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cAMP response element-binding protein activation in ligation preconditioning in neonatal brain.

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ORIGINAL ARTICLES
cAMP Response Element-Binding Protein
Activation in Ligation Preconditioning in
Neonatal Brain
Hsueh-Te Lee, BS,1 Ying-Chao Chang, MD, PhD,2 Lin-Yu Wang, MD,3 Shan-Tair Wang, PhD,4
Chao-Ching Huang, MD,5,6 and Chien-Jung Ho, BS6
Perinatal hypoxic-ischemic (HI) brain injury is a major cause of permanent neurological dysfunction in children. An
approach to study the treatment of neonatal HI encephalopathy that allows for neuroprotection is to investigate the
states of tolerance to HI. Twenty-four-hour carotid-artery ligation preconditioning established by delaying the onset of
hypoxia for 24 hours after permanent unilateral carotid ligation rats markedly diminished the cerebral injury, however,
the signaling mechanisms of this carotid-artery ligation preconditioning in neonatal rats remain unknown. Ligation of
the carotid artery 24 hours before hypoxia provided complete neuroprotection and produced improved performance on
the Morris water maze compared with ligation performed 1 hour before hypoxia. Carotid artery ligation 6 hours before
hypoxia produced intermediate benefit. The 24-hour carotid-artery ligation preconditioning was associated with a robust
and sustained activation of a transcription factor, the cAMP response element–binding protein (CREB), on its phosphorylation site on Ser133. Intracerebroventricular infusions of antisense CREB oligodeoxynucleotides significantly reduced the 24-hour carotid-artery ligation–induced neuroprotection effects by decreasing CREB expressions. Pharmacological activation of the cAMP-CREB signaling with rolipram 24 hours before hypoxia protected rat pups at behavioral
and pathological levels by sustained increased CREB phosphorylation. This study suggests that 24-hour carotid-artery
ligation preconditioning provides important mechanisms for potential pharmacological preconditioning against neonatal
HI brain injury.
Ann Neurol 2004;56:611– 623
Perinatal hypoxic-ischemia (HI) is a major cause of
neonatal mortality and of subsequent neurological disabilities among infants who survive it.1–3 Although the
immature brain is relatively protected from HI by
adaptive mechanisms, severe insults can trigger selfsustaining neurotoxic cascades lasting several days and
result in prominent neuronal injury.1,4,5 Although our
understanding of the pathogenesis of neonatal HI brain
injury has increased considerably, there is still no effective treatment.4,5
In adult animal stroke models, preconditioning by
nonlethal ischemia exhibits protective effects against
neuronal death in vulnerable brain regions after subsequent lethal ischemia. The molecules that mediate the
induction of ischemic tolerance include heat shock proteins, reactive oxygen species, nitric oxide, nuclear
factor-␬B, adenosine A1 receptor, and KATP channels.6 –9
Unilateral carotid artery ligation followed by systemic exposure to hypoxia is a model widely used to
investigate HI brain injury in immature rats.10 Hypoxia preconditioning produces tolerance, through
hypoxia-inducible factor–1 activation, against HI brain
injury in newborn rats.11,12 On the other hand, preconditioning established by delaying the onset of hypoxia for 24 hours after carotid artery ligation also
markedly diminishes the extent of HI injury in the immature brain; tissue adaptation occurs between artery
ligation and the onset of hypoxia,13,14 but the cellular
mechanisms triggered by carotid-artery ligation preconditioning remain poorly understood.
Extracellular stimuli can exert long-lasting effects,
such as promoting cell growth, proliferation, survival,
or death, by triggering signaling cascades that ultimately converge onto nuclear transcription factors.15
From the 1Institute of Basic Medical Science, Medical College, National Cheng Kung University, Tainan, Taiwan; 2Department of
Pediatrics, Chang Gung Memorial Hospital Kaohsiung, Taiwan;
3
Department of Pediatrics, Chi-Mei Hospital, Tainan, Taiwan; Institutes of 4Public Health and 5Molecular Medicine; and 6Department of Pediatrics, Medical College, National Cheng Kung University, Tainan, Taiwan.
Published online Oct 6, 2004 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20259
Address correspondence to Dr Huang, Department of Pediatrics,
National Cheng Kung University Hospital, 138 Sheng-Li Road,
Tainan City 704, Taiwan. E-mail: huangped@mail.ncku.edu.tw
Received Jan 12, 2004, and in revised form May 28 and Jul 18.
Accepted for publication Jul 18, 2004.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
611
One transcription factor, cAMP response element–
binding protein (CREB), is a key mediator of stimulusinduced nuclear responses that underlie learning and
memory, and the plasticity of the nervous system.15,16
CREB is phosphorylated on Ser133 (pCREB) by
cAMP-dependent protein kinase (PKA) or other kinases and consequently binds to the cAMP response
element (CRE) of target genes.15,16 Evidence suggests
that endogenous CREB activation might provide potent survival signals during ischemia,15–18 and during
acquisition of ischemic tolerance in the adult rat
brain.19,20 Whether CREB activation is involved in the
signaling pathway of carotid-artery ligation preconditioning against HI in the immature brain, however, remains undetermined.
