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The Journal of Neuroscience, October 15, 2002, 22(20):8922-8931
Activation of Hypoxia-Inducible Factor-1 in the Rat Cerebral
Cortex after Transient Global Ischemia: Potential Role of Insulin-Like
Growth Factor-1
Juan C.
Chavez1 and
Joseph C.
LaManna1, 2
Departments of 1 Anatomy and 2 Neurology,
Case Western Reserve University, School of Medicine, Cleveland, Ohio
44106
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ABSTRACT |
Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that
regulates the adaptive response to hypoxia in mammalian cells. It
consists of a regulatory subunit HIF-1
, which accumulates under
hypoxic conditions, and a constitutively expressed subunit HIF-1
.
In this study we analyzed HIF-1 expression in the rat cerebral
cortex after transient global ischemia induced by cardiac arrest and
resuscitation. Our results showed that HIF-1 accumulates as early as
1 hr of recovery and persists for at least 7 d.
In addition, the expression of HIF-1 target genes, erythropoietin and
Glut-1, were induced at 12 hr to 7d of recovery. A logical explanation
for HIF-1 accumulation might be that the brain remained hypoxic for
prolonged periods after resuscitation. By using the hypoxic marker
2-(2-nitroimidazole-1[H]-y1)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), we showed that the brain is hypoxic during the first hours of
recovery from cardiac arrest, but the tissue is no longer hypoxic at
2 d. Thus, the initial ischemic episode must have activated other
nonhypoxic mechanisms that maintain prolonged HIF-1 accumulation. One such mechanism might be initiated by insulin-like growth factor-1 (IGF-1). Our results showed that IGF-1 expression was upregulated after
cardiac arrest and resuscitation. In addition, we showed that IGF-1 was
able to induce HIF-1 in pheochromocytoma cells and cultured
neurons as well as in the brain of rats that received intracerebroventricular and systemic IGF-1 infusion. Moreover, infusion
of a selective IGF-1 receptor antagonist abrogates HIF-1 accumulation after cardiac arrest and resuscitation. Our study suggest
that activation of HIF-1 might be part of the mechanism by which IGF-1
promotes cell survival after cerebral ischemia.
Key words:
global cerebral ischemia; hypoxia-inducible factor-1; insulin-like growth factor-1; hypoxia-inducible genes; cardiac arrest; brain hypoxia
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INTRODUCTION |
Mammalian cells are able to sense a
decrease in oxygen tension and respond by activating adaptive
mechanisms, including activation of several hypoxia-inducible genes
such as erythropoietin (Epo) and vascular endothelial growth factor
(VEGF). Many of these genes are regulated by the hypoxia-inducible
factor-1 (HIF-1), a heterodimeric transcription factor consisting of
two subunits, HIF-1 and HIF-1 (Wang and Semenza, 1995
). Both
subunits belong to a family of basic helix-loop-helix transcription
factors and are required for DNA binding and transactivation of target
genes (Wang et al., 1995). HIF-1 is a common binding partner for
other members of the family, and it is constitutively expressed.
HIF-1 is unique to HIF-1 and its expression is primarily regulated
by oxygen tension (Semenza, 1999). During normoxia, the HIF-1 gene
is expressed continuously; however, the HIF-1 protein is rapidly
degraded by the ubiquitin-proteosome system (Salceda and Caro, 1997;
Huang et al., 1998). During hypoxia or in the presence of iron
chelators, degradation of HIF-1 is suppressed, and thereby it
rapidly accumulates in the nucleus (Jiang et al., 1996; Jewell et al.,
2001).
In addition to hypoxia, many reports have shown that some growth
factors such as insulin, insulin-like growth factor (IGF)-1 and IGF-2
can induce HIF-1 accumulation and HIF-1 DNA binding activity in
different cell types (Zelzer et al., 1998; Feldser et al., 1999; Zundel
et al., 2000).
In the adult CNS, IGF-1 is involved in the response of neuronal tissue
to injury and during various neurodegenerative conditions (Torres-Aleman, 2000). The expression of IGF-1 and its binding proteins
are altered in response to brain ischemia (Lee et al., 1996; Schwab et
al., 1997; Beilharz et al., 1998), and exogenous administration of
IGF-1 significantly reduces neuronal loss in different models of
cerebral ischemia (Johnston et al., 1996; Tagami et al., 1997; Guan et
al., 2000; Wang et al., 2000). Furthermore, it has been shown that
IGF-1 protects cultured neurons against diverse forms of injury,
including hypoxia and oxidative stress (Heck et al., 1999; Yamaguchi et
al., 2001).
In this study we analyzed the expression of HIF-1 in the rat
cerebral cortex after transient global ischemia induced by cardiac arrest and resuscitation. Our results showed that HIF-1 accumulates as early as 1 hr of recovery and unexpectedly persists for at least
7 d. We used the in vivo hypoxia marker EF5 to
determine whether brain hypoxia could explain persistent HIF-1
accumulation. In addition, we analyzed the expression of Von Hippel
Lindau protein (pVHL) and the activity of the 26S proteosome to assess
changes in the HIF-1 degradation pathway. Our data revealed that
neither hypoxia nor impairment in the degradation machinery could
explain a sustained expression of HIF-1. Thus, the initial ischemic
episode must have activated other mechanisms that produced prolonged
HIF-1 accumulation. One such mechanism might be initiated by IGF-1
that was found to be upregulated in the brain after cardiac arrest and
resuscitation. To support this hypothesis, we studied the ability of
IGF-1 to induce HIF-1 in vitro and in vivo. In
addition, we tested whether selective inhibition of the IGF-1
receptor (IGF-1R) abrogates sustained HIF-1 accumulation
after transient global ischemia.
