Erythropoietin Improved Neurologic Outcomes in Newborns With

Hypoxic-Ischemic Encephalopathy
Changlian Zhu, Wenqing Kang, Falin Xu, Xiuyong Cheng, Zhan Zhang, Liting Jia,
Ling Ji, Xiaoyan Guo, Hong Xiong, George Simbruner, Klas Blomgren and Xiaoyang
Wang
Pediatrics 2009;124;e218; originally published online July 27, 2009;
DOI: 10.1542/peds.2008-3553

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located on the World Wide Web at:
http://pediatrics.aappublications.org/content/124/2/e218.full.html

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Erythropoietin Improved Neurologic Outcomes in
Newborns With Hypoxic-Ischemic Encephalopathy
WHAT’S KNOWN ON THIS SUBJECT: Erythropoietin has been
shown to be neuroprotective after hypoxic-ischemic brain injury
in animal models, but there is no report on newborn infants with
HIE.
WHAT THIS STUDY ADDS: This prospective study found that
systemic administration of recombinant human erythropoietin
resulted in improved neurologic outcomes after moderate HIE, as
evaluated at up to 18 months of age.
abstract
OBJECTIVE: The purpose of this study was to evaluate the efficacy and
safety of erythropoietin in neonatal hypoxic-ischemic encephalopathy
(HIE), by using a randomized, prospective study design.
METHODS: A total of 167 term infants with moderate/severe HIE were
assigned randomly to receive either erythropoietin (N ⫽ 83) or con-
ventional treatment (N ⫽ 84). Recombinant human erythropoietin, at
either 300 U/kg (N ⫽ 52) or 500 U/kg (N ⫽ 31), was administered every
other day for 2 weeks, starting ⬍48 hours after birth. The primary
outcome was death or disability. Neurodevelopmental outcomes were
assessed at 18 months of age.
RESULTS: Complete outcome data were available for 153 infants. Nine
patients dropped out during treatment, and 5 patients were lost to
follow-up monitoring. Death or moderate/severe disability occurred
for 35 (43.8%) of 80 infants in the control group and 18 (24.6%) of 73
infants in the erythropoietin group (P ⫽ .017) at 18 months. The pri-
mary outcomes were not different between the 2 erythropoietin doses.
Subgroup analyses indicated that erythropoietin improved long-term
outcomes only for infants with moderate HIE (P ⫽ .001) and not those
with severe HIE (P ⫽ .227). No negative hematopoietic side effects were
observed.
CONCLUSION: Repeated, low-dose, recombinant human erythropoietin
treatment reduced the risk of disability for infants with moderate HIE,
without apparent side effects. Pediatrics 2009;124:e218–e226
e218 ZHU et al
Downloaded from pediatrics.aappublications.org by guest on June 17, 2013
CONTRIBUTORS: Changlian Zhu, MD, PhD,a,b Wenqing Kang, MD,c
Falin Xu, MD, PhD,a,b Xiuyong Cheng, MD,a,b Zhan Zhang, MD,
PhD,b,d Liting Jia, MD,b,d Ling Ji, MD,a Xiaoyan Guo, MD,a Hong
Xiong, MD,c George Simbruner, MD,e Klas Blomgren, MD, PhD,f,g
and Xiaoyang Wang, MD, PhDa,b
Departments of aPediatrics and dLaboratory Medicine, Third
Affiliated Hospital of Zhengzhou University, Zhengzhou, China;
bHenan Key Laboratory for Neonatal Brain Injury, Zhengzhou,
China; cDepartment of Pediatrics, Zhengzhou Children’s Hospital,
Zhengzhou, China; eDepartment of Pediatrics IV Medical
University Innsbruck, Innsbruck, Austria; fCenter for Brain
Repair and Rehabilitation, Institute of Neuroscience and
Physiology, University of Gothenburg, Gothenburg, Sweden; and
gDepartment of Pediatric Oncology, Queen Silvia Children’s
Hospital, Gothenburg, Sweden
KEY WORDS
asphyxia, erythropoietin, hypoxic-ischemic encephalopathy,
neonates
ABBREVIATIONS
HIE— hypoxic-ischemic encephalopathy
BBB— blood-brain barrier
MDI—Mental Developmental Index
CSF— cerebrospinal fluid
This trial has been registered at www.clinicaltrials.gov
(identifier NCT008080704).
www.pediatrics.org/cgi/doi/10.1542/peds.2008-3553
doi:10.1542/peds.2008-3553
Accepted for publication Mar 17, 2009
Address correspondence to Changlian Zhu, MD, PhD, Department
of Pediatrics, Third Affiliated Hospital of Zhengzhou University,
Kangfuqain St 7, Zhengzhou 450052, China. E-mail:
zhuc@zzu.edu.cn
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright © 2009 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have
no financial relationships relevant to this article to disclose.

