Estradiol attenuates hyperoxia-induced cell death in the developing white matter

Abstract

Periventricular leukomalacia is the predominant type of brain injury in preterm infants underlying the development of cerebral palsy. Periventricular leukomalacia has its peak incidence at 23 to 32 weeks postconceptional age characterized by extensive oligodendrocyte migration and maturation. Oxygen toxicity has been identified as a possible contributing factor to the pathogenesis of cerebral palsy in survivors of preterm birth. 17beta-estradiol (E2) is important for the development and function of the central nervous system. Furthermore, neuroprotective properties have been attributed to estrogens. We examined the effect of E2 on hyperoxia-induced cell death in the developing white matter in the rat brain.
Six-day-old (P6) rat pups, the immature oligodendroglial cell line (OLN-93), and primary oligodendrocyte cultures were subjected to 80% O(2) in the presence or absence of E2 (600 microg/kg intraperitoneally in vivo, 10(-6)-10(-10)M in vitro). Cell counts and lactate dehydrogenase assay were used to assess cell survival. Immunoblot analysis was used for detection of estrogen receptor expression and investigation of apoptotic signaling pathways. White matter injury was assessed by myelin basic protein immunocytochemistry at P11.
E2 produced significant dose-dependent protection against oxygen-induced apoptotic cell death in primary oligodendrocytes. Treatment with E2 prevented hyperoxia-induced proapoptotic Fas-upregulation and caspase-3 activation. Finally, E2 antagonized hyperoxia-induced inactivation of extracellular signal-regulated kinase 1 and 2 and Akt, key kinases of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase cell survival promoting pathways, respectively. Loss of myelin basic protein labeling was seen in P11 pups after oxygen exposure, and E2 attenuated this injury.
These results suggest a possible role for estrogens in the prevention of neonatal oxygen-induced white matter injury.

Full text

Estradiol Attenuates Hyperoxia-Induced Cell
Death in the Developing White Matter
Bettina Gerstner, MD,
1,2
Marco Sifringer, MSc,
1,3
Mark Dzietko, MD,
1
Alexandra Schu¨ller, MD,
1
Joan Lee, BA,
2
Sinno Simons, MD,
1
Michael Obladen, MD, PhD,
1
Joseph J. Volpe, MD,
2
Paul A. Rosenberg, MD, PhD,
2
and Ursula Felderhoff-Mueser, MD, PhD
1
Objective: Periventricular leukomalacia is the predominant type of brain injury in preterm infants underlying the development
of cerebral palsy. Periventricular leukomalacia has its peak incidence at 23 to 32 weeks postconceptional age characterized by
extensive oligodendrocyte migration and maturation. Oxygen toxicity has been identified as a possible contributing factor to the
pathogenesis of cerebral palsy in survivors of preterm birth. 17␤-estradiol (E2) is important for the development and function
of the central nervous system. Furthermore, neuroprotective properties have been attributed to estrogens. We examined the effect
of E2 on hyperoxia-induced cell death in the developing white matter in the rat brain.
Methods: Six-day-old (P6) rat pups, the immature oligodendroglial cell line (OLN-93), and primary oligodendrocyte cultures
were subjected to 80% O
2
in the presence or absence of E2 (600␮g/kg intraperitoneally in vivo, 10
⫺6
–10
⫺10
M in vitro). Cell
counts and lactate dehydrogenase assay were used to assess cell survival. Immunoblot analysis was used for detection of estrogen
receptor expression and investigation of apoptotic signaling pathways. White matter injury was assessed by myelin basic protein
immunocytochemistry at P11.
Results: E2 produced significant dose-dependent protection against oxygen-induced apoptotic cell death in primary oligoden-
drocytes. Treatment with E2 prevented hyperoxia-induced proapoptotic Fas-upregulation and caspase-3 activation. Finally, E2
antagonized hyperoxia-induced inactivation of extracellular signal-regulated kinase 1 and 2 and Akt, key kinases of the mitogen-
activated protein kinase and phosphatidylinositol 3-kinase cell survival promoting pathways, respectively. Loss of myelin basic
protein labeling was seen in P11 pups after oxygen exposure, and E2 attenuated this injury.
Interpretation: These results suggest a possible role for estrogens in the prevention of neonatal oxygen-induced white matter
injury.
Ann Neurol 2007;61:562–573
Advances in neonatal intensive care have markedly im-
proved survival rates of premature infants. Unfortu-
nately, a substantial proportion of very-low-birth-
weight infant survivors have neurological deficits that
affect motor and cognitive function.
1,2
Long-term neu-
rocognitive impairment restricts quality of life for af-
fected individuals and their families and poses a con-
siderable socioeconomic problem.
3
The neuropathology of perinatal brain injury is com-
plex and involves gray and white matter structures to
varying degrees, depending on the gestational age and
the developmental stage.
4,5
The timing of vulnerability
coincides with the peak of the brain growth spurt,
which starts at about midpregnancy in humans and ex-
tends into the third postnatal year.
6
Periventricular leu-
komalacia (PVL) is the predominant type of injury in
preterm infants and leads to a chronic deficit of white
matter structures. It has its peak incidence during a
well-defined period in human brain development
(23–32 weeks, postconceptional age) characterized by
extensive oligodendrocyte (OL) migration and matura-
tion.
7
Oxygen is widely used in neonatal intensive care.
However, it is implicated in the pathogenesis of neo-
natal chronic lung disease and retinopathy of the pre-
maturity. Our group has recently shown that hyperoxia
is a powerful trigger for widespread apoptotic neuronal
death in the developing brain. Hyperoxia increased the
density of degenerating cells in various brain regions of
7-day-old Wistar rats and C57/BL6 mice such as the
caudate nucleus, layers II and IV of the frontal, pari-
etal, cingulate, and retrosplenial cortices, as well as
From the
1
Department of Neonatology, Charite´ Campus Virchow-
Klinikum, Berlin, Germany;
2
Department of Neurology and Neu-
robiology Program, Children’s Hospital Boston, Boston, MA; and
3
Department of Pediatric Neurology, Children’s Hospital, Medical
Faculty Carl Gustav Carus, Technical University Dresden, Dresden,
Germany.