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MATERIALS AND METHODS |
Induction of transient global ischemia by cardiac arrest
and resuscitation. Transient global cerebral ischemia was produced by a modification of the cardiac arrest model described by Crumrine and
LaManna (1991). Male Wistar rats (300-350 gm) were anesthetized with
2.5% halothane/70% nitrous oxide/30% oxygen. The ventral tail artery
was cannulated to monitor systemic arterial pressure and to obtain
samples of blood gases and pH measurements. In addition, a catheter was
inserted through the external jugular vein into the right atrium, and
body temperature was maintained at 37°C by a feedback-controlled
infrared heat lamp. Animals were allowed to recover completely from
anesthesia for at least 1 hr. Cardiac arrest was induced by a
sequential intra-atrial injection of D-tubocurare (0.3 mg) and ice-cold KCl solution (0.5 M, 0.12 ml/100 gm of body weight). Resuscitation efforts began after 7 min of
arrest. For this purpose, rats were orotracheally intubated for
mechanical ventilation accompanied by chest compression. Once
spontaneous heartbeat returned, a small dose of epinephrine (2 µg)
was administered to achieve a mean arterial blood pressure of at least
80 mmHg. The duration of ischemia was between 11 and 13 min and was
defined as the period between the decrease of blood pressure to zero
and its return to 80% of pre-arrest value. Ventilation was adjusted to
achieve normoxia and normocapnia until rats regained spontaneous respiration. In the sham-operated animals, the same surgical procedure was performed without cardiac arrest and resuscitation. Animals were
killed by decapitation at various durations of recovery.
Immunodetection of the hypoxic marker EF5. To map
brain hypoxic regions after transient global cerebral ischemia, EF5, a
marker for hypoxia, was administered intravenously (10 mg) using an
infusion pump (Harvard Apparatus) at a rate of 150 µl/min for 20 min.
Rat subjected to transient ischemia were infused with EF5 at 15 min or
2 d after resuscitation from cardiac arrest and were killed 45 min
after infusion. Sham-operated rats received a similar treatment. In
addition, a group of rats were infused with EF5 while being exposed to
different oxygen concentrations (21, 16, 14, 12, 10, and 8%
O2) for 4 hr. Once animals were killed, the brains were quickly removed and frozen at 
70°C. To detect EF5 adducts, 10 µm coronal sections were incubated with an ELK3-51 monoclonal Cy3-conjugated antibody (provided by S. Evans, University of
Pennsylvania, Philadelphia, PA) according to the procedure described by
Evans et al. (1995).
Proteasome chymotrypsin-like activity. Chymotrypsin-like
activity of the proteasome was assayed using the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-MCA) (Sigma, St. Louis, MO). After rapid dissection, brain cortex was homogenized in
lysis buffer (10 mM Tris-HCl, pH 7.8, 0.5 mM DTT, 5 mM ATP, 0.035%
SDS, and 5 mM MgCl2).
Assays were performed with 100 µg/100 µl aliquots from the
resulting lysate supplemented with 40 µM of
LLVY-MCA. After incubation at 37°C for 30 min, the substrate was
quenched by addition of 300 µl of ethanol followed by 2.0 ml of
H2O. Proteolytic activity (release of MCA) was
measured using a Perkin-Elmer LS-5B spectrofluorimeter at 380 nm
excitation and 440 nm emission using free MCA as the standard for
quantification. Background fluorescence was determined by incubating
lysates with the proteasome inhibitor lactacystin (50 µM) for 30 min before adding the substrate.
In vivo administration of IGF-1 and IGF-1R antagonist.
Male Wistar rats received systemic (n = 4) or
intracerebroventricular (n = 3) infusion of human IGF-1
(generously provided by A. F. Parlow, National Hormone Peptide
Program) dissolved in PBS. Systemic infusion was performed by using a
subcutaneous mini-osmotic pump (Alzet 2001; at a dose of 50 µg · kg
1 · d1, rate
of infusion 1 µl/hr) for 7 d according to the protocol described
by Carro et al. (2000). At the end of the infusion, blood samples (1 ml) were collected for ELISA analysis of IGF-1. Animals were killed,
and the brains were removed and frozen at 80°C.
Intracerebroventricular IGF-1 was administered by using a stainless
steel cannula (Alzet) implanted into the left lateral ventricle.
Stereotaxic coordinates used were 0.4 mm posterior to bregma, 1.5 mm
lateral to the midline, and 4 mm ventral to the pial surface, as
established by Paxinos and Watson (1997). The cannula was
connected to a subcutaneous osmotic pump (Alzet 2001) filled with IGF-1
(0.25 µg/µl). After 7 d of infusion, animals were killed and
perfused-fixed, and the brains were processed for HIF-1 immunohistochemistry.
In addition, a group of rats were subjected to cardiac arrest and
resuscitation and subsequently received a continuous
intracerebroventricular infusion of saline or 25 µg of JB-1, a
selective antagonist for IGF-1R (Bachem, San Carlos, CA). For this
experiment, a cannula was implanted as described above and connected to
an osmotic pump (Alzet 1003D; delivery rate 1 µl/hr). After 4 d
of infusion, animals were killed, and brains were processed for HIF-1 immunohistochemistry.
IGF-1 sandwich ELISA. Total circulating IGF-1 was measured
using ELISA with a mouse monoclonal anti-IGF-1 antibody (Upstate Biotechnology, Waltham, MA). Microtiter plates were coated overnight with these captured antibodies at 4°C. After the plates were washed with TBS, 25 µl of IGF-1 standards or acid-extracted serum samples were added to each well and incubated at room temperature for 2 hr.
After several washes, 1 µl of biotinylated anti-IGF-1 antibody (R&D
Systems, Minneapolis, MN) with 0.5% normal goat serum was added and
incubated for 2 hr. Immunodetection was accomplished using
streptavidin-HRP (R&D Systems) and the chromogen orthophenylene diamine
(Sigma). Plates were read at 490 nm, and results were expressed as
nanograms of IGF-1 per milliliters of plasma.
Cell cultures and experimental treatments. Primary neuronal
cultures were prepared as described previously (Brewer, 1995) from
embryonic day 18 Wistar rats. Dissociated cortical neurons were plated
in six-well plates (coated with poly-L-lysine)
under serum-free conditions in neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, CA). On the fourth day of plating, one-half of
the medium was changed to B27/neurobasal without glutamate. Experiments
were performed in cells that had been in culture for 6-8 d. Rat
pheochromocytoma (PC12) cells were grown in Roswell Park Memorial
Institute 1640 culture medium (containing
L-glutamine) supplemented with 5% horse serum,
10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin on
collagen-coated dishes. Before IGF-1 treatment, PC12 cells were
detached and seeded in poly-L-lysine (10 µg/ml)-coated plates in a 2% serum medium for 48 hr. PC12 and
primary neurons were exposed to hypoxia (1% O2, 5% CO2, 94% N2) for 4 hr
in Plexiglas modular chambers. Cells incubated under standard normoxic
conditions (5% CO2/95% room air) were used as
controls. To study the effect of IGF-1 on HIF-1 expression, PC12 and
neurons were treated with 100 nM recombinant human IGF-1 (Calbiochem, San Diego, CA) or vehicle for 24 hr.