Perinatal asphyxia-induced brain in-
jury is one of the most common causes
of death and long-term neurologic im-
pairments (cerebral palsy, mental re-
tardation, learning disability, and epi-
lepsy) in term and preterm neonates.1
The current treatment for hypoxic-
ischemic encephalopathy (HIE) is pre-
dominantly supportive, to maintain
physiologic parameters. Multicenter
trials assessing the effects of hypo-
thermia demonstrated improved out-
comes for term neonates with moder-
ate HIE if the infants underwent cooling
within 6 hours.2,3 Hypothermia is not
widely used in China, because efficacy
is uncertain if treatment is delayed ⬎6
hours.4 The safety and efficacy of hypo-
thermia for preterm infants need to be
proven and such treatment may not be
suitable for this group,5 leaving a sub-
stantial proportion of patients with as-
phyxia without effective treatment.
There is a need for effective treat-
ments that can be implemented even
⬎6 hours after the injurious event.
Erythropoietin was originally identi-
fied on the basis of its role in erythro-
poiesis. Clinical trials demonstrated
the safety and efficacy of recombinant
human erythropoietin in the preven-
tion and treatment of anemia of pre-
maturity.6,7 In addition, erythropoietin
may mediate an adaptive tissue re-
sponse to stress and may mediate tis-
sue protection.8 Erythropoietin and its
receptors were upregulated after
brain injury, and the levels of erythro-
poietin in cerebrospinal fluid (CSF)
were correlated positively with out-
comes.9 Systemically administered
erythropoietin was neuroprotective in
neonatal brain injury models.10–13 To
date, there are no reports evaluating
possible effects of erythropoietin on
neonatal HIE. A clinical study using re-
combinant human erythropoietin to
prevent anemia of prematurity found
that elevated serum erythropoietin
concentrations were correlated with
PEDIATRICS Volume 124, Number 2, August 2009
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higher Mental Developmental Index
(MDI) scores.14 Furthermore, a placebo-
controlled trial in adult patients after
stroke, using a dose of 33 000 U per
injection (450 U/kg of body weight),
showed beneficial results.15 However,
erythropoietin doses used in animal
neuroprotection studies (1000 –30 000
U/kg) are invariably much higher than
the doses for anemia treatment (250 –
500 U/kg) and are not considered suit-
able for human neonates because of
potential side effects.16 Because of the
lack of reports on the safety of higher
doses of erythropoietin in neonates, a
low-dose regimen was chosen. The aim
of this study was to investigate
whether systemic administration of
low doses (300 –500 U/kg) of recombi-
nant human erythropoietin could im-
prove neurodevelopmental outcomes
at 18 months for infants with moderate
or severe HIE.
METHODS
Patient Population
Between August 2003 and January
2007, infants who were born at ⬎37
weeks of gestation, with body weights
of ⬎2500 g, and were admitted to the
NICU with clinical evidence of perinatal
HIE were screened for eligibility (Fig 1).
The sample size was determined on
the basis of the following assumptions:
60% of the patients would suffer from
moderate asphyxia and 40% from se-
vere asphyxia, and 60% of the patients
with moderate asphyxia and 90% with
severe asphyxia in the control group
would be dead or disabled after 18
months. If the relative risk would be
reduced 40% in the moderate asphyxia
group and 10% in the severe asphyxia
group with erythropoietin treatment,
then 65 patients would need to be re-
cruited in each group for a signifi-
cance level of 5%, with 80% power and
20% lost to follow-up monitoring. The
inclusion criteria were Apgar scores of
ⱕ5 at 5 minutes after birth or contin-
ARTICLES
ued need for resuscitation (including
endotracheal or mask ventilation) at
10 minutes after birth. The severity of
encephalopathy (moderate or severe)
was assessed by certified examiners
according to the criteria described by
Sarnat and Sarnat,17 consisting of al-
tered state of consciousness (leth-
argy, stupor, or coma) and ⱖ1 of hypo-
tonia, abnormal reflexes (including
oculomotor or pupillary abnormali-
ties), absent or weak sucking, or clini-
cal seizures. Exclusion criteria were
major congenital abnormalities, head
trauma or skull fracture causing intra-
cranial hemorrhage, body tempera-
ture of ⬍34°C, financial problems of
the parents, lack of permanent ad-
dress, and postnatal age of ⬎48 hours.
Written informed consent was ob-
tained from ⱖ1 of the parents for all
patients assigned randomly to eryth-
ropoietin treatment. This study was
conducted in 2 neonatal centers in
Zhengzhou, China, and was approved
by the local ethics committee in accor-
dance with the Helsinki Declaration.
Study Design and Treatment
Procedures
For patients fulfilling the inclusion cri-
teria, complete obstetric history was
obtained. The severity of HIE was deter-
mined as described above, and then
patients were assigned randomly to
either erythropoietin or conventional
