Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma.
ABSTRACT Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneurysm clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.
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ABSTRACT: To evaluate the efficacy of intravenous erythropoietin (EPO) on improvement of visual function and color vision in patients with recent indirect traumatic optic neuropathy (TON).Albrecht von Graæes Archiv für Ophthalmologie 07/2014; 252(8). · 2.33 Impact Factor
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ABSTRACT: Erythropoietin (Epo) improves post-traumatic cerebral blood flow (CBF), pressure autoregulation, and vascular reactivity to l-arginine. This study examines the dependence of these cerebral hemodynamic effects of Epo on nitric oxide generated by endothelial nitric oxide synthase (eNOS).Frontiers in Immunology 10/2014; 5:494.
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ABSTRACT: Background and PurposeRecombinant human erythropoietin (rHuEPO) is currently the mainstay of renal anaemia treatment. Recently, rHuEPO has been shown to provide pleiotrophic tissue protection in various pathological conditions. However, the benefits of rHuEPO beyond anaemia treatment are limited because it increases red blood cell mass. Carbamylated erythropoietin (CEPO) is the first rHuEPO derivative that lacks erythropoietic activity but retains tissue protection properties. Since carbamylation targets lysine residues on rHuEPo, we hypothesized that targeted lysine modifications of rHuEPO may result in a novel non-erythropoietic erythropoietin. Experimental ApproachrHuEPO was subjected to various targeted lysine modifications. In vitro cytoprotection and apoptosis were evaluated using P19 and HEK293 cells. In vivo erythropoiesis was performed by administering the derivatives to animals for 2 weeks. Renoprotection was tested on an ischaemia/reperfusion (I/R) model. Key ResultsWe synthesized a novel derivative, a glutaraldehyde erythropoietin (GEPO). This construct abolished in vivo erythropoiesis. Biochemical characterization showed that GEPO was more electrostatically negative than rHuEPO. Immunoprecipitation experiments revealed that GEPO bound to the IL3RB/EPOR heterotrimeric receptor and ameliorated cellular apoptosis via the activation of Bcl-2. Notably, Bcl-2 activation was suppressed by the JAK2 inhibitor, tyrphostin AG490. In vivo experiments showed that GEPO also ameliorated kidney damage due to I/R injury both functionally and histologically. Conclusions and ImplicationsHerein, we describe a novel lysine-modified rHuEPO, glutaradehyde-EPO (GEPO), obtained from a simple reaction. This derivative has no erythropoietic properties but retains cell-protective characteristics both in vitro and in vivo, with promise for future use as an adjunctive treatment of kidney disease.British Journal of Pharmacology 01/2013; 168(1). · 5.07 Impact Factor
Recombinant human erythropoietin counteracts
secondary injury and markedly enhances
neurological recovery from experimental
spinal cord trauma
Alfredo Gorio*†, Necati Gokmen†‡, Serhat Erbayraktar§, Osman Yilmaz¶, Laura Madaschi*, Cinzia Cichetti*,
Anna Maria Di Giulio*, Enver Vardar?, Anthony Cerami**, and Michael Brines**††
*Laboratory of Pharmacology, Department of Medicine, Surgery and Odontoiatry, Faculty of Medicine, University of Milan, Milan 20142, Italy;
‡Anesthesiology and Reanimation,§Neurosurgery, and¶Animal Research Center, Dokuz Eylu ¨l University School of Medicine, Izmir 35340,
Turkey;?Department of Pathology, SSK Training Hospital, Izmir, Turkey; and **The Kenneth S. Warren Institute, Kitchawan, NY 10562
Contributed by Anthony Cerami, May 14, 2002
Erythropoietin (EPO) functions as a tissue-protective cytokine in
addition to its crucial hormonal role in red cell production. In the
brain, for example, EPO and its receptor are locally produced, are
modulated by metabolic stressors, and provide neuroprotective
and antiinflammatory functions. We have previously shown that
recombinant human EPO (rhEPO) administered within the systemic
circulation enters the brain and is neuroprotective. At present, it is
unknown whether rhEPO can also improve recovery after trau-
matic injury of the spinal cord. To evaluate whether rhEPO im-
proves functional outcome if administered after cord injury, two
rodent models were evaluated. First, a moderate compression of
0.6 N was produced by application of an aneursym clip at level T3
for 1 min. RhEPO (1,000 units per kg of body weight i.p.) admin-
istered immediately after release of compression was associated
with partial recovery of motor function within 12 h after injury,
which was nearly complete by 28 days. In contrast, saline-treated