In this study, we used CREB antisense oligodeoxynucleotides (ODNs) to disrupt CREB protein levels
to examine the effect of CREB on carotid-artery ligation preconditioning. ODNs are preferentially taken
up by neurons in the rodent brain after intracerebral
administration.21,22 Inside the cell, antisense ODN
bases pair to their cognate mRNAs to increase turnover
or block translation of targeted mRNA. On the other
hand, pharmacological activation of the cAMP-PKA
signaling pathway by rolipram, a phosphodiesterase
type IV (PDE4) inhibitor,23–25 or forskolin, an adenylyl cyclase-activator,18,26 could lead to CREB phosphorylation. Therefore, this study aimed to test the following hypotheses in the neonatal rat brain: (1)
preconditioning with carotid-artery ligation protects
against HI injury in a time-dependent manner; (2) preconditioning with 24-hour carotid-artery ligation enhances CREB phosphorylation; (3) CREB expression
mediates the preconditioning effects induced by 24hour carotid-artery ligation; (4) pharmacological preconditioning by activation of the cAMP-CREB signaling pathway is neuroprotective against HI injury.
Materials and Methods
This study was approved by the Animal Care Committee at
National Cheng Kung University. Ten to 12 pups of either
sex per dam were used and housed with a 12-hour-light/12hour-dark schedule in a temperature- and humiditycontrolled colony room. The pups were housed with their
dams until weaning on postnatal (P) day 21 and then housed
in groups of four to five per cage. The male and female rats
was equally distributed between the experimental groups.
In P7 rat pups, unilateral common carotid artery ligation
followed 1 hour later by 8% oxygen hypoxia for 2 hours
produces selective damage in the hemisphere ipsilateral to the
artery occlusion that resembles HI damage to the human
neonatal brain.10
Carotid-Artery Ligation Preconditioning
To examine whether the carotid-artery ligation preconditioning could be established in a time-dependent manner, we
performed carotid artery ligation in rat pups 24 hours (24-
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hour ligation group), 6 hours (6-hour ligation group), or 1
hour (1-hour ligation group) before exposing them to 2
hours of 8% oxygen hypoxia on P7. The animals were anesthetized with 2.5% halothane (balance, room air), and the
right common carotid artery was surgically exposed and permanently ligated with 5-0 surgical silk. After surgery, the
pups were returned to the dam for a 1-, 6-, or 24-hour recovery before hypoxia. On P7, the three groups were placed
in airtight 500ml containers partially submerged in a 37°C
water bath through which humidified 8% oxygen (balance,
nitrogen) was maintained at a flow rate of 3L/min for 2
hours. After completion of hypoxia, the rat pups were returned to their cage. There were two control groups: sham
controls (sham-operated P6 rats without hypoxia) and ligation controls (P6 rats with permanent artery ligation but
without hypoxia).
cAMP Response Element-Binding Protein Antisense
Oligodeoxynucleotide Infusions
The sequence of the CREB antisense and scrambled ODN
were: CREB antisense, 5⬘-TGGTCATCTAGTCACCGGTG-3⬘; and scrambled, 5⬘-GTCTGCAGTCGATCTACGGT-3⬘.21 The antisense sequence perfectly matched the
rat CREB gene corresponding to nucleotides 27 to 46.21,22
The scrambled sequence showed no significant matches in
the GenBank database. P6 rats, anesthetized with 2.5% halothane, were intracerebroventricularly injected with antisense
or scrambled ODN (2nmol in 1␮l) in the right cerebral
hemisphere using a 30-gauge needle on a 10␮l Hamilton
syringe. The location of each injection in relation to the
bregma was 2.0mm posterior, 1.5mm lateral, and 2.0mm
deep to the skull surface.27 In the 24-hour ligation group,
the first ODN infusion was given 3 hours before ligation,
and the second and third were given at 6 and 12 hours after
ligation. Twenty-four hours after ligation (12 hours after the
last ODN infusion), both groups underwent 8% oxygen
hypoxia for 2 hours. An HI control group received three intracerebroventricular injections of scrambled ODN under
the same schedule and underwent HI on P7. On P21, hemispheric weight reduction was measured.
Autoradiographic Determination of Cerebral Blood
Flow
Cerebral blood flow (CBF) was determined in the immature
rats by the iodo-[14C] antipyrine (IAP) autoradiographic
technique.28 Immediately after hypoxia, each rat was injected
subcutaneously with 5␮Ci IAP in 0.1ml normal saline into
the midline back of the rat. The brains of the rats killed 2
minutes after IAP injection were immediately removed, frozen and processed for autoradiography.28 In brief, brain
coronal sections 20␮m thick were cut in cryostat, mounted
on glass slides, dried at 55°C on a hot plate, and subjected to
carbon-14 autoradiography.
Forskolin Preconditioning
P6 rats received a 5␮l intracerebroventricular injection of vehicle [dimethyl sulfoxide (DMSO)] or forskolin (25␮g in
DMSO) as described above.27 Twenty-four hours after injection, the rats underwent HI. On P21, hemispheric weight
reduction was measured. To determine whether DMSO in-
duced a preconditioning effect, we injected P6 rats similarly
with DMSO or saline 24 hours before HI and hemispheric
weight reduction was compared.
Rolipram Preconditioning
P6 rats were administered a single intraperitoneal injection of
0.1ml of rolipram (1 or 3mg/kg) or DMSO; 24 hours later,
they underwent HI. Rectal temperature was measured at 1,
3, and 24 hours after injection and immediately after HI. To
avoid temperature changes, we kept pups and their dam in
an infant incubator maintained at 30 to 31°C during the
first 24 hours after injection.