treatment. Randomization was per-
formed separately in the 2 hospitals,
according to the severity of HIE. In the
first phase, we used 300 U/kg. Because
we did not observe any obvious side
effects, we increased the dose to 500
U/kg for the second phase, speculating
that a higher dose would be more ef-
fective. In the erythropoietin treatment
group, patients were given recombi-
nant human erythropoietin at either
300 U/kg or 500 U/kg, administered
subcutaneously the first time and then
intravenously every other day for 2
weeks. The time to the first erythropoi-
e219
 285 infants
reviewed
176 recrutied
167 enrolled
84 controls 52 300 U of EPO
2 dropped out 5 dropped out
2 lost to follow-up 2 lost to follow-up
80 completed 45 completed
FIGURE 1
Erythropoietin (EPO) trial profile. The schematic flowchart describes the recruitment, random assign-
ment, and follow-up evaluation of the patients. Financial problems means that the parents could not
afford the treatment cost; no permanent address, the parents had no permanent job and likely would
move within 1 year; dropped out, the parents requested that the patient be withdrawn from the study;
lost to follow-up, contact with the family was lost during the follow-up period.
etin injection was 1 to 48 hours after
birth (median: 20 hours for 300 U/kg
and 24 hours for 500 U/kg). Each intra-
venous injection was completed in ⬍2
minutes. The supportive care and
other treatments were identical be-
tween the groups. Additional clinical
variables assessed before and 2
weeks after treatment included liver
and renal function, blood hemoglobin
level, hematocrit level, platelet count,
and reticular red blood cell count.
Outcome Measures
Neurologic signs were assessed and
scored, according to the method de-
scribed by Thompson et al,18 every day
for 7 days, because the evaluation on day
7 is a useful time point with regard to
prognosis.19 The scoring consists of clin-
ical assessment of 9 neurologic signs,
with scores from 0 to 3 and a maximal
score of 22. The higher the score is, the
more the affected infant is at risk of neu-
ropsychomotor disabilities.
The 20-item neonatal behavioral neuro-
logic assessment was performed at
70 mild HIE
18 financial problems
10 no permanent address
6 brain hemorrhage
5 no consent
9 consent cancelled
31 500 U of EPO
2 dropped out
1 lost to follow-up
28 completed
postnatal days 14 and 28.20 Each item is
scored as 0, 1 or 2, and the maximal
score is 40 for normal term infants. The
investigators performing the neonatal
behavioral neurologic assessment un-
derwent the same training program, to
ensure strong interobserver agreement.
All surviving infants were evaluated
regularly every 6 months through
gross neurologic assessment and MDI
testing, with a final examination at 18
months. Assessment of neuromotor
disability was based on the presence
of cerebral palsy and functional dis-
ability, graded according to the 5-level
classification described by Palisano
et al.21 Psychological development was
evaluated by using the Bayley Scales of
Infant Development, Second Edition.22
Criteria for moderate/severe disability
included cerebral palsy, severe hear-
ing loss, blindness, gross motor func-
tion classification levels 3 through 5,
and MDI of ⬍70.
In this study, the doctors and nurses
responsible for treatment were not
blinded but the investigators perform-
ing the short- and long-term outcome
assessments were blinded to the pa-
tients’ group allocation. The final eval-
uation at 18 months of age was per-
formed by doctors from another
department, who were blinded to the
treatment protocol and were not al-
lowed to inquire about the treatment
history from the parents.
Erythropoietin Radioimmunoassay
Samples of blood and lumbar CSF were
collected before (n ⫽ 10) and 3 hours
(n ⫽ 10), 8 hours (n ⫽ 8), or 24 hours
(n ⫽ 7) after subcutaneous adminis-
tration of a single dose of 500 U/kg
recombinant human erythropoietin,
from different patients. Lumbar CSF
samples from untreated healthy new-
borns were not available, for ethical
reasons. The samples were centri-
fuged and the supernatants were fro-
zen at ⫺20°C. CSF samples with blood
contamination (visible or micro-
scopic) were excluded. Erythropoietin
was detected by using a radioimmuno-
assay, according to the manufactur-
er’s protocol (R-20; East Asia Immuno-
technology Institute, Beijing, China).
Statistical Analyses
The outcome differences were ana-
lyzed with Fisher’s exact tests for cate-
gorical variables, and P values are
2-sided. Analysis of variance with Bon-
ferroni correction was used when ⬎2
groups were compared. Data were ex-
pressed as mean ⫾ SD or median and
range, and P values of ⬍.05 were con-
sidered statistically significant.
RESULTS
Erythropoietin Levels in Serum and
CSF
Endogenous erythropoietin was de-
tected in both serum and CSF from the
patients with HIE. The concentration in
serum was 4.6 times higher than that
in CSF (Fig 2). After subcutaneous ad-
ministration, the erythropoietin level