P.A.R. and U.F.-M. contributed equally to this work.
Received Dec 7, 2006, and in revised form Feb 2, 2007. Accepted
for publication Feb 9, 2007.
Published online Apr 11, 2007, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21118
Address correspondence to Dr Gerstner, Department of Neonatol-
ogy, Campus Virchow Klinikum, Universita¨tsmedizin Berlin,
Charite´, Augustenburger Platz 1, 13353 Berlin, Germany.
E-mail: bettina.gerstner@charite.de
562 © 2007 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services

white matter tracts and the periventricular region.
8
This cell death is associated with oxidative stress, de-
creased expression of neurotrophins, decreased activa-
tion of neurotrophin-regulated pathways,
8
and in-
creased levels of proinflammatory cytokines.
9
Recently,
we investigated the effects of hyperoxia on cultured rat
precursor, immature and mature oligodendroglia cells,
derived from the permanent oligodendrocyte cell line
(OLN-93). Hyperoxia initiated the apoptotic cascade
in immature OLs and preoligodendroglial cells, but not
in mature OLs in vitro.
10
New pharmacological ap-
proaches are necessary to limit the neurotoxic effects of
hyperoxia in the developing brain.
17␤-estradiol (E2) is one promising candidate for
neuroprotection. E2 and the estrogen receptor (ER)
play important roles in the development and function
of the central nervous system.
11,12
Its receptors, ER-␣
and ER-␤show a specific distribution with high den-
sities around the ventricles, the region where brain cell
progenitors are generated.
13
Neuroprotective effects of
E2 in the mature brain have been observed in several
models of neurotoxicity involving hypoxia-ischemia
and excitotoxicity.
14,15
Female steroid hormones can
influence the apoptotic cascade at different stages.
16
Moreover, E2 has antioxidative properties that lead to
the reduction of free radicals.
17
E2 also has profound
effects on the function and plasticity of the brain and
proliferation, differentiation, and migration of neurons
are controlled by E2.
18
During pregnancy, E2 levels increase up to 100-fold
in the placenta.
19
The fetus is also exposed to these
increasing hormone levels. At birth, after the umbilical
cord is clamped, the levels of E2 decrease by a factor of
100 within 24 hours.
19
Premature infants experience
this hormone deprivation and simultaneous increase of
the oxgygen tissue tension much earlier than infants
born at term.
In an attempt to characterize measures that will
counteract injury to the developing white matter, this
study focused on the effect of E2 on premyelinating
oligodendrocytes (pre-OL) in established models of ap-
optotic brain damage in vitro and in vivo.
Materials and Methods
Cell Cultures
OLIGODENDROGLIA CELL LINE. The OLN-93 cell line
derived from spontaneously transformed cells in primary rat
brain glia culture was kindly provided by Dr C. Richter-
Landsberg (Institute of Molecular Neurobiology, Oldenburg,
Germany).
20
OLN-93 cells bear the morphological and an-
tigenic properties of 5- to 10-day-old (postnatal time) cul-
tured rat brain OLs. These resemble the intermediate stage
between the pre-OL (O4⫹O1
⫺
MBP
⫺
) and the mature OL
(O4
⫹
O1
⫹
MBP
⫹
). Cells were maintained in Dulbecco’s
minimum essential medium, with 3.7gm/L NaHCO
3
,
25mM HEPES
3
, 4.5gm/L D-glucose, 4.4gm/L NaCl, con-
taining heat-inactivated 10% fetal calf serum. Monolayers
were cultured at 37°C in a humidified, 5% CO
2
atmosphere
with medium replenishment every 2 to 3 days.
OLIGODENDROCYTE PRIMARY CULTURES. Primary rat
OLs were prepared from the cerebral hemispheres of
Sprague–Dawley rats at postnatal days 1 to 2 using a shaking
method
21
with modifications, as described previously.
22
An-
imals were killed by decapitation. Purified OLs were cultured
for 7 to 8 days in a serum-free basal-defined medium: Dul-
becco’s minimum essential medium, 0.1% bovine serum al-
bumin, 50␮g/ml apo-transferrin, 50␮g/ml insulin, 30nM so-
dium selenite, 10nM D-biotin, 10nM hydrocortisone,
200␮ML-cystine, 10ng/ml platelet-derived growth factor,
and 10ng/ml basic fibroblast growth factor. At 7 to 8 days,
the cultures were composed primarily of progenitors and pre-
OLs (O4
⫹
O1
⫺
MBP
⫺
). The purity of OL cultures was con-
sistently greater than 95% OLs with less than 5% astrocyte
contamination. Primary OL cultures and OLN-93 cells were
subjected to 80% O
2
in the presence or absence of E2
(10
⫺6
–10
⫺10
M; Sigma, St. Louis, MO). E2 was given at 0,
2, 4, 6, 12, 24, and 48 hours before oxygen exposure.
IMMUNOCYTOCHEMISTRY. Cells were fixed with 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 10
minutes at room temperature (RT), washed 3 times with
PBS, and blocked with TBST (50mM Tris-HCl, pH 7.4,
150mM NaCl, and 0.1% Triton X-100; Sigma) containing
5% goat serum for 1 hour at RT. The coverslips were incu-
bated with rabbit polyclonal IgG (1␮/ml; Affinity Biore-
agents, Golden, CO) overnight at 4°C. On the following
day, after three washes with PBS for 5 minutes each, the
secondary antibody Alexa Fluor goat anti–rabbit IgG (Mo-
lecular Probes, Eugene, OR) were added to the coverslips
and incubated for 1 hour at RT. After washes with Tris-
buffered saline, nuclei were stained by adding Hoechst
33258 at a final concentration of 2␮g/ml for 1 minute. Cov-
erslips were mounted with FluoroMount (Southern Biotech,
Birmingham, AL) and kept in the dark at 4°C. Cell images
were captured with a fluorescence microscope (Nikon Eclipse
E800; Nikon, Du¨sseldorf, Germany) equipped with a Spot
RT digital camera (Diagnostic Instruments, Sterling Height,
MI).