Outcome Measures
In brief, a circular pool was filled
with water, and an 8 ⫻ 8cm platform was positioned in the
center of one of the quadrants, 1cm below the water surface.
From Days 1 to 4, P32 rats were given 24 training sessions
(six per day) to locate the submerged platform. On Day 5,
the platform was removed, and rats were placed back into
the pool and allowed 60 seconds of free swimming. Escape
latency, escape distance, and swimming patterns of the rats
were monitored by a camera mounted above the pool
(EthoVision, Wageningen, The Netherlands). After the
probe test, rats were submitted to four trials of a visually
cued learning task to locate a green escape platform 2cm
above the water.29
MORRIS WATER MAZE.
On P36, the brains
were removed. After removal of the brainstem and cerebellum, the forebrain was sectioned at the midline, and left and
right hemispheric weights were determined. The percentage
of hemispheric weight reduction measured as (left hemisphere weight ⫺ right hemisphere weight)/left hemisphere
weight was used as the measure of cerebral injury in this
study. Our preliminary data from P7 rats with HI injury
showed that the changes in the hemispheric weight reduction
were highly correlated not only to the changes in the infarct
brain areas (r ⫽ 0.87; p ⬍ 0.0001; n ⫽ 27) according to
plates 17, 20, 23, 28, 31, and 34 in a rat brain atlas,30 but
also to the hemispheric volume changes (r ⫽ 0.91; p ⬍
0.0001; n ⫽ 27) measured on P36. This finding was consistent with another report.31 Therefore, only hemispheric
weight data are shown in this study.
HEMISPHERIC WEIGHT REDUCTION.
Western Blot Analysis
Tissue was homogenized in cold lysis buffers as described
previously.29,32 Fifty-microgram samples were resolved in
10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and blotted electrophoretically to nitrocellulose membranes. Membranes were incubated with primary antibodies,
and immunoreactivity was detected by horseradishconjugated secondary antibody and visualized with enhanced
chemiluminescence. The following primary antibodies (Upstate Biotechnology, Charlottesville, VA) were used: anti–
phospho-Ser133-CREB (pCREB) (1:1,000 dilution), antiCREB (1:1,500), and brain-derived neurotrophic factor
(BDNF; 1:1,000).
cAMP Response Element-Binding Protein
Phosphorylated on Ser133 Immunohistochemistry
Twenty-four hours after hypoxia, 24- and 1-hour ligation
groups were perfused with 4% paraformaldehyde, and the
brains were postfixed for 24 hours and then cryoprotected in
30% sucrose solutions. Coronal sections (20␮m) were incubated with anti–pCREB antibody (1:100) and visualized
with an avidin-biotin system (Vector Laboratories, Burlingame, CA).29 In each pCREB immunohistochemistry staining, negative control was also performed with normal rabbit
serum as a substitute for the primary antibody.
Reverse Transcription Polymerase Chain Reaction of
Brain-Derived Neurotrophic Factor
Total RNA was extracted from each hemisphere according to
the TRIzol protocol (Invitrogen, San Diego, CA). A 5␮g
portion of total RNA and 1.5␮g oligo-dT primer were incubated at 70°C for 10 minutes and gradually cooled to
room temperature. Each RT mixture, containing 25 units of
M-MLV reverse transcriptase (Promega, Madison, WI), 10␮l
5 ⫻ reaction buffer, 0.5mM dNTP, and nuclease-free distilled water, was added to a final volume of 50␮l. The samples were incubated at 37°C for 90 minutes followed by denaturation at 95°C for 10 minutes. Each PCR (20␮l)
contained 2␮l of RT product, 1 unit of Taq DNA polymerase (Viogene, Taiwan), 2␮l 10 ⫻ PCR buffer plus MgCl2,
0.2mM dNTP, and 0.5␮M gene-specific primers (BDNF:
forward 5⬘-GACAAGGCAACTTGGCCTAC-3⬘, reverse 5⬘CTGTCACACACGCTCAGCTC-3⬘, product size: 356bp;
GAPDH: forward 5⬘-ACATTGTTGCCATCAACGAC-3⬘,
reverse 5⬘-ACGCCAGTAGACTCCACGAC-3⬘, product
size: 216bp). Amplified reaction was performed with a thermocycler for a single 3-minute initial denaturation at 94°C
followed 33 cycles (BDNF) or 26 cycles (GAPDH) under
the conditions: 94°C (20 seconds), 55°C (20 seconds), and
72°C (20 seconds) and final extension at 72°C for 4 minutes. The PCR products were separated on 1.5% agarose gels
containing ethidium bromide and quantified by densitometry. The BDNF PCR product was normalized to that of
GAPDH PCR product in each sample.
Statistics
Statistical significance ( p ⬍ 0.05) was determined using oneway analysis of variance (ANOVA) to compare hemispheric
weight reduction and probe test and visual motor test results
of the water maze between experimental groups. Multivariate
analysis of variance (MANOVA) was used to compare escape
time over the learning phase of the water maze. Post hoc
comparisons using Tukey’s method were used in one-way
ANOVA and Bonferroni’s method in MANOVA. Continuous data were represented as mean ⫾ SEM, unless indicated
otherwise.