e220 ZHU et al
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 ARTICLES

TABLE 1 Baseline Characteristics

A 400
Control Erythropoietin, Erythropoietin,
EPO in serum, mU/mL

b (N ⫽ 82) 300 U (N ⫽ 47) 500 U (N ⫽ 29)

300
Gestational age, mean ⫾ SD, wk 39.6 ⫾ 1.6 39.6 ⫾ 1.4 40.0 ⫾ 1.4
Birth weight, mean ⫾ SD, g 3274 ⫾ 478 3310 ⫾ 450 3362 ⫾ 472
200 Gender, n
Female 24 9 8
100 Male 58 38 21
Apgar score at 5 min, mean ⫾ SD 6.8 ⫾ 1.5 6.9 ⫾ 1.6 5.0 ⫾ 3.0
Age at treatment, median (range), h 23 (2–48) 20 (1–48) 24 (1–48)
0 Blood gas values at enrollment
0h 3h 8h 24 h pH, mean ⫾ SD 7.30 ⫾ 0.15 7.33 ⫾ 0.10 7.32 ⫾ 0.12
PCO2, mean ⫾ SD, mm Hg 36.7 ⫾ 12.2 33.9 ⫾ 11.9 36.7 ⫾ 14.0
B 40 Base deficit, mean ⫾ SD, mmol/L ⫺7.7 ⫾ 4.9 ⫺7.8 ⫾ 5.3 ⫺6.9 ⫾ 5.6
Sarnat grade, n
II 61 27 20
a
EPO in CSF, mU/mL

30 III 21 20 9

20
were allocated to the 300 U/kg erythro-
10 poietin subgroup and 29 to the 500
U/kg erythropoietin subgroup. The
0 baseline characteristics of the pa-
0h 3h 8h 24 h tients in the control and recombinant
FIGURE 2 human erythropoietin groups were
Erythropoietin (EPO) concentrations in serum
(A) and CSF (B) after subcutaneous administra- not significantly different (Table 1).
tion. Erythropoietin levels were measured by us- Neurologic improvement attributable
ing a radioimmunoassay. Erythropoietin levels to erythropoietin was observed at
in serum increased and reached a peak 3 hours
after the subcutaneous injection, significantly postnatal day 7, with erythropoietin-
higher than the level before the erythropoietin in- treated patients having significantly
jection. The pattern of erythropoietin level lower Thompson scores, compared
changes in CSF was similar to that in serum. a P
⬍ .05. b P ⬍ .01 with control subjects (Table 2). Persis-
tent beneficial effects of erythropoie-
tin treatment were demonstrated by
in serum increased threefold (P ⫽ higher neonatal behavioral neurologic
.0046), reached a peak 3 hours after assessment scores at 14 and 28 days
administration, and then decreased of age (Table 3).
gradually 8 and 24 hours after admin-
Outcomes at 18 Months
istration (Fig 2A). The changes in CSF
were similar to those in serum. The
Data on outcomes at 18 months were
available for 153 (96.8%) of the 158 pa-
peak CSF level was 23.75 mU/mL
tients. Two patients in the control
(range: 13.75– 43.75 mU/mL) at 3 hours
after injection, which was significantly
group and 3 in the erythropoietin
group (2 in the 300 U/kg erythropoietin
higher than the level before erythro-
poietin injection (Fig 2B).
TABLE 2 Thompson Neurologic Assessment
Baseline Characteristics and Results
Follow-up Data Thompson Score, Mean ⫾ SD
A total of 167 patients were enrolled in Control Erythropoietin, Erythropoietin,
this study; 9 of them dropped out dur- (N ⫽ 82) 300 U (N ⫽ 47) 500 U (N ⫽ 29)
ing treatment, at the parents’ request. 1 d 10.4 ⫾ 2.4 9.7 ⫾ 3.1 10.9 ⫾ 1.9
3 d 8.7 ⫾ 3.4 7.1 ⫾ 3.3 8.6 ⫾ 3.1
Of the remaining 158 patients, 82 were 7 d 3.8 ⫾ 2.7 2.4 ⫾ 2.1a 2.4 ⫾ 1.6a
assigned to the control group and 76 to aP ⬍ .05, compared with control (N ⫽ 81 in the control
the erythropoietin group, 47 of whom group at 7 days).
subgroup and 1 in the 500 U/kg eryth-
ropoietin subgroup) were lost to
follow-up monitoring. Four patients in
the control group died, at 4, 18, and 25
days and 2 months of age. Two of those
patients had severe HIE and 2 moder-
ate HIE. Three patients in the erythro-
poietin group died, at 7 days, 14 days,
and 3 months of age, and all of those
patients had severe HIE (Table 4).
Death was related directly to either
severe brain injury or infection. Death
or moderate/severe disability was
present for 35 (43.8%) of 80 infants in
the control group and 18 (24.6%) of 73
infants in the erythropoietin group
(P ⫽ .017). The rates of disability were
40.8% in the control group and 21.4%
in the erythropoietin group (P ⫽ .013).
The proportions of subjects with MDI
scores of ⬍70 were 17 (22.4%) of 76
infants in the control group and 7
(10%) of 70 infants in the erythropoie-
tin group (P ⫽ .048). The proportions
with cerebral palsy were 14 (18.4%) of
TABLE 3 Neonatal Behavioral Neurologic
Assessment Results
Assessment Score, Mean ⫾ SD
Control Erythropoietin, Erythropoietin,
(N ⫽ 81) 300 U (N ⫽ 46) 500 U (N ⫽ 28)
14 d 35.0 ⫾ 2.1 37.5 ⫾ 1.6a 38.3 ⫾ 1.0a
28 d 37.1 ⫾ 1.9 38.7 ⫾ 1.2a 39.3 ⫾ 0.6a
aP ⬍ .001, compared with control (N ⫽ 79 in the control
group and N ⫽ 45 in the 300-U erythropoietin group at 28
days).