RNase protection assay and Northern blot analysis.
Total RNA was isolated from brain cortex using the RNAgents Isolation
System (Promega, Madison WI) according to the manufacturer's
recommendations. A PCR fragment (468 bp) containing IGF-1 exon 2 and a
portion of exons 1 and 3 was generated by RT-PCR using forward primer 5'-AAGCCTACAAAGTCAGCTCG-3' and reverse primer
5'-GGTCTTGTTTCCTGCACTTC-3'. The resulting cDNA was subcloned into
pGEM-4Z (Promega) and sequenced. To synthesize radiolabeled IGF
antisense riboprobe, the plasmid was linearized with EcoR1
and transcribed with T7 RNA polymerase in the presence of
[32P]UTP using the MAXIscript
in vitro transcription kit (Ambion, Austin, TX). Then, the
probe was treated with Dnase I and purified through PAGE. Rnase
protection assay (RPA) was performed according to the instructions
provided with the RPAIII kit (Ambion), using 20 µg of total RNA.
Protected RNA fragments were resolved by electrophoresis on denaturing
urea-polyacrylamide gels (5%) and visualized by autoradiography. As an
internal control, a riboprobe for -actin was used. Northern blot
analysis was performed as previously described (Chavez et al., 2000)
using 10 µg of total RNA. Specific cDNA probes for rat VEGF and
Glut-1 were purchased from Research Genetics (Huntsville, AL). Epo cDNA
was generated by RT-PCR using the following primers: sense 5'-CTCT
GGGCCTCCCAGTC-3' and antisense 5'-TGTTCGGAGTGGAGCAG-3'.
Western blot analysis and immunoprecipitation. Brain
cortical samples were homogenized in lysis buffer (20 mM HEPES, pH 7.5, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1 M NaCl) supplemented with 0.2 mM dithiothreitol, and protease inhibitors (1 µg/ml leupeptin, 0.5 µg/ml aprotinin, 1.5 µg/ml pepstatin, 0.5 mM PMSF, 1 mM
Na3VO4). After adding NaCl
to a final concentration of 0.45 M, homogenates were centrifuged at 20,000 × g for 15 min at 4°C.
Supernatants were collected and mixed with an equal volume of
homogenization buffer containing 40% (v/v) glycerol. Cultured cells
were washed with PBS, harvested, and centrifuged at 1000 × g for 5 min. The cell pellet was resuspended in lysis buffer
and processed as described above. Brain cortical lysates (200 µg
protein) or cell lysates (50 µg protein) were electrophoresed on
7.5% (HIF-1 and HIF-1) or 15% SDS-polyacrylamide gels (IGF-1,
VHL) and transferred to nitrocellulose membrane by standard procedures.
After blocking with non-fat dry milk, membranes were incubated with the
following primary antibodies: HIF-1 mouse monoclonal antibody (Novus
Biologicals, Littleton, CO), IGF-1 mouse monoclonal antibody (Upstate
Biotechnology), goat polyclonal antibody against -chain of the IGF-1
receptor (Santa Cruz Biotechnology, Santa Cruz, CA), VHL monoclonal
antibody (Pharmigen, Carlsbad, CA), and actin goat polyclonal antibody (Santa Cruz Biotechnology). Crude nuclear extracts of Hep3B cells (American Type Culture Collection, CRL-1830) exposed to 1 or
20% oxygen were used as positive controls (30 µg of protein) in
HIF-1 Western blot analysis.
For immunoprecipitation of the IGF-1R subunit, cell lysates (50 µg) were incubated at 4°C with anti-IGF-1R (2 µg; Santa Cruz
Biotechnology). Protein G-agarose was added, and samples were incubated
for 2 hr at 4°C. The pellet was collected by centrifugation (5000 rpm, 5 min), washed with PBS, and boiled in Laemmli sample buffer.
Samples were resolved in 7.5% SDS-PAGE, and Western blots were
performed using an anti-phosphotyrosine-specific antibody (1:500; Santa
Cruz). These blots were stripped and reprobed with anti-IGF-1R antibody
(1:500, Santa Cruz Biotechnology). Signals were visualized by enhanced
chemiluminescence (Amersham), and autoradiographic results were
quantified by densitometry. Protein concentrations were determined by
Bradford protein assay with bovine serum albumin as standard (Bio-Rad,
Hercules, CA).
HIF-1 and IGF-1 immunohistochemistry.
Anesthetized rats were perfused transcardially with ice-cold PBS
followed by 4% paraformaldehyde. Brains were removed, postfixed in 2%
paraformaldehyde for 24 hr, and embedded in paraffin. Coronal sections
(6 µm) were deparaffinized, hydrated, and subjected to antigen
retrieval at 90°C for 20 min using Target Retrieval Solution (Dako,
Carpinteria, CA). Sections were incubated with 10% normal serum for 2 hr and subsequently were incubated overnight at 4°C with a mouse
monoclonal antibody against HIF-1 (1:200; Novus Biologicals).
HIF-1-positive cells were visualized with biotinylated secondary
anti-mouse antibody (Vector, Burlingame, CA) and streptavidin
conjugated with Oregon Green (Molecular Probes, Eugene, OR).
To identify some of the cells expressing HIF-1, double
immunolabeling using two cellular markers, neuronal-nuclei specific (NeuN) and glial fibrillary acidic protein (GFAP) were performed. The
polyclonal anti-GFAP (1:500; Santa Cruz Biotechnology) and anti-NeuN
(1:500; Chemicon, Temecula, CA) were detected using Texas
Red-conjugated secondary antibodies (Vector). For IGF-1 and HIF-1
double labeling, sections were first processed for HIF-1 staining as
described above, and then for IGF-1 with a monoclonal anti-IGF-1
antibody (1:200; Upstate Biotechnology). Immunodetection was
accomplished using a biotin-conjugated anti-mouse antibody and
streptavidin conjugated with Cy3 (Jackson ImmunoResearch, West Grove, PA).
Statistical analysis. Data are presented as mean ± SD.
Statistical comparisons among groups were made using a one-way ANOVA test with Tukey correction. A p < 0.05 was considered
statistically significant.
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RESULTS |
Cardiac arrest and resuscitation induce HIF-1 accumulation in
the brain cortex and upregulation of HIF-1 target genes
HIF-1 migrates with a characteristic diffuse pattern (~120
kDa) probably corresponding to post-translational modifications of this
protein (Fig. 1A).