IMMUNOBLOTTING. Cells were lysed in 1% sodium dode-
cyl sulfate buffer (pH 7.6, 20mM HEPES containing pro-
tease inhibitor; Roche, Mannheim, Germany), and lysates
were collected and sonicated for 12 seconds. Protein concen-
trations were determined using the Bio-Rad D
c
Protein assay
(Bio-Rad, Hercules, CA). Protein extracts and a biotinylated
molecular weight marker (Cell Signaling Technology, Bev-
erly, MA) were denaturated in Laemmli sample loading
buffer at 95°C, separated by 4 to 20% polyacrylamide gel
electrophoresis, and electrotransferred in transfer buffer to a
polyvinyl diflouride membrane (0.2␮m pore; Bio-Rad). The
membrane was treated with blocking solution (5% nonfat
dry milk in TBST) and incubated overnight at 4°C with
rabbit polyclonal ER-␣(1:200, 1␮g/ml; Santa Cruz Biotech-
nology, Santa Cruz, CA), rabbit polyclonal ER-␤(1:1,000,
1␮g/ml; Affinity BioReagents), rabbit polyclonal Akt
Gerstner et al: E2 Attenuates Brain Injury 563

(1:1,000), rabbit polyclonal phospho-Akt (1:1,000), poly-
clonal rabbit p44/42 mitogen-activated protein (MAP) ki-
nase (1:1,000), mouse monoclonal phospho-p44/42 MAP ki-
nase (1:1,000; all from Cell Signaling Technology, Danvers,
MA), or rabbit polyclonal Actin antibody (1:10,000; Sigma).
Secondary incubations were performed with horseradish per-
oxidase–linked anti–mouse (1:2,000; Bio-Rad) or anti–rabbit
(1:7,500; Amersham Pharmacia Biosciences, Bucks, United
Kingdom) antibodies. Bands were visualized using enhanced
chemiluminescence (PerkinElmer Life Sciences, Boston,
MA), and serial exposures were made to radiographic film
(Denville Scientific, Metuchen, NJ).
OXYGEN EXPOSURE. Cultures were kept in stage-specific
growth hormone–containing medium and transferred to a
humidified chamber filled with 80% O
2
plus 5% CO
2
plus
15% air at 37°C for different time periods. Control plates
were kept under 21% oxygen, indicated as normoxic, plus
5% CO
2
conditions at 37°C.
SURVIVAL ASSAY. After exposure to hyperoxia, cell culture
media were collected and centrifuged, and lactate dehydro-
genase activity was quantified in 50␮l samples of medium
supernatant, using a colorimetric cytotoxicity assay kit ac-
cording to the manufacturer’s instructions (Roche). Absor-
bance data were obtained using a 96-well plate reader (Mo-
lecular Devices, Sunnyvale, CA) with a 450nm filter, and
650nm as reference wavelength. Maximum lactate dehydro-
genase release was determined by cell lysis of one-well un-
treated cells with cell culture media containing 1% Triton
X-100.
ACTIVATED CASPASE-3 ASSAY. Activation of caspase-3 in
living cells was assessed by the caspase-3 activity assay
(Merck, Darmstadt, Germany) following the manufacturer’s
instructions. The assay utilizes a FITC-labeled monoclonal
antibody directed against the cleaved (activated) form of
caspase-3. The FITC label allows for direct detection of ac-
tivated caspase-3 in apoptotic cells by flow cytometry. Sam-
ples were analyzed by using the FL-1 channel of the flow
cytometer.
Animal Studies
All animal experiments were performed in accordance with
the guidelines of Humboldt University. Six-day-old Wistar
rat pups (BgVV, Berlin, Germany) were placed together with
their mothers in an oxygen chamber containing 80% oxygen
for defined time periods up to 24 hours. With beginning of
the exposure animals received a single injection of 600␮g/kg
E2 intraperitoneally, diluted in sterile water, volume 0.1ml/
10g (n ⫽6). Control animals received an injection with ve-
hicle (n ⫽6). Animals were decapitated at the end of expo-
sure at P7 and immediately removed for molecular studies.
For detection of myelin basic protein (MBP), animals were
killed 96 hours later at P11. Brains were immediately re-
moved for molecular studies. Tissue was either collected as
whole brain or was microdissected for corpus callosum and
adjacent white matter structures under the microscope, and
were subsequently snap-frozen in liquid nitrogen and stored
at ⫺80°C until analysis.
IMMUNOBLOTTING OF BRAIN TISSUE. Snap-frozen tissue
(whole brain, white matter) was homogenized in RIPA buffer
(1% NP40, 0,5% sodium deoxycholate, 0,1% sodium dode-
cyl sulfate, 1mM EDTA, 1mM EGTA, 1mM Na
3
VO
4
,
20mM NaF, 0.5mM dithiothreitol (DTT), 1mM phenyl-
methyl sulfonyl fluoride, and protease inhibitor cocktail in
PBS pH 7.4). The homogenate was centrifuged at 1,050g
(4°C) for 10 minutes, and the microsomal fraction was sub-
sequently centrifuged at 17,000g(4°C) for 20 minutes.