PEDIATRICS Volume 124, Number 2, August 2009 e221

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TABLE 4 Outcomes at 18 Months of Age for Erythropoietin and Control Groups 76 infants in the control group and 5
n/N (%) Relative Risk P (6.8%) of 70 infants in the erythropoie-
Erythropoietin Control
(95% Confidence Interval) tin group (P ⫽ .051). Benefit from
(N ⫽ 73) (N ⫽ 80) erythropoietin treatment, as judged on
Primary outcome the basis of death and disability, was
Death 3/73 (4.1) 4/80 (5.0) 0.89 (0.37–2.13) ⬎.999
Disability 15/70 (21.4) 31/76 (40.8) 0.59 (0.38–0.93) .013
seen with moderate HIE (P ⫽ .001) but
Secondary outcome not severe HIE (P ⫽ .227) (Table 4). The
Death or disability 18/73 (24.6) 35/80 (43.8) 0.62 (0.41–0.94) .017 final analysis also revealed that overall
Moderate HIE 3/47 (6.4) 19/59 (32.2) 0.26 (0.09–0.76) .001
Severe HIE 15/26 (57.7) 16/21 (76.2) 0.70 (0.43–1.15) .227
disability was significantly reduced af-
Male 17/57 (29.8) 25/55 (45.5) 0.71 (0.47–1.08) .118 ter erythropoietin treatment for girls
Female 1/16 (6.3) 10/25 (40.0) 0.18 (0.03–1.22) .029 (P ⫽ .029) but not for boys (P ⫽ .118).
MDI of ⬍70 7/70 (10.0) 17/76 (22.4) 0.56 (0.30–1.08) .048
The primary outcomes at 18 months of
Moderate HIE 2/47 (4.3) 9/57 (15.8) 0.38 (0.11–1.34) .106
Severe HIE 5/23 (21.7) 8/19 (42.1) 0.62 (0.29–1.30) .193 age did not show any significant differ-
Cerebral palsy 5/70 (6.8) 14/76 (18.4) 0.51 (0.23–1.11) .051 ences between the 2 erythropoietin
Moderate HIE 1/47 (2.1) 8/57 (14.0) 0.23 (0.04–1.47) .039 dose groups (Table 5).
Severe HIE 4/23 (17.4) 6/19 (31.6) 0.67 (0.30–1.52) .468