Under normoxic conditions, HIF-1 is continually synthesized but
rapidly degraded by the ubiquitin-proteosome system. Accordingly,
HIF-1 was barely detected in brain cortical samples of sham-operated
animals. Transient global cerebral ischemia induced by cardiac arrest
and resuscitation led to a rapid accumulation of HIF-1 in the brain
cortex. This accumulation was observed at 1 hr after resuscitation,
persisted for at least 7 d, and subsided by 14 d of recovery.
On the other hand, the levels of the constitutively expressed HIF-1
subunit were not affected by cardiac arrest and resuscitation.
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Figure 1.
HIF-1 accumulation and induction of HIF-1
target genes in the cerebral cortex of rats subjected to transient
global cerebral ischemia. A, Western blots showing
HIF-1 and HIF-1 protein levels in cortical samples from
sham-operated (C) and rats subjected to ~12 min
of cardiac arrest and resuscitation and allowed to recover for 1 hr or
up to 30 d (1h-30d). Crude nuclear
extracts from Hep3B cells exposed to 21% oxygen () or 1%
O2 (+) were used as a negative and positive control,
respectively. -actin immunoblot was used to document equal protein
loading. B, Northern blots showing transient induction
of Epo and Glut-1 mRNA levels in the brain cortex of sham-operated rats
(C) and cardiac arrest/resuscitated rats
(1h-30d). Northern blot of -actin served as the
sample-loading control.
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In addition, we analyzed the expression of two HIF-1 target genes, Epo
and Glut-1, by Northern blot analysis. Our results showed that both
targets were induced at 12 hr and remained elevated up to 7 d of
recovery. At 14 d of recovery, when HIF-1 was no longer
present, mRNA levels of both targets returned to control levels (Fig.
1B).
Immunolocalization of HIF-1
The results shown in Figure 2
demonstrate that HIF-1 immunoreactivity was nondetectable in control
brain sections (Fig. 2A). Intense nuclear
immunostaining was observed at 1 d after cardiac arrest, mainly in
neurons (Fig. 2B), as indicated by colocalization with the neuronal-specific marker NeuN (Fig.
2C,D). In addition, immunopositive flat nuclei
probably corresponding to endothelial cells were also observed in some
blood vessels (Fig. 2C). A similar pattern of neuronal
staining was observed after 1 hr and 2 and 4 d of recovery from
cardiac arrest (data not shown). Although we did not observe obvious
colocalization of HIF-1-positive cells with GFAP, we could not rule
out the possibility that glial cells are also expressing HIF-1 after
cardiac arrest (data not shown).
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Figure 2.
HIF-1 immunostaining in the cerebral cortex at
1 d of recovery from cardiac arrest. A, HIF-1
was not detected in sham-operated rat brain. B,
Positive HIF-1 immunostaining (green)
in the brain cortex at 1 d of recovery in cells with rounded
nuclei characteristic of neurons. C, D, Double
staining for HIF-1 (green) and the
neuronal-specific marker NeuN (red) showed partial
colocalization. In addition, flat nuclei associated with small blood
vessels stained for HIF-1, probably corresponding to endothelial
cells (C, arrow). Scale bar, 100 µm.
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In vivo analysis of brain hypoxia after cardiac
arrest and resuscitation
Because HIF-1 accumulation is triggered primarily by hypoxia,
it was necessary to determine whether the brain remained hypoxic for a
prolonged period after transient global ischemia. For this purpose we
used the nitroimidazole EF5 as an in vivo hypoxia marker. In
our model of transient global cerebral ischemia, we found a strong EF5
adduct binding at 1 hr of recovery from cardiac arrest, indicating the
presence of tissue hypoxia at that time. EF5 binding was especially
prominent in cortex, hippocampus, and white matter. In contrast, no
hypoxic regions were found in the brain at 2 d after cardiac
arrest (Fig. 3). This result showed that
the tissue remained hypoxic by the first hour of recovery, probably
because of a decreased cerebral blood flow reported previously.
However, at 2 d of recovery, the brain was no longer hypoxic,
suggesting that the HIF-1 accumulation that is found at that time
cannot be caused by continued tissue hypoxia.
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Figure 3.
Immunohistochemistry for the in
vivo hypoxia marker EF5 showing weak signal
(red) in the sham-operated rat brain but strong signal
at 1 hr of recovery from cardiac arrest and resuscitation. At 48 hr of
recovery, EF5 adduct binding was comparable with the sham-control. The
same sections were immunostained for the neuronal marker NeuN
(green). The nitroimidazole compound EF5 was
injected intravenously as described in Materials and Methods. Scale
bar, 1 mm.
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To test the sensitivity of EF5 as a hypoxic marker, we compared
HIF-1 levels and EF5 binding in the brain of rats exposed to
different oxygen concentrations. Our results showed that EF5 binding
appears to be graded with hypoxic severity. EF5 signal was detected
only at 12% oxygen exposure or below, whereas no signal was detected
at 14% (data not shown) or 21% O2 (Fig.
4A). In the same
samples, significant brain HIF-1 accumulation was detected only
after exposure to 12% O2 or below (Fig.
4B). Thus, our result demonstrates that EF5
sensitivity correlates with HIF-1 expression induced by hypoxia.
Taken together, these experiments show that after a transient ischemic
episode the rat brain does not remain hypoxic for a prolonged period,
at least not to an extent that could explain sustained HIF-1
accumulation. Although hypoxia can be considered the primary activator
of HIF-1 during the early phase of recovery, it is likely that a
nonhypoxic mechanism is responsible for the sustained HIF-1
accumulation after 2 d of recovery from cardiac arrest.
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Figure 4.
Detection of the hypoxia marker EF5 and HIF-1
accumulation in the brain of animals exposed to varying oxygen
concentrations. A, Increasing brain EF5 adduct binding
(red) with increasing severity of hypoxia (4 hr exposure
at 21, 12, or 8% oxygen). The same sections were immunostained for
NeuN (green). Scale bar, 1 mm. B,
Immunoblots showing increasing HIF-1 accumulation but unchanged
HIF-1 expression in the brain cortex with decreasing oxygen
concentrations. -actin immunoblot shows equal protein loading.