Twenty micrograms of the resulting cytosolic protein extracts
were heat denaturated in Laemmli sample loading buffer,
separated by 10.5% sodium dodecyl sulfate polyacrylamide
gel electrophoresis, and electrotransferred onto a nitrocellu-
lose membrane. Nonspecific protein binding was prevented
by treating the membrane with 5% nonfat dry milk in Tris-
buffered saline/0.1% Tween 20 for 2 hours at RT. Thereaf-
ter, the membrane was incubated overnight at 4°C with pri-
mary antibody. For analysis of ER-␣/-␤rabbit polyclonal
antibodies (ER-␣; 1:500, ER-␤; 1:1,000; Affinity BioRe-
agents), of Fas a rabbit polyclonal antibody (1:250; Stress-
gen, Ann Arbor, MI), and of the mitogen-activated extracel-
lular kinase pathway, a rabbit polyclonal phosphorylation
state-independent extracellular signal-regulated kinases 1 and
2 (ERK1/2) antibody (1:1,000) and a mouse monoclo-
nal phospho-p44/42 ERK1/2 antibody (Thr202/Tyr204;
1:1,000) were used. The Akt pathway was analyzed by using
a rabbit polyclonal Akt antibody (1:1,000) and a rabbit poly-
clonal phospho-Akt antibody (Ser473; 1:1,000; all from Cell
Signaling). Secondary incubations were performed with
horseradish peroxidase–linked anti–mouse (1:2,000; Dako,
Glostrup, Denmark) or anti–rabbit (1:2,000; Amersham
Pharmacia Biosciences) antibody. Bands were visualized us-
ing enhanced chemiluminescence (Amersham Pharmacia
Biosciences), and serial exposures were made to radiographic
film (Hyperfilm enhanced chemiluminescence; Amersham
Pharmacia Biosciences). Densitometric analysis of the blots
was performed with an image analysis program (BioDocAna-
lyze; Whatman Biometra, Goettingen, Germany). For strip-
ping, membranes were incubated with stripping buffer
(100mM ␤-mercaptoethanol, 2% sodium dodecyl sulfate,
62.5mM Tris-HCl, pH 6.7) at 50°C for 30 minutes, then
washed, blocked, and reprobed overnight at 4°C with mouse
anti–␤-actin monoclonal antibody (1:5,000; Sigma-Aldrich).
SEMIQUANTITATIVE REVERSE TRANSCRIPTASE POLYMER-
ASE CHAIN REACTION ON BRAIN TISSUE. Total cellular
RNA from snap-frozen whole brains was isolated by acidic
phenol/chloroform extraction
23
and DNase I treated
(Roche); 500ng RNA was reverse transcribed with Moloney
murine leukemia virus reverse transcriptase (Promega, Mad-
ison, WI) in 25␮l of reaction mixture. The resulting com-
plementary DNA (1␮l) was amplified by polymerase chain
reaction. Primers to amplify rat Fas (GenBank sequence
D26112) were 5⬘-CCGACAACAACTGCTCAGA-3⬘(sense
primer, positioned at nucleotide 174) and 5⬘-GCACCTG-
CACTTGGTATTC-3⬘(antisense primer, positioned at nu-
cleotide 430). The primers to amplify the internal standard
18S ribosomal RNA (GenBank sequence M11188) were 5⬘-
AACGAGGATCCATTGGAG-3⬘(sense primer, positioned
at nucleotide 596) and 5⬘-ATGCCAGAGTCTCGTTCG-3⬘
564 Annals of Neurology Vol 61 No 6 June 2007

(antisense primer, positioned at nucleotide 1409). Comple-
mentary DNA was amplified in 30 cycles, consisting of dena-
turing over 30 seconds at 94 °C, annealing over 45 seconds at
55°C, and primer extension over 45 seconds at 72°C. Ampli-
fied complementary DNA was subjected to 5% polyacryl-
amide gel electrophoresis, subsequent silver staining, and den-
sitometric analysis with the image analysis program
BioDocAnalyze (Whatman Biometra).
Statistical Analysis
Data were analyzed using GraphPad Prism version 4.00 for
Windows (GraphPad Software, San Diego, CA). Unless oth-
erwise indicated, the results shown are one experiment rep-
resentative of three to six separate experiments that were per-
formed. Either Student’s ttest, one-way analysis of variance,
or two-way analysis of variance with the Tukey–Kramer post
hoc analysis for multiple comparisons were performed for
statistical analyses. Statistical significance was determined at
p⬍0.05.
Results
Estrogen Receptor-
␣
and -
␤
Are Expressed in
Oligodendroglial Cells during Development
To determine the expression of ER-␣and -␤during
oligodendroglial cell development, we assessed protein
levels of both receptors in developing primary OL cul-
tures (Fig 1A). ER-␣and -␤were preferentially ex-
pressed in immature OLs and showed a downregula-
tion in mature OLs. Furthermore, the intracellular
distribution of ER-␣was examined in pre-OLs by im-
munocytochemistry (see Fig 1B). ER-␣was predomi-
nantly located in the nucleus, with diffuse cytoplasmic
staining.
Time Dependency of Hyperoxia-Induced Toxicity to
Primary Oligodendrocytes
To estimate the time course of oxygen toxicity, pri-
mary pre-OLs were incubated with 80% oxygen for 0,
6, 12, and 24 hours. Cell viability was assessed by lac-
tate dehydrogenase release. Survival of developing OLs
decreased with time of exposure (Fig 2). After 12 hours
of incubation, cell survival was reduced to 25%, and
after 24 hours almost no viable cells were detected.
Control cells were kept in 21% oxygen for 0 to 24
hours and displayed a constant amount (8–10%) of
cell death for each time period (data not shown).
17
␤
-Estradiol Protects against Hyperoxia-Mediated
Cell Death in Developing Oligodendrocytes
To investigate whether E2 treatment can reduce
hyperoxia-mediated cell death in OLs, we administered
E2 or vehicle to primary pre-OL cultures at 0, 2, 4, 6,
12, 24, and 48 hours before 12 hours exposure to 80%
oxygen. To address whether the neuroprotective effect
of E2 is dose dependent, we incubated pre-OLs with
80% oxygen without (Fig 3A), with 1␮M E2 (see Fig
3B), or with 1nM E2 (see Fig 3C) 12 hours before
oxygen exposure. Representative transmission light-
phase contrast photomicrographs of primary OL cell
cultures showed that after 12 hours of hyperoxia, de-
veloping OLs showed signs of cell death including
plasma membrane blebbing and nuclear condensation.