Safety
TABLE 5 Outcomes at 18 Months of Age for Erythropoietin Subgroups Erythropoietin was well tolerated, and
n/N (%) Relative Risk P neither allergic reactions nor venous
(95% Confidence Interval) thromboses were observed with this
Erythropoietin, Erythropoietin,
300 U (N ⫽ 45) 500 U (N ⫽ 28) treatment protocol. Liver and renal
Primary outcome functions, as well as electrolyte levels,
Death 2/45 (4.4) 1/28 (3.6) 1.09 (0.48–2.47) ⬎.999 were not different between the eryth-
Disability 9/43 (20.1) 6/27 (22.2) 0.97 (0.61–1.54) ⬎.999
Secondary outcome ropoietin and control groups. Hemo-
Death or disability 11/45 (24.4) 7/28 (25.0) 0.99 (0.65–1.51) ⬎.999 globin and reticulocyte levels de-
Moderate HIE 2/27 (7.4) 1/20 (5.0) 1.17 (0.51–2.72) ⬎.999 creased significantly at 2 weeks after
Severe HIE 9/18 (50.0) 6/8 (75.0) 0.73 (0.45–1.21) .395
MDI of ⬍70 4/43 (9.3) 3/27 (11.1) 0.92 (0.47–1.80) ⬎.999 birth in the control group, but this de-
Moderate HIE 1/27 (3.7) 1/20 (5.0) 0.87 (0.21–3.54) ⬎.999 crease was prevented in the erythro-
Severe HIE 3/16 (18.8) 2/7 (28.6) 0.83 (0.38–1.80) .621 poietin groups (Table 6).
Cerebral palsy 3/43 (7.0) 2/27 (7.4) 0.98 (0.46–2.05) ⬎.999
Moderate HIE 1/27 (3.7) 0/20 (0) 1.77 (1.37–2.28) ⬎.999
Severe HIE 2/16 (12.5) 2/7 (28.6) 0.68 (0.25–1.88) .557 DISCUSSION
The results of our investigation dem-
onstrated that administration of re-
combinant human erythropoietin at a
TABLE 6 Blood Analysis Values Before and After Erythropoietin Treatment dose of 300 or 500 U/kg every other day
Control Erythropoietin, Erythropoietin, for 2 weeks was safe and resulted in
(N ⫽ 81) 300 U (N ⫽ 46) 500 U (N ⫽ 28) improved neurologic outcomes, as as-
Before treatment sessed at 18 months of age, for pa-
Hemoglobin level, mean ⫾ SD, g/L 197 ⫾ 28 193 ⫾ 26 190 ⫾ 30 tients with moderate but not severe
RBC count, mean ⫾ SD, 109 cells per L 6.0 ⫾ 0.9 5.8 ⫾ 0.7 5.5 ⫾ 0.8
Hematocrit level, mean ⫾ SD, % 58 ⫾ 10 56 ⫾ 14 53 ⫾ 9 HIE. This study raises the following
Reticulocyte level, mean ⫾ SD, % 3.7 ⫾ 1.0 3.6 ⫾ 1.1 3.5 ⫾ 0.5 questions. (1) Can the patient popula-
Platelet level, mean ⫾ SD, 109 cells per L 219 ⫾ 90 203 ⫾ 65 181 ⫾ 51 tion in this study be compared readily
After treatment
Hemoglobin level, mean ⫾ SD, g/L 169 ⫾ 26a 174 ⫾ 22 180 ⫾ 23
with those in the hypothermia trials?
RBC count, mean ⫾ SD, 109 cells per L 5.1 ⫾ 0.8 5.2 ⫾ 0.6 5.4 ⫾ 0.6 (2) What are the pharmacokinetic
Hematocrit level, mean ⫾ SD, % 50 ⫾ 8 55 ⫾ 8 55 ⫾ 5 characteristics of erythropoietin, in-
Reticulocyte level, mean ⫾ SD, % 2.7 ⫾ 0.9b 3.7 ⫾ 0.6c 3.6 ⫾ 0.5d
Platelet level, mean ⫾ SD, 109 cells per L 236 ⫾ 86 221 ⫾ 69 213 ⫾ 54
cluding issues of dosage and timing,
RBC indicates red blood cell. and does administered erythropoietin
a P ⬍ .001, compared with before treatment.
cross the blood-brain barrier (BBB)?
b P ⬍ .01, compared with before treatment.