animals exhibited only poor recovery. In the second model used,
rhEPO administration (5,000 units per kg of body weight i.p. given
once 1 h after injury) also produced a superior recovery of function
compared with saline-treated controls after a contusion of 1 N at
level T9. In this model of more severe spinal cord injury, secondary
inflammation was also markedly attenuated by rhEPO administra-
tion and associated with reduced cavitation within the cord. These
observations suggest that rhEPO provides early recovery of func-
tion, especially after spinal cord compression, as well as longer-
latency neuroprotective, antiinflammatory and antiapoptotic
requiring substantial long-term health care expenditures. Cur-
rently, methylprednisolone administered at high dose within 8 h
after injury is the only therapy with any recognized benefit (1),
which, unfortunately, is relatively minor. Any new treatment of
TSCI that allows for major recovery of function would be a
significant advance in clinical care.
Injury of the nervous system provokes a complex cascade of
proinflammatory cytokines and other molecules that ultimately
result in apoptosis and necrosis of neurons, oligodendrocytes,
and endothelial cells (2–4). Recent studies have demonstrated
that one general response of the brain to injury is the increased
local production of the erythropoietin (EPO) and its receptor (5,
6). These proteins are members of the cytokine type I super-
family that provide beneficial effects including inhibition of
apoptosis, reduction of inflammation, modulation of excitability
(7–11), and mobilization and proliferation of neuronal stem cells
(12). Prior study has shown that recombinant human EPO
(rhEPO) administered directly into the brain dramatically re-
duces hypoxic or ischemic injury and conversely, that neutral-
ization of endogenous EPO amplifies injury (8). We have
raumatic spinal cord injury (TSCI) occurs frequently and is
devastating for the individual patient and costly to society by
extended these observations by showing that systemically ad-
ministered rhEPO is not strictly excluded by the blood–brain
barrier, as predicted on size considerations, and effectively
prevents cellular injury and inflammation when given after
ischemic and mechanical trauma (11).
Although a substantial appreciation for the multiple activities
of EPO in the brain has accumulated, relatively little is known
about its potential role(s) within the spinal cord. Immunohisto-
chemical analyses of the normal spinal cord document abundant
expression of EPO and EPO receptor protein (13, 14), especially
by motor neurons and myelinated axons. We have recently used
a rabbit model to show that rhEPO acts in spinal cord ischemia
as it does within the brain, effectively rescuing neurons from
apoptosis when administered intravenously as a bolus injection
immediately after restoration of blood flow (14).
Physical injuries to the nervous system produce a secondary
inflammatory reaction that tends to expand the ultimate size of
the lesion. Much of the devastating motor and sensory paralysis
after TSCI occurs because of a delayed and widespread oligo-
dendrocyte apoptosis and demyelination of long spinal tracts
(reviewed in ref. 4) In this study, we compare the effects of
systemically administered rhEPO on spinal cord injury produced
by either application of an aneurysm clip or by contusion. We
find that even a single dose of rhEPO given 1 h after injury is
associated with early improvement of motor function, leading to
a near complete functional recovery at 28 days. In contrast, the
control animals remained severely affected throughout the
study. The dramatic difference in outcome between the two
treatment groups is largely explained by the prevention of
oligodendrocyte apoptosis and preservation of the white matter
tracts involving the site of injury.
Methods and Materials
Experimental Design. Experimental studies were designed to eval-
uate acute or subacute beneficial effects on behavioral (motor)
assessments related to the systemic administration of rhEPO in
the setting of TSCI. In the impact model, we also undertook
histopathologically analyses of the damaged spinal cord, espe-
cially within the cuneate fasciculus. Compressive injury was
produced by transient extradural application of an aneurysm clip
that produces a moderately severe injury with a distinct vascular
component and relatively little hemorrhage (15). In contrast, an
impact (concussive) model was used to mechanically disrupt
Abbreviations: EPO, erythropoietin; rhEPO, recombinant human EPO; TSCI, traumatic
spinal cord injury; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end
labeling; bw, body weight.