Cells that were pretreated with E2 display less cell
death that was dose dependent with a median effective
concentration (EC
50
)of4.7⫻10
⫺10
M (see Fig 3D).
Significant protection with 1␮M E2 was found, if the
drug was given to the cell culture at least 4 hours be-
fore exposure to 12 hours of 80% oxygen (see Fig 3E).
Fig 1. (A) Western blot analysis of estrogen receptors (ER)-
␣
and -
␤
expression in premyelinating (O4
⫹
) and mature
(MBP
⫹
) oligodendrocytes showing a specific band at 66kDa
for the ER-
␣
and a specific band at 55kDa for the ER-
␤
whose intensities were found to be decreased in mature OLs in
comparison with the
␤
-actin band (42kDa). Cell lysates were
immunoblotted with antibodies against ER-
␣
, ER-
␤
, and
␤
-actin, as indicated. (B) Immunocytochemistry of ER-
␣
in
immature oligodendrocytes (O4
⫹
). ER-
␣
(green) was mostly
nuclear (Hoechst 33258, blue), but also showed a diffuse dis-
tribution in the cytoplasm. Scale bar ⫽10
␮
m. MBP ⫽mye-
lin basic protein.
Fig 2. Hyperoxia causes cell death in developing oligodendro-
cytes. Primary oligodendrocyte cell viability, as measured by lac-
tate dehydrogenase release, after 0, 6, 12, and 24 hours incuba-
tion with 80% oxygen (means ⫾standard error of the mean of
3 independent experiments). *p⬍0.05; ***p⬍0.001, one-
way analysis of variance with Tukey’s multiple-comparison tests.
Gerstner et al: E2 Attenuates Brain Injury 565

Fas Death Receptor Is Involved in Apoptotic Cell
Death in Developing Oligodendrocytes
Fas is a key molecule serving in most cases as a death
signal within the apoptotic machinery.
24
Increase in
expression of Fas after 80% oxygen exposure in the ol-
igodendroglia cell line was determined at different time
points after insult. Immunoblotting demonstrated an
increase of Fas at 6 and 12 hours after 80% oxygen
exposure in the oligodendroglia cell line. Pretreatment
with 100nM E2 attenuated this upregulation, signifi-
cant after 6 hours of oxygen exposure (Fig 4A). Previ-
ously, we reported on the distribution pattern and type
of neurodegeneration induced by hyperoxia in the de-
veloping brain of Wistar rats. Degenerating cells were
determined in the frontal, parietal, cingulate, and ret-
rosplenial cortex, caudate nucleus, nucleus accumbens,
corpus callosum and adjacent white matter, thalamus,
hippocampal dentate gyrus, subiculum, and hypothal-
amus.
8
Application of E2 intraperitoneal substantially
reduced cumulative cell death scores in the P6 rat brain
after exposure to 80% oxygen for 24 hours.
25
In this study, an increase in expression of Fas in the
brains of 6-day-old rats after 80% oxygen exposure was
determined at defined time points after insult using re-
verse transcriptase polymerase chain reaction and West-
ern blotting. Reverse transcriptase polymerase chain re-
action and immunoblotting was performed in samples
from whole brain and white matter preparations. Poly-
acrylamide gels (Fas in reference to 18S ribosomal
RNA) and blots (Fas in reference to ␤-actin band)
Fig 3. (A–C) Neuroprotective effect of 17
␤
-estradiol (E2) against hyperoxia-mediated cell death. Representative transmission light-
phase contrast photomicrographs of primary oligodendrocyte cell cultures after 12 hours incubation with either 21% (data not
shown) or 80% oxygen. After incubation with 80% oxygen, immature oligodendrocytes (OLs) without (A), with 1
␮
M E2 (B), and
with 1nM E2 (C) treatment. Hyperoxia-exposed immature OLs show signs of cell death including plasma membrane blebbing and
nuclear condensation. Cells that were pretreated with E2 display less cell death that is dose dependent. (D) Dose-dependent effect of
E2. Treatment of primary OL cell cultures with E2 at different concentrations (10
⫺6
,10
⫺7
,10
⫺8
,10
⫺9
,10
⫺10
M, 12 hours be-
fore treatment), followed by exposure to 80% oxygen for 12 hours. Cell toxicity was measured by lactate dehydrogenase (LDH) re-
lease (optical density ratio). The protective effect of E2 dose dependent with an median effective concentration (EC
50
)of4.7⫻
10
⫺10
M. Means ⫾standard error of the mean (SEM) of three independent experiments. **p⬍0.01, ***p⬍0.001 in one-way
analysis of variance with Tukey’s multiple-comparison tests. (E) Pretreatment of primary OL cell cultures with E2 0, 4, 6, 12, 24,
and 48 hours before hyperoxia. Percentage of survival was measured by LDH release. The protective effect of E2 was significant
after pretreatment of at least 4 hours. Means ⫾SEM of three independent experiments. ***p⬍0.001; *p⬍0.05, one-way anal-
ysis of variance with Tukey’s multiple-comparison tests.
566 Annals of Neurology Vol 61 No 6 June 2007

were subjected to densitometric analysis, and density
ratios were statistically analyzed. Hyperoxia triggered
an increase of Fas messenger RNA (see Fig 4B) and Fas
protein (see Figs 4C, D) after 12 and 24 hours in the
P6-day-old rat brain. Treatment of P6-day-old rats
with E2 (600␮g/kg intraperitoneally) before hyperoxia
Fig 4. (A) Effect of 17
␤
-estradiol (E2) on apoptosis signaling proteins in immature oligodendrocytes (OLN-93) exposed to hyper-
oxia. Immunoblot densitometric quantification of protein levels for Fas shows increased Fas-expression after 6 and 12 hours of hy-
peroxia. ***p⬍0.001, **p⬍0.01, comparison between normoxia (0 hours) or oxygen exposure for 2, 6, 12, or 24 hours (analy-
sis of variance). Treatment with estrogen (100nM) eliminates Fas-upregulation. Blot is representative of a series of blots of three
independent experiments. Graphs represent means ⫾standard error of means (SEMs) of the density ratios of Fas to
␤
-actin. #p⬍
0.05, comparison between vehicle and E2-treated cells, Student’s ttest. (B) Fas messenger RNA (mRNA) expression in P7-day-old
whole rat brains 12 or 24 hours after 80% oxygen exposure and in P6-day-old whole rat brains treated with E2 before hyperoxia.