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Von Hippel Lindau protein expression and 26S
proteosome activity
To determine whether the metabolic pathway that degrades HIF-1
is affected by transient cerebral ischemia, we studied pVHL expression
and the 26S proteasome activity. During normoxia, pVHL in association
with elongin B and elongin C, binds to HIF-1 subunits, and targets
them for ubiquitination and subsequent degradation by the proteosome
(Maxwell et al., 1999; Cockman et al., 2000). Hence, changes in pVHL
expression or impairment of the proteasome can potentially affect the
rate of HIF-1 degradation. Immunoblot analysis showed that pVHL
expression was not affected by cardiac arrest and reperfusion in the
brain cortex (Fig. 5A).
Chymotrypsin proteasome activity was transiently and moderately
decreased by ~25% at 1 hr, 12 hr, and 1 d of recovery from
cardiac arrest compared with control cortical samples. However,
proteasome activity was no longer impaired at 2-7 d of recovery (Fig.
5B). Despite normal levels of pVHL protein expression, these
results suggest that the transient inhibition of proteosome activity
may contribute to HIF-1 accumulation only in the early phase of
recovery from cardiac arrest.
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Figure 5.
Cardiac arrest and resuscitation did not affect
pVHL expression but induced transient reduction in proteosome activity.
A, Western blot showing pVHL protein levels in the
cerebral cortex of control (C) and resuscitated
animals (1h-30d recovery after cardiac
arrest). B, Graph representing changes in the
chymotrypsin-like activity of the proteosome after transient cerebral
ischemia (1h-7d recovery). Results are
shown as mean ± SD of three different samples per time point
(*p < 0.05 compared with control).
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Induction of brain IGF-1 expression but not IGF-1R after cardiac
arrest and resuscitation
Although hypoxia is considered to be the main stimulus for
HIF-1 accumulation, other nonhypoxic-related factors such as
insulin, IGF-1, IGF-2, and EGF have been shown to increase HIF-1
protein levels in certain cell types (Zelzer et al., 1998; Feldser et al., 1999; Zhong et al., 2000; Zundel et al., 2000).
We analyzed the expression of IGF-1 that was previously reported to be
responsive to ischemia/reperfusion brain injury. Ribonuclease protection assay was used to quantify the levels of IGF-I mRNA. The
antisense riboprobe used for this experiment protects a region common
to IGF-1A and IGF-1B isoforms, generating a single protected fragment.
Low basal levels of IGF-1 mRNA and protein were detected in control
cortical samples. IGF-1 mRNA levels were not affected during the first
12 hr of recovery from cardiac arrest but were markedly induced at 1 and 2 d of recovery and started to decrease at 7 d (Fig.
6A). IGF-1 protein
levels were also induced, and as expected, this induction was delayed
compared with the mRNA response. A nearly fourfold increase was
observed at 2-7 d of recovery, but this increase subsided at 14 d
of recovery (Fig. 6B). In addition, our results
showed no changes in the expression of the IGF-1 receptor subunit
during the recovery period (Fig. 6B).
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Figure 6.
Upregulation of IGF-1 mRNA and protein levels in
the cerebral cortex after cardiac arrest and resuscitation.
A, Ribonuclease protection assay for IGF-1 and -actin
(loading control) showing a significant induction of IGF-1 mRNA in the
cerebral cortex at 1-7 d of recovery compared with control
(C). B, Western blot showing
increased IGF-1 protein levels in the cortex at 2-7 d of recovery from
cardiac arrest. -actin was used as a control for equal protein
loading. Comparable IGF-1R -subunit protein levels in the cerebral
cortex of control and resuscitated animals
(1h-7d of recovery). C,
Graph representing the levels of endogenous circulating rat
IGF-1 measured by ELISA in the plasma of rats exposed to cardiac arrest
and resuscitation. The apparent increase in IGF-1 levels in the
experimental samples (1h-4d of recovery) did not
reach statistical significance when compared with the control samples.
Results are shown as the mean ± SD of duplicate samples from
three different animals.
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To determine whether an increased uptake of IGF-1 from the plasma could
contribute to our findings showing an increase in brain IGF-1, we
measured endogenous circulating IGF-1 levels using an ELISA assay in
rats subjected to cardiac arrest and allowed to recover for up to
4 d. Our results showed that the IGF-1 plasma levels did not
change significantly after cardiac arrest compared with controls (Fig.
6C). Although we cannot rule out a contribution from the
IGF-1 circulating pool, the fact that its mRNA levels are significantly
elevated suggests that the greater part of the increase in brain IGF-1
is likely caused by an upregulation of its expression in the tissue
itself. Taken together, our results showed that the delayed brain IGF-1
induction after transient global ischemia temporally correlated with
HIF-1 accumulation.
The mechanism underlying the delayed upregulation of IGF-1 is not
known; however, we can speculate that hypoxia is not involved because
by 2 d of recovery, EF5 binding was not detected. In fact, we
found that the expression of IGF-1 (mRNA and protein) does not change
in the brain of rats exposed to hypobaric hypoxia [0.5 atmosphere
(ATM), equivalent to 10% O2] for 1 hr to
7 d (Fig. 7A,B).
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Figure 7.
Expression of IGF-1 in the rat cerebral cortex
after prolonged hypoxia. A, Ribonuclease protection
assay showing that hypobaric hypoxia (0.5 ATM, equivalent to 10%
O2 for 1 hr-7 d) did not affect IGF-1 mRNA levels in the
brain cortex compared with normoxic control (C).
B, Immunoblot showing no changes in IGF-1 protein levels
in the brain cortex during hypobaric hypoxia
(1h-7d).
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IGF-1 colocalized with HIF-1
Control sections showed weak IGF-1 immunoreactivity in the cortex
mainly associated with neurons (data not shown). This immunostaining was enhanced after 4 d of recovery from cardiac arrest and
resuscitation. No apparent staining of glial cells was observed (Fig.
8A). Double immunostaining for IGF-1 and HIF-1 indicates that most neurons expressing IGF-1 also express HIF-1 (Fig.
8A,B). However, many HIF-1-positive neurons did not stain for IGF-1. No obvious
colocalization of IGF-1 and HIF-1 immunostaining was observed in
small blood vessels.
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Figure 8.
IGF-1 immunostaining of the rat cerebral cortex
after transient global cerebral ischemia. A, IGF-1
expression (red) was apparently restricted mainly to
neurons at 4 d of recovery from cardiac arrest. B,
Double staining for HIF-1 (green) showing
colocalization with some IGF-1-positive neurons
(arrows). Scale bar, 100 µm.