†A.G. and N.G. contributed equally to this work.
††To whom reprint requests should be addressed. E-mail: email@example.com.
July 9, 2002 ?
vol. 99 ?
white and gray matter as well as the microvasculature resulting
in significantly more intraparenchymal hemorrhage (16).
Aneursym Clip Model. Wistar rats (female) weighing 180–300 g
were used in this study. Animals were housed under standard
conditions in the Animal Research Laboratory at Dokuz Eylu ¨l
University. The study protocol was approved by the Animal
Research Committee of Dokuz Eylu ¨l University. Animals were
maintained in a 12-h light?dark cycle with water and food freely
The animals were fasted for 12 h before surgery, humanely
restrained and anesthetized with an i.p. injection of thiopental
sodium [40 mg/kg of body weight (bw)]. Preoperatively, imi-
penem (10 mg/kg of bw) was administered intramuscularly for
prophylaxis of infection. Rectal temperatures were maintained
at 37–38°C throughout the operative procedure by exposing the
animal to a heat lamp as needed until they completely recovered
from anesthesia. The animals were positioned in the prone
position and surgery performed under sterile conditions. After
infiltration of the skin (bupivacaine 0.25%), a complete single
level (T3) laminectomy was performed through a 2-cm incision
with the aid of a dissecting microscope. TSCI was induced by the
extradural application of a temporary aneurysm clip exerting a
0.6-N closing force on the spinal cord for 1 min. After removal
of the clip, the skin incision was closed and the animals allowed
to recover fully from anesthesia and returned to their cages. The
rats were monitored continuously with bladder palpation at least
twice daily until spontaneous voiding resumed.
Forty eight animals were randomly divided into four groups.
Animals in the sham group (n ? 6) underwent the surgical
procedure, but their spinal cords were not clipped. In a control
group, animals (n ? 14) received normal saline (via i.p. injec-
tion) immediately after the incision was closed. One of the active
treatment groups (n ? 14) received rhEPO (1,000 units/kg of bw
i.p.; Eprex, Cilag, Zug, Switzerland) immediately after the
incision was closed. A final treatment group (n ? 14) received
three sucessive daily single doses of 1000 units/kg of bw i.p.
Motor neurological function of the rats was evaluated by using
the locomotor rating scale of Basso et al. (17, 18). In this scale,
animals are assigned a score ranging from 0 (no observable
hindlimb movements) to 21 (normal gait). The rats were tested
for functional deficits at 1, 12, 24, 48, and 72 h, and then at 1, 2,
3, and 4 weeks after injury by the same examiner who was blind
to the treatment each animal had received.
Contusion Model. Adult Sprague–Dawley rats (females) weighing
240–260 g were maintained in the animal facilities under stan-
dard housing conditions (22 ? 2°C, 65% humidity, artificial
lights from 06.00–20.00 h). A standard dry diet and water were
available ad libitum. All experimental protocols were approved
by the Review Committee of the University of Milan, and met
the Italian guidelines for laboratory animals that conform to the
European Communities Directive of November 1986 (86?609?
An impaction device was developed initially at the University
of Trieste (UTS) by modification of a small materials testing unit
(see Description of UTS-Impactor and Fig. 5, which are published
The core of the UTS-Impactor is a 2.3-mm end-diameter stain-
less steel rod that is precisely driven into the spinal cord with a
specified velocity and displacement. To accomplish this, the rod
is placed at the desired height over the animal’s spinal cord,
which is immobilized in a frame, and the impact is monitored by
means of a miniaturized piezoelectric dynamometer, present
within a section of the impacting rod. The device is linked to a
computer that records and manages the data.
Fourteen animals were assigned to each experimental group.
No animal died during the 4 weeks of evaluation after TSCI.