There was an increase in mRNA levels for Fas 12 and 24 hours after hyperoxia. Treatment of P6-day-old rats with E2 before hy-
peroxia exposure attenuated Fas upregulation in comparison with Fas-mRNA levels of untreated littermates. The results of densito-
metric analysis of the gels are presented in reference to 18S ribosomal RNA (rRNA). Data represent the ratio (%) of the density of
the Fas band to the 18S rRNA band ⫾SEM (n ⫽4). *p⬍0.05, comparison between P7-day-old whole rat brains 12 or 24
hours after 80% oxygen exposure and P7-day-old whole rat brains treated with E2 before hyperoxia (analysis of variance). (C, D)
Hyperoxia triggered an increase of Fas protein after 12 and 24 hours in lysates from P7-day-old whole rat brain (WB) and white
matter preparation (WM). Treatment of P6-day-old rats with E2 (600
␮
g/kg intraperitoneally) before hyperoxia exposure attenuated
Fas upregulation in comparison with Fas levels of untreated littermates. Blots are representative of a series of four blots. Densitomet-
ric quantification of the blots is presented. Columns represent means ⫾SEMs of the density ratios of Fas to
␤
-actin (n ⫽4 per
group). ***p⬍0.001, comparison between vehicle and E2, Student’s ttest.
Gerstner et al: E2 Attenuates Brain Injury 567

exposure attenuated Fas upregulation in comparison
with Fas levels of untreated littermates.
Hyperoxia Induces Activation of Caspase-3 in the
Oligodendroglia Cell Line OLN-93 That Is
Attenuated after Pretreatment with 17
␤
-Estradiol
Caspase-3 has been shown to play a key role in medi-
ating the effector stage of apoptosis by initiating the
process of DNA fragmentation.
26
The activation of the
effector caspase-3 was assessed by intracellular binding
of a FITC-labeled monoclonal antibody specific to the
activated (cleaved) form of the enzyme using flow cy-
tometry. As described previously, after 24 hours of ox-
ygen incubation, OLN-93 cell viability is reduced to
80%, compared with 10 to 20% in primary OLs, and
only after 96 hours oxygen exposure, almost no
OLN-93 cells are viable.
10
Primary OLs are much
more susceptible toward oxygen exposure (see Fig 2).
The percentage of OLs displaying activated caspase-3
was found to increase up to twofold after a 24-hour
incubation period with 80% oxygen (Fig 5). Pretreat-
ment with 100nM E2 significantly reduced caspase-3
activation by approximately 30%.
Hyperoxia Reduces Levels of Phosphorylated
Extracellular Signal–Regulated Kinase 1/2 and Akt
in Primary Oligodendroglia Cultures and in the
Brains of 7-Day-Old Rats That Is Reversed
by 17
␤
-Estradiol
The kinases ERK1/2 and Akt (protein kinase B) are
members of two important pathways that control neu-
ronal cell survival, the MAP kinase and the phosphati-
dylinositol 3 (PI3) kinase pathways. Activated ERK1/2
recently was shown to phosphorylate and inhibit
caspase-9, a key caspase in activation of the intrinsic ap-
optotic pathway, which is initiated by release of cyto-
chrome cinto the cytoplasm.
27
Moreover, it has been
shown that activated Akt phosphorylates Bad, a pro-
apoptotic molecule, and causes its inactivation. This
leads to liberation and activation of the antiapoptotic
molecule Bcl-2.
28
To investigate the impact of treatment
with E2 on levels of phosphorylated (active) ERK1/2
and Akt, we performed Western blot analysis on protein
samples obtained from primary oligodendroglia cultures
(Figs 6A, B) and white matter preparations (see Figs 6C,
D) from P6 rats subjected to 80% oxygen for various
time points or room air. This analysis indicated that ex-
posure to 80% oxygen decreased levels of the active,
phosphorylated isoforms of the ERK1/2 (pERK1/2) (see
Figs 6B, D) and of the serine-threonine kinase Akt
(pAkt) (see Figs 6A, C), which both mediate intracellu-
lar signaling after plasma membrane-associated tyrosine
receptor kinase autophosphorylation by growth fac-
tors.
29
E2 ameliorated hyperoxia-induced decreases of
the phosphorylated active forms of pERK1/2 and pAkt
in primary developing OLs and white matter prepara-
tion of the P6 rat brain.
TREATMENT OF 17〉-ESTRADIOL ATTENUATES WHITE
MATTER INJURY IN VIVO. Our group recently found
that oxygen-induced cell death in vivo is age depen-
dent.
8
Rats at postnatal ages P0, P3, P7, P14, and P20
were subjected to 80% oxygen over a 24-hour period.
After oxygen exposure, degenerating cells were quanti-
fied in sections stained by the DeOlmos cupric silver
method inter alia in the corpus callosum and adjacent
white matter.
8
Here we investigated whether oxygen
influences the developing process of OLs in vivo by
subjecting P6 rat pups to 80% oxygen over a 24-hour
period and killing the animals at P11. At the beginning
of the hyperoxia exposure, rat pups were treated intra-
peritoneally with E2 (600␮g/kg) or vehicle (2-
hydroxypropyl-␤-cyclodextrin). The injury was assessed
by two blinded observers for degree of MBP loss in the
cerebral white matter (Fig 7A–D). There was an atten-
uation of lesion severity in rats treated with E2, as
compared with vehicle-treated control animals (see Figs
7C, D).