Results
Carotid-Artery Ligation–Induced Preconditioning
Effects of Ligation Preconditioning
During water-maze training, there was a significant
group difference between the sham and ligation control
Lee et al: pCREB in Brain Preconditioning
613
groups, and the 1-, 6-, and 24-hour ligation groups
( p ⬍ 0.001). Post hoc multiple comparisons showed
that 1-hour ligation group spent significantly more
time finding the submerged platform ( p ⬍ 0.001), less
time in the target quadrant ( p ⬍ 0.05), and more time
reaching the visible platform ( p ⬍ 0.001), than the
other groups (Fig 1A–C). There were no significant
differences in time spent finding the submerged platform, staying in the target quadrant, or reaching the
visible platform between the sham control, ligation
control, 6-hour, and 24-hour ligation groups.
The degree of brain injury, as measured by the degree of cerebral hemispheric weight reduction, was also
significantly greater in the 1-hour ligation group than
in the other groups (all p ⬍ 0.001; see Fig 1D). There
was no significant difference in the degree of brain injury among the 6-, 24-hour ligation groups, the sham
and ligation-control groups, although the neuroprotection in the 6-hour ligation group was not as complete
as the other three groups.
Immediately after hypoxia, CBF was reduced in the
cortex and lateral parts of the striatum ipsilateral to the
carotid artery ligation compared with the contralateral
hemisphere in the 1-hour ligation group. In contrast,
no hemispheric difference in CBF was seen in the 24hour ligation group (see Fig 1E).
Fig 1. Carotid-artery ligation preconditioning induced neuroprotection against hypoxic-ischemic brain injury at behavior and pathological levels in a time-dependent manner. (A) During water-maze training, the 1-hour ligation group spent significantly more time
finding the platform than the other groups (all p ⬍ 0.001). There were no significant differences between the 6-, 24-hour ligation,
sham and ligation control groups. (B) On the probe test, the 1-hour ligation group spent significantly less time in the target quadrant than each of the other groups (all p ⬍ 0.05). There were no significant differences between the 6-, 24-hour ligation, sham
and ligation control groups. (C) In the visual motor performance, the 1-hour ligation group spent significantly more time than each
of the other groups (all p ⬍ 0.001). In contrast, there was no significant difference among the 6-, 24-hour ligation, sham and
ligation control groups. (D) The degree of brain injury, measured by hemispheric weight reduction, in the 1-hour ligation group
was significantly greater than in the other groups (all p ⬍ 0.001). There was no significant difference among the sham, ligation
control groups, 6-, and 24-hour ligation groups. (#p ⬍ 0.001, ✽p ⬍ 0.05). (E) Posthypoxia cerebral blood flow (CBF) examination by iodo-[14C] antipyrine autoradiography showed reduced CBF in the hemisphere ipsilateral to artery ligation compared with
the contralateral hemisphere in the 1-hour ligation group. In contrast, no hemispheric CBF difference was seen in the 24-hour ligation group (R ⫽ right hemisphere, L ⫽ left hemisphere).
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Ligation Preconditioning and cAMP Response
Element-Binding Protein Phosphorylated on Ser133
We performed Western blot analyses to determine the
temporal profile of pCREB in the cortex and hippocampus ipsilateral to the artery ligation. Hippocampal pCREB levels increased and reached a peak at 6
hours after ligation preconditioning; those levels decreased but remained elevated 24 hours after preconditioning. Cortical pCREB levels increased, with one
peak at 3 hours and another at 24 hours after preconditioning. Total CREB levels remained unchanged (Fig
2A, B).
We next examined the temporal profile of pCREB
posthypoxia. In the 1-hour ligation group, hippocampal pCREB levels were already high 3 hours after
hypoxia, when first measured, and then progressively
decreased to control level by 24 hours after hypoxia.
In the 24-hour ligation group, hippocampal pCREB
levels progressively increased 6 to 24 hours after hypoxia. Cortical pCREB levels in the 1-hour ligation
group increased 3 to 12 hours after hypoxia and decreased to control level 24 hours after hypoxia. In the
24-hour ligation group, increased cortical pCREB levels persisted 24 hours after hypoxia. Total CREB remained unchanged (see Fig 2C, D).
Immunohistochemistry of cAMP Response ElementBinding Protein Phosphorylated on Ser133
There were few pCREB-immunopositive cells in the
cortex and hippocampus in the sham control (Fig 3A,
D). Twenty-four hours after hypoxia in the 24-hour
Fig 2. The temporal profile of cAMP response element–binding protein phosphorylated on Ser133 (pCREB) levels in the ipsilateral
hippocampus and cortex before and after hypoxia. The pCREB levels was robustly increased and sustained in the hippocampus (A)
and cortex (B) during the 24 hours of ligation preconditioning. Hippocampal pCREB levels increased and reached a peak at 6
hours after preconditioning, and the levels decreased but remained elevated 24 hours after preconditioning. Cortical pCREB levels
increased, with one peak at 3 hours and another at 24 hours after preconditioning. Total CREB remained unchanged in both
groups. (C) In the 1-hour ligation group, hippocampal pCREB levels were already high 3 hours after hypoxia and then progressively
decreased to control level by 24 hours after hypoxia. In the 24-hour ligation group, hippocampal pCREB levels progressively increased 6 to 24 hours after hypoxia. (D) Cortical pCREB levels in the 1-hour ligation group increased 3 to 12 hours after hypoxia
and decreased to control level 24 hours after hypoxia. In the 24-hour ligation group, increased cortical pCREB levels persisted 24
hours after hypoxia. Total CREB remained unchanged.