Animals were anesthetized by inhalation of halothane, and a
laminectomy was performed at the T9 vertebral level under
aseptic conditions. With the aid of an operating microscope, the
rat was placed under the impounding piston positioned 1 mm
above the exposed cord and set for a 3-mm excursion. A force
of 1 N was applied for 1 s, followed by automatic return of the
rod. The extent of piston and spinal cord movements were
precisely recorded. The animals were administered buprenor-
phine (0.03 mg/kg) for pain control before awakening and
penicillin G (10,000 units/kg) as an antimicrobial after surgery.
After TSCI, rats were housed two per cage and had manual
bladder evacuation, if required, three times daily. No urine
infections were observed in the study.
Animals received rhEPO (epoietin ?; Ortho Biotech, Raritan,
NJ) via an i.p. injection one hour after impaction. One group
received only a single dose of 5,000 units/kg of bw administered
1 h after injury. A second group received daily injections of 5,000
units/kg of bw daily for 7 days. A fourth group received saline 1 h
after injury. Three scorers assessed independently all outcome
measures in a blinded fashion. Neurological function was eval-
uated 24 h after injury and then twice a week (17, 18), as well as
by a swimming test (19). In this assessment, a rat was placed in
the center of a round tub of water (40 cm diameter) filled to a
depth of 30 cm with a wire mesh ladder attached to the side. The
animal was rated 0 when neither hind limb was used for
swimming and climbing out, 1 when there was a partial use of the
hind limbs, 2 when both hind limbs were used normally.
Half of the animals in each treatment group were killed at 7
days for anatomical studies by use of halothane anesthesia
followed by transcardial perfusion with 10% paraformaldehyde
in isotonic phosphate buffer saline at pH 7.4. The spinal cord
encompassing the injury site was further postfixed with the same
paraformaldehyde-containing solution for 3 days, and segments
of the spinal cord were then embedded in paraffin and 8-?m
sections were cut transversely. Every twentieth section obtained
was stained with hematoxylin and eosin. Cross-sections contain-
ing the lesion epicenter and the extent of total T9 segment
cavitation were analyzed with computer-assisted image analysis
(Leica DG 100 mounted on a Zeiss microscope). The percent
cavitation was calculated as the area of cavitation divided by the
total cross-sectional area at the level of the injury.
Apoptosis of oligodendrocytes within the fasciculus cuneatus
was determined after 7 days by using the terminal deoxynucle-
otidyltransferase-mediated dUTP end labeling (TUNEL) meth-
odology using 10-?m-thick sections obtained at a distance of 2.5
mm rostrally to the center of the impact site. Briefly, sections
were deparifinized and then treated with proteinase-K (20 ?g/ml
in 10 mM Tris?Cl, pH 7.6, for 15 min at room temperature),
blocked in 3% H2O2in methanol for 10 min, permeabilized for
2 min in 0.1% Triton X-100?sodium citrate at 4°C, and treated
with TUNEL reaction mixture according to the manufacturer’s
protocol (In Situ Cell Death Detection kit, Roche Diagnostics).
Positive neurons were identified after development for 15 min in
diaminobenzidine, dehydration, and application of cover slips.
Terminal transferase was omitted as a negative control. Sections
obtained from treated and control animals were examined by
using light microscopy, and the total number of TUNEL-positive
cells was determined by an observer blinded to the treatment.
Data are expressed as the mean ? SD of at least 6 measure-
ments. Multiple group comparisons of the differences in quan-
titative measurements were made by ANOVA followed by
Dunnett’s t test. Statistical significance was accepted at P ? 0.05.
Aneurysm Clip Model. Animals receiving saline injections imme-
diately after aneurysm clip removal suffered a flaccid paraplegia
for the first 3 days after injury (mean motor score of 1.2), but
Gorio et al.
July 9, 2002 ?
vol. 99 ?
no. 14 ?
recovered some function (mean motor score of 6; slight move-
ment of one or two joints) by day 7 (Fig. 1). In contradistinction,
animals receiving either a single dose of rhEPO (1,000 units/kg
of bw) or three daily doses of rhEPO followed a superior clinical
course despite an equivalent clinical score 1 h after clip removal.