c P ⬍ .001, compared with control. (3) How do the effectiveness, side ef-
d P ⬍ .01, compared with control. fects, and potential of erythropoietin

e222 ZHU et al
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therapy compare with those of in-
duced hypothermia?
Death or moderate/severe disability
was present for 35 (43.8%) of 80 in-
fants in the control group and 18
(24.6%) of 73 infants in the erythropoi-
etin group (P ⫽ .017). The death and
disability rate for the control group
was lower than that in the hypother-
mia study (66%),2 probably because of
the small proportion of severe HIE in
our study (26.25%, compared with
37.7%).2 Consequently, these results
demonstrated a distinctly beneficial
effect on neurodevelopmental out-
comes even when erythropoietin was
administered up to 48 hours after
birth, at a dosage similar to those used
for treatment for anemia. Erythropoie-
tin had no effect in reducing the mor-
tality rate, but the rate of disability was
reduced from 40.8% to 21.4% (P ⫽
.013). The overall effect of erythropoie-
tin was to halve the incidence of death
or disability. However, a statistically
significant effect was demonstrated
only for infants with moderate HIE. This
is analogous to the results noted after
head cooling, which reduced signifi-
cantly the rate of disability in infants
with less-severe amplitude-integrated
electroencephalographic changes.3 The
rate of disability after treatment with
erythropoietin was similar to that seen
after induced hypothermia,2,3,23 with
reduction in the rate of disability
among survivors from 46% to 40% in
the control group compared to 31% to
27% in the hypothermia group. We re-
port a similar or greater reduction in
the rate of disability, from 40.8% to
21.4%, for infants treated with eryth-
ropoietin (Table 4). In reducing the
proportion of infants with MDI
scores of ⬍70, erythropoietin had an
effect similar to that of induced hy-
pothermia, reducing the proportion
from 22.4% in the control group to
10%.
Compared with induced hypothermia,
PEDIATRICS Volume 124, Number 2, August 2009
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erythropoietin treatment is technically
much simpler to perform and has
fewer side effects. Another major ad-
vantage of the erythropoietin treat-
ment is that it seems to be effective
even when started ⬎6 hours but ⬍48
hours after birth, which indicates, at
least in part, different mechanisms of
action. Erythropoietin treatment, as
proposed in our study, should be con-
sidered an alternative to or a treat-
ment to be combined with hypother-
mia. Important questions concern
dosage and timing, erythropoietin
transfer across the BBB, and the mech-
anisms of action. Reported neuropro-
tective doses of recombinant human
erythropoietin in animal models range
from 1000 to 30 000 U/kg.10,12,24–26 In
adult stroke patients, a dose of 33 000
U/day (⬃470 U/kg) for 3 consecutive
days resulted in improved outcomes at
30 days.15 However, a phase II study ad-
ministering 40 000 U/day intrave-
nously for 3 days to adult stroke pa-
tients was stopped because of an
increased mortality rate, which was
attributed at least in part to intracra-
nial hemorrhage.27 In the current
study, we chose a dose that was in the
range (per kilogram of body weight) of
the approved doses for anemia treat-
ment and close to that used for treat-
ing adult stroke patients. Repeated ad-
ministration of erythropoietin, at both
doses, every other day for 2 weeks re-
sulted in improved neurologic out-
comes for newborn infants with HIE.
The 2 doses tested (300 and 500 U/kg)
had indistinguishable effects and were
in the same range as those used for
anemia treatment (250 –500 U/kg).
However, further investigations are re-
quired to elucidate the dose-response
relationship.
Erythropoietin is larger than the mo-
lecular weight threshold for lipid-
mediated transport across the BBB.
Whether it is able to cross the BBB is an
important issue for any systemically
ARTICLES
administered neuroprotective agent.
Animal and human studies with high-
dose erythropoietin treatment sug-
gested that recombinant human eryth-
ropoietin can enter the brain either
through a receptor-facilitated pro-
cess8 or in a manner dependent on
time, dose, and peak serum concentra-
tion.28,29 Endogenous erythropoietin
levels are known to increase after
hypoxic-ischemic insults,30 and the in-
creased erythropoietin concentra-
tions measured after HIE reflect the
combined contributions of endoge-
nous and exogenous erythropoietin.
However, judging from the time course
and from published data, we can safely
infer that the increases are at least
partly related to erythropoietin admin-
istration. First, the time course of
erythropoietin concentrations in CSF
was mirrored by that in serum. Sec-
ond, erythropoietin concentrations in
serum and CSF in one study were re-
ported to be increased after HIE, com-
pared with control values, but levels
peaked ⬃3 days after asphyxia, with
the erythropoietin concentrations in
serum being twice as high as those in
CSF.31 In contrast, in our investigation