Lee et al: pCREB in Brain Preconditioning
615
ligation group, there was a marked increase of
pCREB immunoreactivities in the cortex and hippocampus on the lesioned hemisphere (see Fig 3B,
E), which contrasted with the very weak pCREB immunoreactivity in the 1-hour ligation group (see Fig
3C, F).
Effect of Oligodeoxynucleotides on Ligation
Preconditioning and cAMP Response ElementBinding Protein
In the 24-hour ligation group, we used antisense ODN
specific for CREB to diminish CREB expression to see
whether CREB expression was necessary for the neuroprotective effects in that group. Rats were unilaterally
infused with 5⬘ biotinylated antisense ODN 3 hours
before being killed to examine the localization of ODN
delivery. Dark staining showed ODN in the ipsilateral
striatum, cortex, and hippocampus (Fig 4A). The degree of brain injury was significantly different between
the three groups, including the 1-hour ligation group
pretreated with scrambled ODN and the 24-hour ligation group with scrambled or CREB antisense ODN
infusions ( p ⬍ 0.05; Fig 4B). Post hoc multiple comparisons showed that the antisense ODN-infused rats
had significantly greater brain injury than the scrambled ODN-infused rats in the 24-hour ligation group
( p ⬍ 0.001). The 1-hour ligation group pretreated
with scrambled ODN had significantly greater brain
injury than did the antisense ODN-infused rats ( p ⬍
0.001) or scrambled ODN-infused rats in the 24-hour
ligation group ( p ⬍ 0.001). The antisense ODN infusions reduced CREB protein expression by 60% 6
hours after the last antisense ODN infusion; CREB
levels returned to control level 12 hours after the last
infusion (at the time of hypoxia; see Fig 4C). There
was no significant difference in the degree of brain injury between CREB-antisense-ODN–treated (0.4 ⫾
0.2%, n ⫽ 19) and scrambled-ODN–treated (0.7 ⫾
0.4%, n ⫽ 18) rat pups not exposed to HI, which
excluded the potentially deleterious effects of repeated
antisense ODN infusions in the developing brain.
Effects of Forskolin Pharmacological Preconditioning
The degree of brain injury was significantly lesser in the
forskolin-pretreated rats than in the DMSO-pretreated
rats ( p ⬍ 0.05; see Fig 4D). Although DMSO has many
biological effects that could be neuroprotective,33 there
was no significant difference in the degree of brain injury between DMSO- (38.8 ⫾ 2.2%, n ⫽ 26) and
saline- (35.5 ⫾ 4.1%, n ⫽ 17) pretreated groups. Compared with vehicle, forskolin injection caused sustained
increases of pCREB in the hippocampus and cortex 6 to
Fig 3. Anatomical distribution of cAMP response element–binding protein phosphorylated on Ser133 (pCREB) immunoreactivity in
the cortex and hippocampus 24 hours after hypoxia. There were few pCREB-immunopositive cells in the cortex (A) and hippocampus (D) in the sham control. A marked increase of pCREB-immunoreactive cells was seen in the cortex (B) and hippocampus (E)
on the lesioned hemisphere in the 24-hour ligation group. Inset shows high-power image of cornu ammonis 1 region of the hippocampus (CA1) (arrow) with increased pCREB immunoreactivities mainly located in the nucleus of the CA1 cells. In contrast,
very weak pCREB immunoreactivity was found in the cortex (C) and hippocampus (F) in the 1-hour ligation group (n ⫽ 4 per
group). Scale bars ⫽ 200␮m in A–C, 500␮m in D–F, and 50␮m in inset of E.
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Fig 4. cAMP response element–binding protein (CREB) antisense oligodeoxynucleotide (ODN) infusions diminished 24-hour ligation preconditioning effect by reducing CREB protein; in contrast, forskolin preconditioning 24 hours before hypoxia induced neuroprotection by CREB activation. (A) Antisense ODN (2nmol in 2␮l) with an added 5⬘ biotinylated group was infused into the
right hemisphere to demonstrate the location of ODN delivery. Representative brain sections (30␮m) showed dark stained biotinylated ODN in the ipsilateral striatum, cortex, and hippocampus. (B) The degree of brain injury was significantly different between
the three groups, including the 1-hour ligation group treated with scrambled ODN, and the 24-hour ligation group with scrambled
or CREB antisense ODN infusions (p ⬍ 0.05). In the 24-hour ligation group, three times of CREB antisense or scrambled ODN
infusions were performed with the first ODN infusion given 3 hours before ligation and the second and third infusion at 6 and 12
hours after ligation. Twenty-four hours after ligation, both groups underwent 8% oxygen hypoxia for 2 hours. Post hoc comparisons
showed that the CREB antisense ODN-infused rats had significantly greater brain injury than scrambled ODN-infused rats
(21.2 ⫾ 2.8% vs 0.7 ⫾ 0.6%, p ⬍ 0.001). The 1-hour ligation animals pretreated with scrambled ODN had significantly
greater brain injury (43.9 ⫾ 3.1%) than did the 24-hour ligation group pretreated with scrambled ODN (p ⬍ 0.001) or with
antisense ODN (p ⬍ 0.001). (C) The CREB protein expression was determined 6 and 12 hours after the last ODN infusion. The
CREB protein expression was reduced by 60% 6 hours after the last antisense ODN infusion, and the levels returned to basal level
12 hours after the last infusion (at the time of hypoxia). (D) The degree of brain injury was significantly lesser in the forskolinpretreated rats than in the vehicle-pretreated rats (25.3 ⫾ 3.0% vs 35.8 ⫾ 2.7%, p ⬍ 0.05). (E) Compared with vehicle, forskolin injection caused sustained increases of pCREB levels in the hippocampus and cortex 6 to 24 hours after injection. There was
no difference in total CREB between the two groups. CREB ⫽ cAMP response element–binding protein; pCREB ⫽ CREB phosphorylated on Ser133; ICV ⫽ intracerebroventricular.