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IGF-1 induces HIF-1 accumulation and HIF-1 target genes in PC12
cells and primary cortical neurons
We examined the ability of IGF-1 to induce HIF-1 activation in
PC12 cells and primary cortical neurons. Treatment of these cells with
IGF-1 (100 nM, 24 hr) under normoxic conditions induced a
significant accumulation of HIF-1 (n = 3). However,
the effect of IGF-1 on HIF-1 accumulation was less compared with
hypoxia (1% O2). In contrast, neither hypoxia
nor IGF-1 affected HIF-1 expression in either cell type (Fig.
9A). In addition, we analyzed the expression of two HIF-1 target genes, Glut-1 and VEGF, by Northern
blot analysis. Our results showed that both targets were significantly
induced by IGF-1 treatment. This induction was less pronounced compared
with hypoxia (Fig. 9B). To show that IGF-1 was biologically
active, we measured ligand-induced tyrosine phosphorylation of IGF-1R
in PC12 and primary neurons by immunoprecipitation with IGF-1R
subunit antibody followed by immunoblotting with either anti-phosphotyrosine or anti-IGF-1R antibodies (n = 3). We found an increase in tyrosine-phosphorylated IGF-1R subunit
in PC12 and primary neurons treated with IGF-1, demonstrating the
activation of the receptor. No changes in IGF-1R levels were
observed under these conditions (Fig. 9C).
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Figure 9.
IGF-1 induces HIF-1 accumulation and expression
of Glut-1 and VEGF in PC12 cells and primary cultures of cortical
neurons. A, Western blot showing that hypoxia (1%
O2 for 24 hr) and IGF-1 (100 nM, 24 hr)
treatment were able to induce HIF-1 accumulation in PC12 and primary
cortical neurons. HIF-1 and -actin protein levels were not
affected by either treatment. B, Northern
blot analysis showing induction of Glut-1 and VEGF mRNA levels by
hypoxia (1% O2 for 24 hr) or IGF-1 treatment
(100 nM, 24 hr) in PC12 and cortical neurons. C,
To determine whether IGF-1 treatment (100 nM, 24 hr) results in the activation of the IGF-1R, samples were immunoprecipitated with
anti-IGF-1R antibody and Western blot was performed with a
phosphotyrosine-specific antibody (pTyr). The same
membrane was used for IGF-1R Western blot. D, Western
blot analysis of HIF-1 in PC12 cells exposed to normoxia
(N; 21% O2), hypoxia (H;
1%O2 for 4 hr), or IGF-1 (100 nM for 24 hr) with or without anti-IGF-1 antibody
(Ab; 0.25 µg/ml for 24 hr).
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To demonstrate that IGF-1 is not involved in the hypoxia-dependent
induction of HIF-1, we exposed PC12 cells to hypoxia (1% O2, 24 hr) or IGF-1 (100 nM, 24 hr)
in the absence or presence of an anti-IGF-1 antibody (0.25 µg/ml).
This neutralizing antibody did not affect the accumulation of HIF-1
during hypoxia, but it reduced significantly the expression of HIF-1
in response to IGF-1 (Fig. 9D).
Peripheral infusion of IGF-1 induces HIF-1 accumulation and
expression of HIF-1 target genes in the rat brain
Considering that peripheral administration of recombinant IGF-1
exerts potent therapeutic effects in several models of brain damage,
including brain ischemia, we studied the effects of systemic administration of IGF-1 on HIF-1 accumulation in the brain of normal
rats. As shown in Figure
10A, subcutaneous
administration of recombinant IGF-1 with an osmotic minipump for 7 d resulted in a significant accumulation of HIF-1 in the cerebral
cortex. Conversely, the animals infused only with the vehicle did not show HIF-1 accumulation. The induction of HIF-1 and HIF-1 target genes by IGF-1 was comparable to the effect of transient global ischemia.
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Figure 10.
Effect of peripheral infusion of human
recombinant IGF-1 on HIF-1 accumulation and the expression of target
genes in the rat cerebral cortex. A, Western blots
showing HIF-1 protein levels in the cerebral cortex after hypoxia
(H; 4 hr of 8%O2), 4 d of
recovery after cardiac arrest (CA), and 7 d of
continuous IGF-1 infusion (IGF). HIF-1 protein
levels did not change in response to either treatment. -Actin was
used as a control for equal protein loading. C, Control
samples for each experimental treatment. B, Northern
blot analysis of Epo and Glut-1 mRNA levels in the brain cortex after
hypoxia (H), transient global ischemia
(CA), and IGF-1 infusion at conditions similar to those
described in A.
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To demonstrate that HIF-1 is transcriptionally active after IGF-1
treatment, we analyzed the expression of the HIF-1 target genes, Epo
and Glut-1, by Northern blot analysis. Our results showed that both
targets were significantly induced in the brain of rats treated with
IGF-1 (Fig. 10B). The dose used in this study resulted in a twofold increase in total circulating IGF-1 from 174.17 ± 25.02 to 366.65 ± 62.11 ng/ml of plasma after
7 d of infusion (p < 0.05). This amount
was comparable to previous studies using similar doses, and those
levels of IGF-1 were enough to induce other effects in the rat brain
(Fernandez et al., 1998).
Infusion of IGF-1 in the lateral ventricle induced HIF-1
accumulation in the rat brain
Although peripherally infused IGF-1 has been shown to cross the
blood-brain barrier (Reinhardt and Bondy, 1994), we could not rule out
a systemic effect of IGF-1 that can secondarily affect HIF-1
expression in the brain. To demonstrate a direct effect of IGF-1 on
HIF-1 expression in the rat brain, we infused IGF-1 in the lateral
ventricle of normal rats and analyzed HIF-1 expression in the
periventricular area by immunohistochemistry. Our analysis revealed
that HIF-1-positive cells were present in the parenchymal tissue
surrounding the lateral ventricular on the infused side (Fig.
11B) but not on the
contralateral site, indicating that IGF-1 did not diffuse
contralaterally. The number of HIF-1 positive-cells decreased as a
function of distance from the lateral ventricle. Double labeling with
NeuN showed that most of the cells positive for HIF-1 were neurons
(Fig. 11C,D).
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Figure 11.
Effect of intraventricular infusion of IGF-1 on
HIF-1 accumulation. A, Schematic coronal section
showing site of IGF-1 infusion into the lateral ventricle.
B, Immunofluorescence staining of HIF-1 showing
positive cells (green) in the striatum parenchyma
adjacent to the infused lateral ventricle. C, Double
staining for the neuronal marker NeuN (red) of the
section shown in B. D, Merged images from
B and C showing that most
HIF-1-positive cells were neurons (yellow).