RhEPO-treated animals exhibited improvement of motor func-
tion by 12 h (mean motor scores of 4.8 and 3.8, respectively,
corresponding to movement at all three joints of the hindlimb,
P ? 0.001 compared with control). At 7 days after injury, the
rhEPO-treated animals improved greatly, which was only slightly
less for the 1,000 units dose compared with the 3,000 units dose
(mean motor scores of 11 versus 14 respectively; P ? 0.05).
Clinical recovery continued over the successive observation
times. At the termination of the study at 28 days, the final motor
scores were quite poor (10) for the saline group, but near normal
(18) for both rhEPO-treated groups (P ? 0.001). As expected,
sham-operated animals did not exhibit motor deficits (n ? 6;
score of 21; data not shown).
Contusion Model. Like the aneurysm clip model, all animals
subjected to spinal cord contusion were profoundly affected
immediately after injury, as assessed by the open field testing
(Fig. 2; mean motor score of 0 for all groups). In contrast to the
aneurysm clip model, significant recovery was not evident in any
group until the 4th day after injury. Between the 4th and 12th
postoperative day, rhEPO-treated animals exhibited a marked
entire observational period. Equivalent recovery was observed
in the groups receiving a single dose of rhEPO 1 h after injury
or multiple doses of rhEPO for 7 days. The group receiving 500
units/kg of bw recovered function to a degree intermediate
between the high-dose rhEPO and saline. Motor evaluation
obtained by the swim test provided similar results with rhEPO
(5,000 units/kg of bw ? 7 days) exhibiting some recovery by 4
days (Fig. 3). In contrast to the open field testing, however, a
dose of 500 units of rhEPO administered daily for 7 days was no
better than saline treatment.
rhEPO treatment daily for 1 week was also associated with a
significant reduction in frank damage to the spinal cord at the
lesion epicenter as assessed by the reduction of cavitation
volume by about 25% compared with control throughout the
removal of aneurysm clip in open field testing show significant improvement
earlier for animals receiving either a single (1,000 units/kg bw i.p.) or 3 doses
of rhEPO (3,000 units/kg of bw total; n ? 14 each group). (B) Neurological
that animals that received rhEPO exhibited nearly normal motor function. In
contrast, saline-treated animals regained much less function (n ? 7 each
group;*, P ? 0.05;**, P ? 0 01,***, P ? 0.001).
(A) Neurological (motor) scores of animals assessed over 72 h after
as 5,000 units/kg of bw i.p. once was as effective as 7 doses of rhEPO.
Significant recovery of neurological function was observed by 4 days in the
(B) Recovery of neurological function steadily continued over 28 days for
animals given a single dose of rhEPO (5,000 units/kg of bw i.p.), such that
nearly full recovery was obtained. In contrast, saline-treated animals did not
materially improve after 14 days following injury. (n ? 6 each group;**, P ?
0.01;***, P ? 0.001).
www.pnas.org?cgi?doi?10.1073?pnas.142287899Gorio et al.
spinal cord at T9 (Fig. 4 and Table 1; P ? 0.01). Notably, the
sparing could be accounted for by a remarkable preservation of
white matter at the light microscopy level. Myelinated axons
appeared histologically normal in rhEPO-treated animals,
whereas in animals receiving saline, widespread degeneration
and swollen myelin sheaths were observed. TUNEL labeling in
the fasciculus cuneatus 2.5 mm rostral to the lesion epicenter
revealed a mean number of TUNEL-positive cells of 12 ? 3 for
saline-treated animals, whereas for animals given rhEPO (5,000
units/kg of bw ? 7 days), no TUNEL-positive cells were ob-
served (n ? 12 each group; P ? 0.001). Qualitatively, infiltration
by inflammatory cells also appeared greatly reduced in animals
receiving rhEPO therapy.