24 hours after injection (see Fig 4E). There was no difference in total CREB between the two groups.
Effects of Rolipram
During water-maze training, the escape times were significantly different between the controls and rats preconditioned with rolipram or vehicle ( p ⬍ 0.001; Fig
5A). The vehicle-pretreated rats spent significantly
more time finding the platform than did roliprampreconditioned rats ( p ⬍ 0.01) and controls ( p ⬍
0.001), and that controls spent less time than did
rolipram-preconditioned rats (all p ⬍ 0.05). On the
probe test, there were significant differences ( p ⬍
0.005) between the four groups in time spent in the
Lee et al: pCREB in Brain Preconditioning
617
target quadrant (see Fig 5B). Vehicle-pretreated rats
spent significantly less time than did the 3mg/kg rolipram–preconditioned ( p ⬍ 0.05) and control rats
( p ⬍ 0.001). There was no significant difference between 1mg/kg rolipram–preconditioned and vehiclepretreated rats, or between 1 or 3mg/kg rolipram–preconditioned rats and controls. In the visual motor test,
there were significant differences ( p ⬍ 0.001) between
the four groups in time spent reaching the visible platform. Vehicle-pretreated rats spent significantly more
time reaching the platform than 1mg/kg rolipram–preconditioned ( p ⬍ 0.05), 3mg/kg rolipram–preconditioned ( p ⬍ 0.001), and control rats ( p ⬍ 0.001; see
Fig 5C). There was no significant difference between
1mg/kg rolipram–preconditioned, 3mg/kg rolipram–
preconditioned, and control rats.
The degree of brain injury between the four groups
showed significant differences ( p ⬍ 0.0001): it was
greater in the vehicle-pretreated group than in the
other groups (all p ⬍ 0.001; see Fig 5D). There was no
difference between 1mg/kg rolipram–preconditioned,
3mg/kg rolipram–preconditioned, and control rats, although a greater degree of brain injury was observed in
the 1mg/kg rolipram–preconditioned group.
The neuroprotective effects of rolipram observed
here could not be attributed to hypothermia: the temperature of the 1 and 3mg/kg rolipram–preconditioned
animals did not differ from that of vehicle-pretreated
Fig 5. Rolipram preconditioning induced neuroprotection at behavioral and pathological levels. (A) During water-maze training,
the vehicle-pretreated rats spent significantly more time finding the submerged platform than did rolipram-preconditioned rats (all p
⬍ 0.01) and control (p ⬍ 0.001). The controls also spent less time than did rolipram-preconditioned rats (all p ⬍ 0.05). (B) On
the probe test, vehicle-pretreated rats spent significantly less time in the target quadrant than did the 3mg/kg rolipram–preconditioned (p ⬍ 0.05) and control rats (p ⬍ 0.001). There was no significant difference between 1mg/kg rolipram–preconditioned and
vehicle-pretreated rats, 1mg/kg rolipram–preconditioned and controls, or between 3mg/kg rolipram–preconditioned rats and controls.
(C) In the visual motor test, vehicle-pretreated rats spent significantly more time than 1mg/kg rolipram–preconditioned (p ⬍ 0.05),
3mg/kg rolipram–preconditioned (p ⬍ 0.001), or control rats (p ⬍ 0.001). In contrast, there was no significant difference between
1mg/kg rolipram–preconditioned, 3mg/kg rolipram–preconditioned, and control groups. (D) The degree of brain injury in the
vehicle-pretreated group was significantly greater than the other groups (each p ⬍ 0.001). There was no difference between 1mg/kg
rolipram–preconditioned, 3mg/kg rolipram–preconditioned, and control groups, although a greater degree of brain injury was observed in the 1mg/kg rolipram–preconditioned group (#p ⬍ 0.001, ✽p ⬍ 0.01, p ⬍ 0.05).
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animals before (3 hours after injection: 34.4 ⫾ 0.2°C,
34.2 ⫾ 0.3°C, and 34.3 ⫾ 0.3°C; 24 hours after injection: 34.6 ⫾ 0.2°C, 34.3 ⫾ 0.3°C, and 34.6 ⫾
0.2°C) or after hypoxia (34.8 ⫾ 0.3°C, 34.6 ⫾ 0.4°C,
and 34.6 ⫾ 0.3°C).
Rolipram and cAMP Response Element-Binding
Protein Phosphorylated on Ser133
Compared with vehicle, a single rolipram preconditioning, especially in the 3mg/kg group, caused robust
and sustained increases in pCREB for 24 hours in the
hippocampus (Fig 6A, B) and cortex (see Fig 6D, E).