LV, Lateral ventricle. Scale bar, 100 µm.
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Selective inhibition of IGF-1R after cardiac arrest and
resuscitation abrogates HIF-1 accumulation
To confirm the role of IGF-1 in the delayed and persistent
activation of HIF-1 in the brain after transient global cerebral ischemia, the biological activity of the IGF-1R was neutralized by the
intracerebroventricular administration of the selective antagonist
JB-1. Figure 12 depicts representative
examples of HIF-1 immunoreactivity in the brain of rats that were
subjected to cardiac arrest and resuscitation and subsequently received
continuous intracerebroventricular infusion of saline or JB-1 for
4 d during the recovery phase. The brain of the saline-treated
rats exhibited widespread neuronal HIF-1 staining in the cerebral
cortex, the caudoputamen, hippocampal and septal formation, and
other parenchymal regions in both hemispheres (Fig.
12A). Injection of the selective IGF-1R antagonist
into the lateral ventricle caused a significant reduction of HIF-1
immunoreactivity in regions ipsilateral to the site of the injection.
This inhibitory effect of JB-1 was highest in the right caudoputamen,
in the cerebral cortex, and in regions neighboring the ventricle walls
(Fig. 12B). The effect of JB-1 decreased in distant
structures from the site of injection, which may be attributed to the
slow diffusion or to a decreased half-life of this peptide in the
CSF.
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Figure 12.
Effect of intracerebroventricular infusion of
JB-1 on HIF-1 accumulation in the rat brain after cardiac arrest and
resuscitation. A, HIF-1 immunostaining in the
parenchyma adjacent to the lateral ventricle after
intracerebroventricular infusion of saline after cardiac arrest and
resuscitation. B, HIF-1 immunostaining in a similar
region as in A showing absence of HIF-1-positive
cells after selective inhibition of the IGF-1R by
intracerebroventricular infusion of JB-1 for 4 d after cardiac
arrest and resuscitation. C, D, Double
staining for NeuN of the same sections shown in A and
B, respectively. LV, Lateral ventricle.
Scale bar, 100 µm.
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DISCUSSION |
In our model of cardiac arrest, rats regained spontaneous
respiration within 3 hr after resuscitation and regained consciousness before 12 hr. Resuscitated rats were usually able to move and feed
themselves by 24 hr. The major pathophysiological effects of ~12 min
of global ischemia induced by cardiac arrest and resuscitation in the
rat have been reported previously (Crumrine and LaManna, 1991). These
include an initial arterial acidosis, hypertension, and
hemoconcentration that returned to pre-ischemic levels between 30 and
180 min after reperfusion. In addition, changes in brain blood flow
include an initial hyperemia followed by a secondary hypoperfusion
(Crumrine and LaManna, 1991; Lauro et al., 1999).
Transient global cerebral ischemia induced by cardiac arrest and
resuscitation leads to delayed neuronal death as indicated by a loss of
CA1 hippocampal neurons (Crumrine and LaManna, 1991). In response to
this metabolic stress, the expression of VEGF is upregulated as early
as 1 hr of recovery and lasts for up to 4 d (Pichiule et al.,
1999; Jin et al., 2000). This gene has a hypoxic response element in
its promoter and is regulated by the transcription factor HIF-1
(Forsythe et al., 1996). To determine whether activation of HIF-1 was
responsible for an increased expression of VEGF, we studied the
expression of the regulatory subunit HIF-1. Our results showed that
HIF-1 accumulates within 1 hr after resuscitation from cardiac
arrest, and unexpectedly, it remains elevated for >1 week. In addition
to VEGF induction, we showed that other HIF-1 target genes such as Epo
and Glut-1 are also transiently induced. Thus, reversible global
ischemia leads to the activation of HIF-1 and the expression of its
target genes.
Next, we attempted to determine the mechanism responsible for prolonged
HIF-1 accumulation. The most logical explanation would be that HIF-1
is responding to a tissue hypoxic signal. In fact, cerebral blood flow
was found severely reduced in the first hour (16% of control) and
remained approximately half of the control value at 6 hr of recovery
(Crumrine and LaManna, 1991). This hypoperfusion is likely to decrease
oxygen delivery that might induce HIF-1 activation in the early period
of recovery. Nevertheless, it is unlikely that cerebral blood flow
remained low during longer recovery periods (2 d or more). To determine whether the brain remained hypoxic for a prolonged period after transient ischemia, we used the hypoxic marker EF5. This
2-nitroimidazole drug has been used extensively to detect tissue
hypoxia because the rate of its bioreductive metabolism is inversely
dependent on oxygen partial pressure. Intracellular metabolism of EF5
leads to its covalent binding with several molecules. These bound
adducts can be detected with specific antibodies (Evans et al., 1995). Our results showed EF5 binding in the brain at 1 hr but not at 2 d
of recovery from cardiac arrest.
To test the sensitivity of EF5 as a hypoxic marker in the brain, we
exposed rats to different oxygen concentrations and compared the brain
EF5 signal with HIF-1 accumulation. We found that EF5 binding
correlates with HIF-1 accumulation at 12% or lower oxygen concentrations. However, both EF5 binding and HIF-1 were barely detected at 14-21% oxygen. Thus, the lack of EF5 binding at 2 d
of recovery indicates that the brain was no longer hypoxic. Other
nonhypoxic mechanisms must be responsible for the unexpected prolonged
HIF-1 induction.
Considering that impairment of the HIF-1 degradation machinery can
lead to HIF-1 accumulation, we analyzed the activity of the 26S
proteosome complex as well as the expression of the tumor suppressor
pVHL. We found a slight transient reduction of the proteosome activity
in the first hours of recovery from cardiac arrest as indicated by a
25% decrease in chymotrypsin-like activity, whereas pVHL protein
levels in the cerebral cortex were unchanged after cardiac arrest.
These observations suggest that HIF-1 accumulation in the first
hours of recovery might be caused by hypoxic stress caused by a reduced
brain blood flow in addition to a transient inhibition of the 26S
proteosome. However, none of these conditions persisted for >2 d after
cardiac arrest.