the erythropoietin concentration
peaked 3 hours after erythropoietin
administration and reached 5- to 10-
fold higher levels than before treat-
ment. The erythropoietin concentra-
tion in blood was lower than that in a
report on premature newborns,
showing a peak concentration of 740
mU/mL 4 to 12 hours after adminis-
tration.32 Because we did not assess
erythropoietin concentrations be-
tween 3 and 8 hours after erythropoi-
etin administration, the peak concen-
tration might have occurred between
those time points. Investigations with
labeled erythropoietin would be neces-
sary to elucidate the pharmacokinetic
features and relative contributions of
exogenous and endogenous erythro-
poietin. In this study, we noted peak
erythropoietin concentrations in CSF
e223
ranging from 13.75 to 43.75 mU/mL, 3
hours after subcutaneous administra-
tion, similar to or higher than the con-
centration of 15 mU/mL in a human
stroke trial.15 The peak concentrations
of erythropoietin in CSF were reported
to occur 8 to 24 hours after adminis-
tration for adults and 4 to 12 hours
after administration for preterm in-
fants.29,32,33 These differences may be
attributable to differences in brain de-
velopmental stages,34 the integrity of
the BBB, and/or the administration
route and dose.32,35 However, optimal
dosage regimens remain to be deter-
mined for newborn infants treated for
HIE.
Erythropoietin at a dose of 5000 U/kg
has been shown to prevent neuronal
apoptosis,36 to promote neurogen-
esis,26 to increase cell proliferation,37
and to reduce the inflammatory re-
sponse in the brain.38 Apoptotic cell
death contributes relatively more to
ischemic injury in the immature brain
than in the adult brain,39 persists for
several weeks after hypoxia-ischemia,
and is correlated with the prognosis
after brain injury.40–42 Neurogenesis is
a slow process and may contribute
to functional recovery after brain in-
jury.43,44 These delayed processes of
degeneration and regeneration after
an insult indicate that neuroprotective
therapy for HIE should continue during
the recovery phase. Consequently, we
think that it was rational to continue
treatment for 2 weeks and that this
prolonged treatment contributed to
the neuroprotective effects observed.
We speculate that shorter or longer
treatment periods would have re-
sulted in different outcomes.
Erythropoietin has been widely used to
prevent anemia of prematurity, with-
out toxic effects even after months of
administration.6,7,45 We did not find any
obvious hematopoietic effect after 2
weeks of erythropoietin treatment,
which may be attributable in part to
frequent blood sampling or HIE induc-
ing an intrinsic response with in-
creased endogenous erythropoietin
levels, making the contribution of ex-
ogenous erythropoietin seem smaller.
However, decreased hemoglobin and
reticulocyte levels in the control
group, compared with unchanged pa-
rameters in the erythropoietin group,
indicated strongly that administered
erythropoietin did induce a hemato-
poietic response. In adult patients,
erythropoietin increased the risk of hy-
pertension and thrombosis through
enhanced platelet reactivity.16 In this
investigation, we did not find any obvi-
ous erythropoietin-related toxicity,
and the doses used (300 –500 U/kg),
with administration for a period of 2
weeks, for term infants seemed to in-
flict no short-term hazards or risks.
Because erythropoietin has been ad-
ministered to a large number of pre-
mature infants for decades, negative
side effects outweighing the positive
effects observed here seems an un-
likely scenario.
CONCLUSIONS
We found that systemic administration
of recombinant human erythropoietin
at a dose of 300 or 500 U/kg every other
day for 2 weeks was safe and resulted
in improved neurologic outcomes for
patients with moderate HIE, as as-
sessed at 18 months of age. We specu-
late that erythropoietin treatment
could be an important adjunct or alter-
native to therapeutically induced
hypothermia.
ACKNOWLEDGMENTS
This work was supported by the Medi-
cal Science Academy of Henan, a grant
from the Department of Education of
Henan Province, the National Natural
Science Foundation of China, the
Ministry of Education of China (211
Project), and the Swedish Research
Links Program through the Swedish In-
ternational Development Cooperation
Agency.

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 Erythropoietin Improved Neurologic Outcomes in Newborns With
Hypoxic-Ischemic Encephalopathy
Changlian Zhu, Wenqing Kang, Falin Xu, Xiuyong Cheng, Zhan Zhang, Liting Jia,
Ling Ji, Xiaoyan Guo, Hong Xiong, George Simbruner, Klas Blomgren and Xiaoyang
Wang
Pediatrics 2009;124;e218; originally published online July 27, 2009;
DOI: 10.1542/peds.2008-3553

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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly

publication, it has been published continuously since 1948. PEDIATRICS is owned, published,
and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk
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