Total CREB remained unchanged by rolipram treatment.
the bilateral hippocampus (see Fig 6C) and cortex (see
Fig 6F) at 24 and 72 hours after hypoxia. Total CREB
was unchanged between the two groups. Sustained
CREB phosphorylation may activate downstream targets such as BDNF by binding to CRE located in the
promoter region of the BDNF gene.15,17 We next examined whether posthypoxia rolipram-induced sustained CREB phosphorylation could increase BDNF
expression at the transcriptional and translational levels. Compared with vehicle, 3mg/kg rolipram preconditioning caused enhanced BDNF mRNA (Fig 7A, C)
and protein expression (see Fig 7B, D) in the bilateral
hippocampus and cortex 24 and 72 hours after hypoxia.
Effects of Rolipram on Posthypoxia cAMP Response
Element-Binding Protein Phosphorylated on Ser133
and Brain-Derived Neurotrophic Factor Expression
Compared to vehicle, 3mg/kg rolipram preconditioning caused robust and sustained increases of pCREB in
Discussion
This study showed that carotid-artery ligation preconditioning can time-dependently provide neuroprotection against hypoxic-ischemic brain injury in neonatal
Fig 6. Effects of rolipram preconditioning on the temporal expression of pCREB levels before and after hypoxia. Compared with
vehicle, a single rolipram preconditioning, especially in the 3mg/kg group, caused robust and sustained increases in pCREB levels for
24 hours in the hippocampus (A, B) and cortex (D, E). Total CREB remained unchanged by rolipram treatment. Compared with
vehicle, 3mg/kg rolipram preconditioning caused robust and sustained increases of pCREB expression in the bilateral hippocampus
(C) and cortex (F) 24 hours and 72 hours after hypoxia. Total CREB was unchanged between the two groups. L ⫽ left hemisphere; R ⫽ right hemisphere; V ⫽ vehicle; DMSO ⫽ dimethylsulfoxide; CREB ⫽ cAMP response element–binding protein;
pCREB ⫽ CREB phosphorylated on Ser133.
Lee et al: pCREB in Brain Preconditioning
619
rats, and that complete behavioral and pathological
neuroprotection requires 24-hour ligation preconditioning. The neuroprotective mechanism of 24-hour
carotid-artery ligation preconditioning is associated
with robust and sustained CREB phosphorylation.
Pharmacological activation of cAMP-CREB signaling
cascades by sustained CREB phosphorylation, especially with rolipram, 24 hours before hypoxia protected
rat pups against brain injury. These findings provide
evidence that persistent CREB activation is an important step in the signal transduction that underlies
carotid-artery ligation preconditioning against HI injury in the immature brain.
In rats, occlusion of one common carotid artery in
the absence of subsequent hypoxia causes only minor
alteration in cerebral blood flow in the ipsilateral cerebral hemisphere.14 When combined with systemic hypoxia, however, unilateral carotid artery ligation is associated with decreased CBF and ischemic brain damage
in the ipsilateral hemisphere.10,13,14,28,34 In addition,
delaying the onset of hypoxia for 24 hours after carotid
artery ligation prevents the depletion of ATP and
markedly diminishes the extent of brain injury in the
neonatal rats.13,14 Our findings suggest that the neuroprotection induced in the 24-hour ligation preconditioning is associated with compensatory cerebrovascular
adaptation in the ipsilateral hemisphere during a subsequent episode of hypoxia.
Synapse-to-nucleus signaling leading to CREBmediated transcription is important for neuronal synaptic plasticity, memory function, regeneration, and
survival in response to various stresses.15,16 Because of
its responsiveness to multiple intracellular cascades and
to multiple external signals, CREB is well positioned to
participate in intricate nuclear computations.15 Ser133
of CREB is a convergence point for phosphorylation
by many kinase-signaling cascades and is necessary for
a molecular switch that controls gene expressions. In
Fig 7. Effects of rolipram preconditioning on the temporal expression of brain-derived neurotrophic factor (BDNF) mRNA and protein posthypoxia. Compared with vehicle (DMSO), 3mg/kg rolipram preconditioning caused increased BDNF mRNA (A, C) and
protein (B, D) expression in the bilateral hippocampus and cortex 24 hours and 72 hours after hypoxia. L ⫽ left hemisphere; R ⫽
right hemisphere. DMSO ⫽ dimethylsulfoxide; GAPDH glyceraldehyde 3-phosphate dehydrogenase.
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neurons, CREB is phosphorylated under hypoxia or
oxidative stress, suggesting that activation of CREBdependent survival programs in response to stimuli
might represent a cellular form of defense.15,17–20 Differential susceptibility to hypoxia-induced neuronal
death correlates well with the ability to sustain CREB
phosphorylation. In the hippocampus, temporary
global ischemia induces CREB phosphorylation that is
transient in cornu ammonis 1 region of the hippocampus (CA1) neurons but prolonged in dentate gyrus
neurons.17,18 Remarkably, whereas CA1 neurons are
drastically depleted after the ischemic insult, dentate
gyrus neurons are substantially spared.17,20 In addition,
in an adult rat focal ischemia model, persistent CREB
phosphorylation was found in the periischemic area of
the cortex, where neurons remained viable, whereas
only a transient increase in CREB phosphorylation was
noted in the ischemic core with subsequent neuronal
death.18 These findings suggest that neuronal survival
is closely associated with persistent rather than transient CREB phosphorylation in ischemia-vulnerable regions.