To attempt to explain the accumulation of HIF-1 after 2 d of
recovery, we focused our attention on IGF-1. It was reported previously
that this growth factor activated HIF-1 in certain cell types under
normoxic conditions (Zelzer et al., 1998; Feldser et al., 1999; Zundel
et al., 2000). In the CNS, IGF-1 is of particular interest because it
is locally synthesized, and systemic IGF-1 can cross the blood-brain
barrier (Reinhardt and Bondy, 1994; Pan and Kastin, 2000). IGF-1 and
its receptor are known to be present and active in the mature nervous
system. However, brain IGF-1 levels are low compared with peripheral
tissues such as the liver (Daughaday and Rotwein, 1989). Previous
reports have shown that IGF-1, its receptors, and binding proteins are
all upregulated in response to ischemia and other neurodegenerative conditions (Schwab et al., 1997; Beilharz et al., 1998; Fernandez et
al., 1998). This upregulation appears to provide neuroprotection, because exogenous IGF-1 both in vivo and in vitro
protects neurons from different types of injury (Tagami et al., 1997;
Heck et al., 1999; Guan et al., 2000; Wang et al., 2000; Yamaguchi et
al., 2000).
In this study, we have shown that IGF-1 gene expression was transiently
upregulated in the cerebral cortex after cardiac arrest and
resuscitation. Induction of IGF-1 expression occurred between 2 and
7 d of recovery and temporally correlated with the persistent accumulation of HIF-1 during the same period. In contrast, the circulating levels of IGF-1 did not change significantly.
To test the ability of IGF-1 to induce HIF-1 activation in neural
cells, we studied the effect of IGF-1 on the accumulation of HIF-1
and the expression of HIF-1 target genes in vitro and in vivo. Our results showed that IGF-1 induced HIF-1
accumulation in cultured neurons and PC12 cells. In addition, we showed
that peripheral and intracerebroventricular infusion of IGF-1 in rats resulted in accumulation of HIF-1 in the brain. Furthermore, we
showed that the HIF-1 target genes, VEGF, Epo, and Glut-1, are all
induced after IGF-1 treatment, indicating that HIF-1 is transcriptionally active. These lines of evidence demonstrate that
IGF-1 can induce activation of HIF-1 in the CNS. Accordingly, IGF-1
might be responsible for the sustained HIF-1 accumulation after
cardiac arrest and resuscitation. To prove this, we used an IGF-1
analog that selectively inhibits the autophosphorylation of the IGF-1R.
This antagonist, JB-1, has been used previously to inhibit the IGF-1
pathway in the rat brain and in cell culture systems
(Pietrzkowski et al., 1992; Quesada and Etgen, 2002). Our
results showed that intracerebroventricular infusion of JB-1 during the
recovery phase from cardiac arrest and resuscitation inhibited HIF-1
accumulation. Thus, the delayed induction of IGF-1 expression and the
subsequent activation of the IGF-1R receptor after cerebral ischemia
seem to be required for the long-lasting HIF-1 activation.
Our study does not provide evidence for the mechanism underlying the
induction of IGF-1 after brain ischemia; however, this induction seems
not to be oxygen dependent because by 2 d of recovery the brain is
no longer hypoxic, as indicated by EF5 binding analysis. Consistent
with this notion, even prolonged exposure to hypoxia (up to 7 d,
~10% oxygen), previously found to induce HIF-1 activation (Chavez et
al., 2000), does not induce IGF-1 in the rat brain. Interestingly, in
cultured PC12 cells, a neutralizing anti-IGF-1 antibody did not affect
the hypoxia-induced HIF-1 accumulation, whereas it blocked the
IGF-1-mediated HIF-1 accumulation, as expected. Thus, IGF-1 and
hypoxia activate two distinct and independent mechanisms of HIF-1 activation.
Whether activation of HIF-1 has a role in neuronal survival or
contributes to cell death during ischemia and other CNS insults is
still a matter of discussion. The picture emerging from studies with
different models of CNS injury is that activation of HIF-1 is part of
an adaptive mechanism that might allow cell survival during hypoxia and
after an ischemic insult. HIF-1 target genes that may mediate neuronal
survival after ischemia include glycolytic enzymes, erythropoietin,
Glut-1, and VEGF (Bergeron et al., 1999; Zaman et al., 1999;
Marti et al., 2000; Digicaylioglu and Lipton, 2001). Additional
evidence supporting the neuroprotective role of HIF-1 comes from
studies using iron chelators and cobalt chloride. These compounds seem
to exert neuroprotective effects against oxidative stress in neural
cells in part by activating HIF-1 and its target genes (Zaman et al.,
1999; Bergeron et al., 2000). In contrast, some studies have
documented HIF-1 pro-apototic effects in embryonic stem cells or
cortical neurons under hypoxic conditions. It appears that HIF-1
mediates upregulation as well as stabilization of p53 and
downregulation of Bcl-2 leading to cell death (An et al., 1998;
Carmeliet et al., 1998; Halterman et al., 1999). These apparent
contradictory observations suggest that the effect of HIF-1 induction
may depend on the cell type, the developmental stage of the cell, or
the death stimulus.
In conclusion, after transient global cerebral ischemia, the sustained
HIF-1 activation seems to be regulated through hypoxia-dependent and
-independent mechanisms. The subsequent induction of HIF-1 target genes
may be part of the intrinsic neuroprotective mechanism aimed at
attenuating damage as a result of oxidative stress and disruption of
energy metabolism in the brain. Moreover, our results suggest that
IGF-1 might exert its neuroprotective effects in different types of CNS
injury at least in part by activating HIF-1 and its target genes.
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FOOTNOTES |
Received April 19, 2002; revised July 22, 2002; accepted July 31, 2002.
This work was supported by National Institute of Neurological Disorders
and Stroke Grants NS-38632 and NS-37111. We thank Dr. Cameron Koch
(University of Pennsylvania) for his advice and assistance with the EF5
staining. We also thank Dr. W. David Lust (Department of Neurological
Surgery, Case Western Reserve University) for suggestions and
critical reviews of this manuscript and Sandy Hufeisen for technical
assistance with the cell culture work. The human recombinant IGF-1 was
generously provided by A. F. Parlow from the National Institute of
Diabetes and Digestive and Kidney Diseases National Hormone and Peptide
Program (Harbor-University of California Los Angeles Medical Center).
EF5 was a gift from S. Evans (University of Pennsylvania).
Correspondence should be addressed to Dr. Joseph C. LaManna, Department
of Neurology (BRB525), Case Western Reserve University, School
of Medicine, 10900 Euclid Avenue, Cleveland OH 44106-4938. E-mail:
JCL4{at}po.cwru.edu.
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ABSTRACT
INTRODUCTION
