Ca2?]ichanges and cell death in neonatal RON glial cells
experiments performed in various solutions, detected by loss of FURA-FF
fluorescence. No cell death was found under control conditions (aCSF)
over the 90 min period of the experiments. The degree of cell death found
during ischemia was significantly higher when LPS (1 ?g/ml) was included
in the perfusing solution. Removing extracellular Ca2?(?50 ?M EGTA)
significantly reduced the cell death evoked by ischemia and blocked the
increase in ischemic cell death produced by LPS. The ROS scavengers
TEMPO and catalase had no effect either upon the cell death produced by
ischemia, or ischemia ? LPS. Percentage cell death is calculated as the
number of cells that died of the total number of cells for each experiment
type. Error bars ? SEM, n ? total number of cells/number of RONs; ?,
p ? 0.01 vs ischemia alone.
Histogram showing the incidence of cell death in ischemia
159The Journal of Immunology
LPS binds to microglia and astrocytes in situ
TLR-4 (?CD14) is the only known binding site for LPS (34). Cell
culture studies report that microglia express TLR-4, whereas oli-
godendrocyte precursors do not (17), and astrocytes either do not
(17) or express very low levels unless pre-exposed to LPS (35).
LPS binding has not previously been examined in situ. We report
extensive LPS binding in neonatal RON and brain. LPS binding
was particularly marked around blood vessels. Microglial cells
were shown to bind LPS, but the majority of LPS-bindings cells
were GFAP?astrocytes in both RON and brain. The acute cyto-
toxic effect of LPS upon immature oligodendrocytes is therefore
mediated by other glial cell types, presumably mainly astrocytes.
LPS toxicity is delayed and is Ca2?dependent
The acute cell death evoked by LPS reported in this study was
preceded by a delayed sudden increase in [Ca2?]iand was pre-
vented by removing extracellular Ca2?, demonstrating the Ca2?
dependence of the injury. The delayed nature of the [Ca2?]irise is
consistent with LPS having initial effects on astrocytes and/or mi-
croglial cells, which then mediate a delayed and lethal rise in ol-
igodendrocyte [Ca2?]i. If this is correct, the initial effects that pre-
cede the oligodendrocyte [Ca2?]irise take between 60 and 120
min and constitute the priming steps in an injury cascade.
The role of cytokines
The incidence of PVL in premature infants is correlated with el-
evated cytokine levels in umbilical cord blood, amniotic fluid and
neonatal blood (33, 36, 37). We report that TNF-? and IL-1?
directly evoke acute cell death in RON glia, with the degree of cell
death not significantly different from that produced by LPS. Al-
though implicated in the inflammatory response to LPS (38), IL-6
was not found to cause acute glial cell death. Combined IL-1?,
IL-6, and TNF-? exposure did not increase the degree of cell
death, and it appears that maximal injury was achieved at the cy-
tokine concentrations used in this study.
LPS-mediated cell death was reduced to the level seen in control
experiments by the addition of a neutralizing Ab for TNF-?, or of
IL-1ra. Therefore, production of both cytokines is necessary for
the acute cytotoxic effects of LPS. Maternal venous LPS injection
elevates fetal brain TNF-? and IL-1? mRNA within 60 min (39).
In cell culture, LPS can induce TNF-? release from microglia
within 60–120 min and IL-1? within 120–180 min (the earliest
time points measured) (40–43). Cultured astrocytes are also ca-
pable of cytokine release (44), with LPS-induced TNF-? release
requiring the prior release of IL-1? (45–47). This is consistent
with the current finding that 120 min of exposure to TNF-? was
toxic but IL-1? required 180 min of exposure for toxicity to be-
come apparent. If astrocytes are the major source of the cytokine
release induced by LPS, as the LPS binding data would suggest,
this would explain the protection provided by blocking either
TNF-? or IL-1?.
LPS-stimulated production of glial TNF-? and IL-1? involves
the activation of the transcription factor NF-?B, which is evident
within 60 min of LPS exposure in cell culture (48). Cytokines and
ROS are also strong activators of NF-?B in glia (48, 49), suggest-
ing a feed-forward loop involving exposure to LPS, activation of
NF-?B, cytokine and ROS generation and further activation of
NF-?B (Fig. 8). This would be consistent with the protective effect
of ROS scavengers seen in the results and with studies showing
LPS evoked ROS generation in cultured microglia and astrocytes
The toxic effect of TNF-? upon oligodendroglia is well charac-
terized (e.g., Refs. 18 and 54), and IL-1? toxicity of oligodendro-
glia has also been reported (55). It is unclear whether such toxicity
is a direct pathway to the glial injury described in the current
results, because TNF-?-mediated oligodendroglial cell death is
thought to be slow. However, significant death of cultured rat ol-
igodendroglia has been reported within 120 min of exposure to
TNF-? (18), and the generally slower oligodendrocyte death seen
in other studies may be due to the low sensitivity to TNF-?-me-
diated injury in the mature cells used (54). However, a direct con-
tribution of ROS to the oligodendroglial injury cannot be ruled out.
LPS and ischemia
Prior reports suggest that bacterial endotoxin- and ischemia-medi-
ated immature white matter injury is additive (31). Bacterial en-
dotoxins such as LPS evoke hypotension and can be predicted to
induce ischemia in fetal white matter in vivo (19, 20). The current
study shows additive injury by LPS and ischemia in isolated im-
mature white matter, independent of any hemodynamic factors that
may come into play in vivo. This additive effect was Ca2?depen-
dent; whereas both LPS- and ischemia-induced injury of RON oli-
godendrocytes is mediated by Ca2?influx (29). However, the po-
tentiation of ischemic injury by LPS occurred over a shorter time
course (60 min) than was required for direct LPS toxicity, and was
not affected by ROS scavengers that blocked direct toxicity. There-
fore, it may be that interactions between LPS and ischemia involve
different pathways than those operating during direct LPS toxicity.
The authors have no financial conflict of interest.
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161The Journal of Immunology