Discussion
This study demonstrates that E2 treatment is protec-
tive in hyperoxia-induced white matter injury in the
developing rat brain. E2 acts in a neuroprotective fash-
ion by downregulation of Fas death receptor, inhibi-
tion of caspase-3 activation, restoration of survival sig-
nals transmitted by MAP kinases and protein kinase B,
and attenuation of MBP loss in developing OLs in
vivo.
Recently, we reported that exposure of the brain to
high concentrations of oxygen cause apoptotic cell
Fig 5. 17
␤
-Estradiol (E2) decreases activation of caspase-3.
Activation of the effector caspase-3 was assessed by intracellular
binding of a fluorescein isothiocyanate–labeled monoclonal
antibody specific for the activated (cleaved) form of the en-
zyme, using flow cytometry. OLN-93 cells were incubated un-
der hyperoxic (80% oxygen) conditions for 24 hours.
Means ⫾standard error of the mean of three independent
experiments. **p⬍0.01 in Student’s ttest.
568 Annals of Neurology Vol 61 No 6 June 2007

death during a specific period of development in vitro
and in vivo. Cultured pre-OLs (O4
⫹
O1
⫺
MBP
⫺
) and
immature OLs (O4
⫹
O1
⫹
MBP
⫺
), derived from the
OLN-93 cell line, display susceptibility toward hyper-
oxia, whereas mature OLs (MBP
⫹
) remain viable.
30
Moreover, we reported that hyperoxia induces wide-
spread cell death in brains of 3- to 6-day-old rats and
mice, both in gray and white matter regions.
8
Vulner-
ability to oxygen-induced cell death was age dependent
with a maximum during the first week of life in both
OL cell cultures and animals.
Several studies have highlighted that female sex hor-
mones represent potential neuroprotective agents
against damage produced by acute and chronic injuries
in the adult brain. Estrogens have been shown to pro-
mote survival and differentiation of several neuronal
populations maintained in culture
31
and to reduce cell
death associated with excitotoxicity,
15
oxidative
stress,
32
or exposure to ␤-amyloid.
33
The neuroprotec-
tive effects of estrogen have been widely documented
in animal models of neurological disorders, such as
Parkinson’s
34
and Alzheimer’s diseases,
35
as well as ce-
rebral ischemia.
14
Both ER-␣and -␤are found in various regions of
the human and rodent brain, including the hypothala-
mus, hippocampus, cerebral cortex, midbrain, brain-
Fig 6. (A, B) 17
␤
-Estradiol (E2) reversed hyperoxia-induced reduction of phosphorylated isoforms of extracellular signal–regulated ki-
nase 1 and 2 (pERK1/2) and pAkt levels in primary oligodendrocytes (OLs). pAkt (A) and pERK1/2 (B) levels were reduced in O4
⫹
OLs after hyperoxia exposure. Total ERK1/2 and Akt levels remained unaltered. Administration of E2 increased pERK1/2 and pAkt
levels compared with cells that were treated with vehicle. Blots are representative of a series of three blots for each antibody and each
treatment condition. Densitometric quantification of the blots is presented. Columns represent means ⫾standard error of the means
(SEMs) of the density ratios of the protein of interest to
␤
-actin (n ⫽4 per group). Treatment with E2 increased levels of pERK1/2
and pAkt in comparison with vehicle-treated oligodendrocytes and leaves levels of ERK1/2 and Akt unaltered. ***p⬍0.001; **p⬍
0.01; *p⬍0.05, comparison between vehicle and E2 (B), Student’s ttest. (C, D) E2 reversed hyperoxia-induced reduction of phos-
phorylated isoform of pAkt (C) and pERK1/2 (D) levels in the white matter of P7-day-old rats. Administration of E2 (600
␮
g/kg in-
traperitoneally) increased pAkt and pERK1/2 levels significantly and leaves levels of Akt and ERK1/2 unaltered. Blots are representative
of a series of six blots for each antibody. Densitometric quantification of the blots is presented. Columns represent means ⫾SEMs of
the density ratios of the protein of interest to
␤
-actin (n ⫽4– 6 per group). (C) ***p⬍0.001; **p⬍0.01; *p⬍0.05, comparison
between vehicle and E2, Student’s ttest. (D) ***p⬍0.001; **p⬍0.01, comparison between vehicle and E2; ##p⬍0.01, compar-
ison between control and hyperoxia treated P7-day-old rat pups, Student’s ttest.
Gerstner et al: E2 Attenuates Brain Injury 569

stem, and forebrain, and ER-mediated effects are
thought to provide neuroprotection.
11,36
These studies
defined the neuroanatomic localization of each recep-
tor, but did not specify cell type. Here, we report that
ER-␣and -␤are expressed in oligodendroglial cells.
Possible mechanisms underlying estrogen’s protective
effects include the activation of nuclear, cytoplasmic,
and membrane-localized ER. Interestingly, our data
demonstrate that ER-␣protein expression displays a
downregulation during the maturation process of oli-
godendroglial cells, suggesting an important role of
ER-␣during brain development and in models of neo-
natal brain injury. In an ovariectomized/ischemia adult
mouse model of stroke, estradiol treatment protects
wild-type and ER-␤–null mice from brain injury,
whereas this protection is abolished in ER-␣–null ani-
mals, suggesting a crucial role for ER␣in providing
neuroprotection.
37
However, another study in which
stroke was induced by reversible middle cerebral artery
occlusion found no increase in tissue damage in ER-
␣–null mice, suggesting that non–ER-mediated path-
ways, involving antioxidant effects, interaction with
membrane binding sites, and modulation of neuro-
transmitter systems, may be involved.
15,32,38
Therefore,
protection by estrogen may involve both ER-dependent
classic and nonclassic genomic responses, and ER-
independent mechanisms. Thus, further studies are
warranted to better characterize the modes of action of
estrogen in the brain.