The results of these experiments demonstrate a major neuro-
logical benefit associated with the systemic administration of
rhEPO after TSCI produced either by transient compression or
blunt trauma. In both models evaluated, a single dose of rhEPO
was associated with a markedly superior clinical course of
recovery of motor function compared with placebo, character-
ized by an earlier and more complete normalization of function
over a 28-day period of study. Further, injury produced by the
aneurysm clip improved significantly within 12 h after injury, a
time at which saline-treated animals remained completely par-
alyzed. After compressive TSCI, a single dose of rhEPO was
associated with the same excellent outcome as three rhEPO
doses given on successive days. Data obtained from both models
suggest that much of the beneficial effect of rhEPO treatment
occurs within the first week after injury, as characterized by an
earlier recovery of motor function after injury. Thereafter,
neurological recovery occurred at the same rate in both saline-
and rhEPO-treated animals, despite the dramatic differences
observed at the histological level after 7 days.
The observed differences in temporal responses to rhEPO
between the models could be explained by the magnitude of
delayed injury. Specifically, the aneurysm clip model produces a
compressive?occlusive injury characterized by transient vascular
occlusion and pressure-related block of axonal conduction, but
often with minimal disruption of myelin. Restoration of axonal
conduction is likely to occur sooner for an axon possessing an
intact myelin sheath. In contrast, the impactor model, being
primarily mechanical in nature, differentially injures large my-
elinated fibers, with the most severe damage occurring at the
nodes of Ranvier, and tends to spare smaller axons (20).
Additionally, significant hemorrhage occurs from the severe
parenchymal disruption caused by absorption of kinetic energy.
Therefore, in the later stages of response to injury caused by
contusion, cellular debris and grossly disrupted axons elicit
pronounced inflammatory and degenerative processes that ini-
tiate a secondary phase of injury.
The very early recovery observed in the aneurysm clip model
could depend on a beneficial effect of rhEPO on the restoration
of adequate blood flow after injury. After experimental TSCI, a
profound reduction in spinal cord blood flow occurs, which
progressively worsens (21) and may last for 24 h (22). In a recent
study assessing the effects of EPO on the clinical course after
subarachnoid hemorrhage in a rabbit model, rhEPO treatment
dramatically attenuated the intense intracerebral arterial spasm
tions obtained at the lesion epicenter 7 days after injury. (Lower) Saline-
treated. (Upper) rhEPO-treated (5,000 units/kg bw i.p. daily for 7 days).
Extensive white matter preservation can be observed in the rhEPO-treated
group. In contrast, cavitation (asterisks) occupied the central cord of saline-
treated animals, surrounded by swollen and fragmented axons. Numerous
inflammatory cells were also present throughout (not seen at this magnifica-
tion). Cavitation volume was reduced in the rhEPO-treated animals by ?25%.
Many TUNEL-positive nuclei corresponding to oligodendrocytes were ob-
served in the fasciculus cuneatus only in the saline-treated animals (see text).
Representative hemotoxylin-and-eosin-stained rat spinal cord sec-
Table 1. Percentage of spared tissue
TreatmentLesion epicenterThroughout T9
41.0 ? 3.7
52.1 ? 2.6**
50.4 ? 3.1
59.6 ? 3.5**
**, P ? 0.01 compared to saline; n ? 12 each condition.
In contrast, a lower dose of rhEPO (500 units/kg of bw i.p.) was no more
effective than saline. (n ? 12 each group;**, P ? 0.01).
Gorio et al.
July 9, 2002 ?
vol. 99 ?
no. 14 ?