In the adult rat model of mild focal ischemia, enhanced CREB phosphorylation was observed in the
hippocampal CA1 neurons that were subjected to
mild ischemia but did not show apparent histological
damage. These findings suggest that mild ischemia
was sufficient to trigger CREB phosphorylation in response to intracellular cAMP or Ca2⫹ influx.15,16,18,20,35 Our study shows that unilateral ligation of the carotid artery itself in newborn rats does
not cause significant long-term behavioral or morphological changes. Instead, this form of artery ligation
evoked rapid and robust sustained CREB phosphorylation in the ipsilateral cortex and hippocampus. Our
findings confirm that the time interval between ligation and hypoxic insult is important for the degree of
cerebral damage in the neonatal rat brain.14,34 The
interval of 1 to 2 hours was established in the classical
Levine rat pup model.10 Although rapid CREB phosphorylation could be elicited 1 to 3 hours after ligation, it afforded no neuroprotection to hypoxic injury
during this interval. In contrast, we showed significant neuroprotection at 6 hours and complete neuroprotection at 24 hours. The degree of neuroprotection induced by 24-hour ligation correlated with the
duration of CREB phosphorylation. These findings
strongly suggest that the signaling pathways leading
to sustained CREB phosphorylation and CREBmediated downstream gene expression constitute an
important neuroprotection mechanism against HI in
the neonatal brain.
Our study also demonstrated that the duration of
CREB phosphorylation determined the degree of neuroprotection posthypoxia. Marked and persistent
pCREB expression occurred 24 hours after hypoxia in
the 24-hour ligation group that later showed complete
neuroprotection; in contrast, pCREB was back to control levels 24 hours after hypoxia in the 1-hour ligation
group that showed obvious brain damage. Our results
are consistent with study findings that CREB phosphorylation may not always be sufficient for transcription activation, and that the duration of CREB phosphorylation after stimulation is one key determinant of
whether a stimulus can activate CREB-mediated neuroprotective gene transcription.15,17–19,35,36
Because reduction of CREB expression during HI
potentially could aggravate the degree of brain damage, the duration of decreased CREB by antisense
ODN infusions for the 24-hour ligation group was
limited to 6 hours before hypoxia. This might explain
why the neuroprotection induced by 24-hour ligation
preconditioning is not totally eliminated by blocking
CREB. We found that, in the 24-hour ligation group,
neuroprotection was significantly reduced in the antisense ODN-infused rats compared to the scrambled
ODN-infused rats, and that antisense ODN infusion
alone in the P6 rat pups did not cause significant
hemispheric weight reduction compared with scrambled ODN infusions. The latter strongly argues
against the short course of antisense ODN initiating a
sequence-specific cytotoxic effect in the developing
brain. Taken together, these findings provide evidence that CREB is potentially required for the preconditioning effect induced in the 24-hour ligation
group.
Studies have shown that cAMP-mediated signal
transduction is closely associated with neuronal survival
in acute cerebral ischemia.18 Our finding that forskolin
injection 24 hours before HI induced significant neuroprotection by persistently increasing CREB phosphorylation suggests that activation of cAMP-CREB
signaling is an important neuroprotective cascade in
the immature brain. Furthermore, our outcome study
showed that a single 3mg/kg rolipram injection 24
hours before hypoxia provides profound neuroprotection at both morphological and behavioral levels. In
addition, compared with the vehicle-pretreated rats,
rolipram-preconditioned rats showed higher and sustained pCREB expression and its downstream gene
(BDNF mRNA) and protein, in the hippocampus and
cortex 24 and 72 hours after hypoxia. In the central
nervous system, BDNF plays an important role in regulating the survival and differentiation of neurons during development.15–17 BDNF is also markedly neuroprotective against neonatal HI injury.27 The BDNF
gene is a CREB family target whose protein product
functions at synapses to control adaptive neuronal responses. Taken together, these findings strongly suggest
that preconditioning by activating the cAMP-CREB
signaling pathway is sufficient to provide neuroprotection in vivo against HI in the immature brain. Further
Lee et al: pCREB in Brain Preconditioning
621
study will be needed to determine whether the cAMPCREB signaling pathway is neuroprotective when activated after hypoxia.
Acquiring tolerance to HI through pharmacological
preconditioning, we believe, has clinical implications
in perinatal medicine. Drug treatment that accelerates
the induction of neonatal hypoxic tolerance in highrisk pregnancy may be a potential procedure, but it
should be strong enough to show robust protection
without long-term side effects. We found that 24hour ligation preconditioning induced sustained
CREB phosphorylation, completely protected the
neonatal rat brain against HI, and concurrently preserved brain function. Furthermore, preconditioning
by pharmacologically activating cAMP-CREB signaling cascades with rolipram also provided marked neuroprotection at pathological and behavioral levels.
This study suggests that CREB activation in vivo is
an important event in neuroprotection against HI injury in the immature brain. More important, drugs
that mimic the beneficial effects of carotid-artery ligation preconditioning by activating cAMP-CREB
signaling cascades potentially would provide novel
therapeutic approaches for the treatment of HI brain
injury in high-risk newborns.
This study was supported by grants from the Taiwan National Science Counsel (NSC: 92-2314-B-006-068, 92-2314-B-006-069,
C.C.H.), National Health Research Institute (NHRI-EX929131NN, C.C.H.), Chi-Mei Medical Research Foundation
(L.Y.W.), and National Cheng Kung University Hospital (90-05,
C.C.H.).
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