Encouraged by the aforementioned work of others in
neuronal cultures and adult animal models of brain in-
jury, we set out to study the effect of estradiol in the
neonatal brain, focusing on the white matter. Espe-
cially during the last trimester of pregnancy, migration
Fig 7. (A, B) Normal myelin basic protein (MBP) expression in P11 rat pup that was kept under normoxic conditions. (C, D)
Bilateral loss of MBP is seen in the P11 pup in the white matter tract after hyperoxia for 24 hours at P6. (E, F) Protective effect
of 17
␤
-estradiol in vivo, demonstrating attenuation of MBP injury at P11 with systemic 17
␤
-estradiol treatment during hyperoxia
exposure (n ⫽3 per group).
570 Annals of Neurology Vol 61 No 6 June 2007

and differentiation of OLs take place.
39
Late OL pro-
genitors (O4
⫹
O1
⫺
MBP
⫺
) and immature OLs
(O4
⫹
O1
⫹
MBP
⫺
) are the predominant OL stage in
human cerebral white matter during the peak incidence
of PVL.
40
When the umbilical cord is clamped, high
placental estrogen supply is no longer available for the
premature neonate. This may in itself lead to disadvan-
tages or a greater risk for development of PVL and
later motor and cognitive deficits. However, based on
Nilsen and colleagues’ data,
41
proapoptotic effects of
E2 in the immature brain cannot be excluded.
One potential mechanism for the protective effect of
E2 includes changes in gene expression in the develop-
ing brain and in oligodendroglial cultures; that is, ac-
tivation of cell survival–promoting signaling pathways,
such as the mitogen-activated extracellular kinase-
ERK1/2 and the PI3 kinase-Akt pathways. As de-
scribed previously, hyperoxia reduced levels of the ac-
tive forms of ERK1/2 and Akt in a time-dependent
fashion in thalamus, striatum, and cortical structures.
8
ERK1/2 and Akt are key players in these two path-
ways, which are activated by tyrosine kinase receptors
on binding of growth factors to their receptors. These
changes reflect impairment of survival promoting sig-
nals
42
and imbalance between neuroprotective and
neurodestructive mechanisms in the brain, which dur-
ing the physiological, developmental elimination of
brain cells will likely promote apoptotic death. Here
we demonstrate that the hyperoxia-mediated down-
regulation of the phosphorylated forms of ERK and
Akt occurs also in the white matter of neonatal rats
and in oligodendroglial cells, indicating an important
role of these pathways in the pathophysiology of white
matter injury induced by hyperoxia.
Activation of ERK1/2, and activation of Akt, can
ameliorate hyperoxia-induced apoptotic cell death in
lungs and retina.
43,44
Other groups have shown that
the PI3 kinase cascade is involved in the neuroprotec-
tive mechanism stimulated by estrogen.
25,45
The con-
cept that attenuation of pAkt loss may contribute to
the protective effect of E2 is well-founded; however,
previous data on pERK in neonatal and adult brain
injury indicate that this MAP kinase has both proapop-
totic and antiapoptotic actions.
46
This study shows
that E2 counteracts the inactivation of ERK1/2 and
Akt pathways, and therefore protects against apoptotic
cell death. A previous in vivo study supports the fact
that oxygen causes cell death by inactivation of survival
signaling proteins Ras, ERK-1/2, and protein kinase B
(Akt). Furthermore, synRas-transgenic mice overex-
pressing constitutively activated Ras and phosphory-
lated kinases ERK-1/2 in the brain were protected
against oxygen neurotoxicity.
8
Another pathway that accounts for the antiapoptotic
action of E2 is its influence on the expression level of
the proapoptotic molecule Fas. Here we present evi-
dence that hyperoxia leads to increased expression of
Fas in the P7 rat brain and in premyelinating OLs.
Treatment of P6 rats with E2 before hyperoxia expo-
sure attenuated Fas-messenger RNA upregulation in
comparison with Fas-messenger RNA levels of un-
treated littermates and decreased Fas protein expression
in the oligodendroglial cell line. Others have shown
that female steroid hormones can influence the apopto-
tic cascade at different stages. Injury models relying on
apoptotic signaling mechanisms have demonstrated
neuroprotection via estrogen inhibition of proapoptotic
factors, including caspases,
47,48
cytochrome c,
49
and ac-
tivation of antiapoptotic gene transcription, such as
Bcl-2 and Bcl-XL.
50,51
Furthermore, E2 reduces the
proapoptotic calcium influx into neuronal cells.
16
Moreover, E2 has antioxidative properties that can lead
to the reduction of free radicals.
17
Bittigau and co-
workers
52
showed in a P7 model of antiepileptic drug–
induced brain injury that injection of E2 significantly
reduces neurodegeneration by activation of antiapop-
totic signaling pathways involving phosphatidylinositol
3-Akt and MAP kinases.
In this study, we demonstrated the protective impact
of E2 on the developing white matter. Our results raise
the possibility that specific hormonal replacement
treatment for premature infants may improve neuro-
logical outcome. These results raise the interesting hy-
pothesis that E2 might be suitable as a preventive agent
in premature infants and sick term infants, who need
to be exposed to oxygen for the purposes of treatment.
However, determination of an exact time window is
highly warranted as a focus of further studies. E2 re-
placement therapy in extremely low-birth-weight in-
fants has been introduced in some centers with the
goal to improve bone mineralization, and no adverse
side effects have been observed so far.
53
Therefore, we
suggest that maintaining placental E2 plasma levels
may be effective to protect neonates from brain injury.
More data are urgently needed concerning the safety
and feasibility of estrogen supplementation in the neo-
natal period.
This work was supported by the Ernst Schering Research Founda-
tion, the German Federal Department of Education and Research
(BMBF; 01 ZZ 0101), the Sonnenfeld-Stiftung, the European
Commission (Sixth Framework Program, contract no LSHM-CT-
2006-036534), and the NIH (NS28475, HD18655, PO1NS38475,
J.J.V., P.A.R.).
We are grateful to Prof Dr Richter-Landsberg, who kindly provided
us with OLN-93 cells.
Gerstner et al: E2 Attenuates Brain Injury 571

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