secondary to the irritative effects of blood infused into the
subarachnoid space (23). Further, a single dose of rhEPO given
peripherally has been shown to preserve autoregulation of
cerebral blood flow (24). Thus, rhEPO both actively reduces the
cerebral ischemia after hemorrhage by maintaining tissue per-
fusion, and directly provides neuroprotection for metabolically
stressed neurons. The mechanism of this vascular effect has not
been directly evaluated, but the potent vascular effects of rhEPO
noted within the systemic circulation appears to depend on the
modulation of inducible nitric oxide synthase activity (25). In
addition to the constrictive effects of compression on circulation
within the spinal cord, injury-related neurological dysfunction
itself typically produces severe hypotension and bradycardia in
both humans (4) and animals (26), further worsening the effects
cardiovascular alterations that occur immediately after spinal
cord injury (27). Because one mechanism explaining the neu-
roprotective effect of rhEPO has been shown to depend on
inhibition of nitric oxide production (28), it is reasonable to
hypothesize that similar mechanisms may be relevant within the
In the contusion model, the early recovery observed in the
rhEPO-treated groups could arise from a primary effect of EPO
through prevention of cell death, increasing the rate of recovery,
or both. Further studies will be needed to distinguish between
these possibilities. Interestingly, a 500 units/kg of bw dose of
rhEPO appeared to be of intermediate effectiveness in open
field testing but was ineffective in the swim test. This finding
differs from our observations obtained from a 3-vessel reversible
middle artery cerebral ischemia model in the rat for which 500
units/kg of bw was as effective as higher doses of rhEPO, but
lower dosages were ineffective (11). One difference between
these studies may be a decreased rate of absorption of rhEPO
from the i.p. injection site and mobilization into the injured
region of the spinal cord as a result of the profound hemody-
namic changes that occur in TSCI.
Secondary injury contributes significantly to spinal cord pa-
histological examination of the spinal cord 7 days after contusion
showed a dramatic reduction in the volume of cavitation asso-
ciated with rhEPO treatment. This decrease was associated with
an obvious reduction in the number of inflammatory cells in and
around the region of injury. We have previously observed a
similar effect in a mouse model of contusive brain injury (11).
Interestingly, the relatively small reduction in cavitation volume
measured (?25%) is nonetheless associated with a very large
difference in neurological function. Motor score readouts are
more sensitive than histological assessment in this model, as
white matter may not appear greatly damaged but still may not
support transmission of afferent and efferent electrical activity.
The presence of apoptotic oligodendrocytes as determined by
TUNEL labeling within the region of the fasciculus cuneatus are
supportive of this concept. Specifically, no apoptotic nuclei were
observed in this region in the rhEPO-treatment group, whereas
multiple apoptotic nuclei morphologically consistent with oligo-
dendroglia were observed in the spinal cords of the saline-
treated group. Methylpredinisolone and ganglioside GM1, two
agents that are partially effective in the contusion model, do not
reduce the infiltration of neutrophils immediately after spinal
cord injury (30). Inflammatory cells are involved in the late
damage that occurs to the oligodendrocytes that provide the
myelin for axons within the spinal cord (31). rhEPO appears to
reduce the inflammatory infiltrate, and in this manner likely
reduces the contribution of late injury to the neurological deficit.
One way the rhEPO could affect inflammation is through
modulation of members of the nuclear factor (NF)-?B family
that are principal regulators of inflammatory genes (32, 33).
Previous work has shown that NF?B itself is strongly up-
regulated after TSCI, produced by macrophages?microglia, en-
dothelial cells, and neurons (29). One NF?B-dependent gene
product is inducible nitric oxide synthase, which has been shown
to increase the first day after injury, and peaks by day 7 (34).
Recently, EPO has been shown to signal through the NF?B
pathway (35) as well as by the janus kinase-2?signal transducers
and activators of transcription-5 system (36–38). Further study
will be necessary to determine whether the prominent effects on
secondary injury depends in part on regulation of NF?B.
To date, the only pharmacotherapeutic with demonstrated
effectiveness in TSCI is methylprednisolone. This agent must be
given within the first 8 h after spinal cord injury. However, the
beneficial effect is frequently only moderate even in animal
models (39). In contrast, dramatic and significant effects of
rhEPO systemically administered immediately after injury on
neurological outcome suggests that this well-tolerated agent may
provide a large therapeutic benefit in the setting of TSCI arising
from either compression or contusion. The observation that a
single dose of rhEPO after injury is highly efficacious suggests
that this agent or its analogues may be an ideal immediate
treatment after acute injury. Further study will be needed to
determine how extensive the time window for effective treat-
ment is and whether there are restorative benefits of rhEPO
administered in the setting of chronic spinal cord injury. The
outstanding safety record of the use of rhEPO for the treatment
of anemia should encourage an early evaluation of the use of this
agent in the setting of TSCI.
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