Spastic Paresis After Perinatal Brain Damage in Rats Is Reduced by Human Cord Blood Mononuclear Cells

Abstract

Brain damage around birth may cause lifelong neurodevelopmental deficits. We examined the therapeutic potential of human umbilical cord blood-derived mononuclear cells containing multipotent stem cells to facilitate motor recovery after cerebral hypoxic-ischemic damage in neonatal rats. Left carotid artery ligation followed by 8% O(2) inhalation for 80 min was performed on postnatal d 7, succeeded by intraperitoneal transplantation of human umbilical cord blood-derived mononuclear cells on postnatal d 8 in a sham-controlled design. Histologic and immunohistochemical analysis on postnatal d 21 revealed that neonates developed severe cerebral damage after the hypoxic-ischemic insult. These animals also suffered from contralateral spastic paresis, as evidenced by their locomotor behavior. After transplantation of human umbilical cord blood-derived mononuclear cells, spastic paresis was largely alleviated, resulting in a normal walking behavior. This "therapeutic" effect was accompanied by the fact that mononuclear cells had entered the brain and were incorporated around the lesion without obvious signs of transdifferentiation. This study demonstrates that intraperitoneal transplantation of human umbilical cord blood-derived mononuclear cells in a rat model of perinatal brain damage leads to both incorporation of these cells in the lesioned brain area and to an alleviation of the neurologic effects of cerebral palsy as assessed by footprint and walking pattern analysis.

Full text

Spastic Paresis After Perinatal Brain Damage in Rats Is Reduced
by Human Cord Blood Mononuclear Cells
CAROLA MEIER, JOHANNES MIDDELANIS, BIANCA WASIELEWSKI, SANDRA NEUHOFF, ASTRID ROTH-HAERER,
MARKUS GANTERT, HUBERT R. DINSE, ROLF DERMIETZEL, AND ARNE JENSEN
Department of Neuroanatomy and Molecular Brain Research [C.M., B.W., S.N., A.R.-H., R.D.], Department of Neuroinformatics [H.R.D.],
Ruhr-University Bochum, D- 44801 Bochum, Germany; Department of Gynecology and Obstetrics [J.M., M.G., A.J.], Ruhr-University
Bochum, D- 44892 Bochum, Germany
ABSTRACT: Brain damage around birth may cause lifelong neu-
rodevelopmental deficits. We examined the therapeutic potential of
human umbilical cord blood– derived mononuclear cells containing
multipotent stem cells to facilitate motor recovery after cerebral
hypoxic-ischemic damage in neonatal rats. Left carotid artery ligation
followed by 8% O
2
inhalation for 80 min was performed on postnatal
d 7, succeeded by intraperitoneal transplantation of human umbilical
cord blood– derived mononuclear cells on postnatal d 8 in a sham-
controlled design. Histologic and immunohistochemical analysis on
postnatal d 21 revealed that neonates developed severe cerebral
damage after the hypoxic-ischemic insult. These animals also suf-
fered from contralateral spastic paresis, as evidenced by their loco-
motor behavior. After transplantation of human umbilical cord
blood– derived mononuclear cells, spastic paresis was largely allevi-
ated, resulting in a normal walking behavior. This “therapeutic”
effect was accompanied by the fact that mononuclear cells had
entered the brain and were incorporated around the lesion without
obvious signs of transdifferentiation. This study demonstrates that
intraperitoneal transplantation of human umbilical cord blood–
derived mononuclear cells in a rat model of perinatal brain damage
leads to both incorporation of these cells in the lesioned brain area
and to an alleviation of the neurologic effects of cerebral palsy as
assessed by footprint and walking pattern analysis. (Pediatr Res 59:
244–249, 2006)
Each year, thousands of children incur perinatal brain
damage that potentially results in lifelong sequelae (1,2).
Depending on the extent and location of the insult these
children may develop spastic paresis, choreo-athetosis, ataxia,
and disorders of sensorimotor coordination (1). In the United
States, estimates of the costs to society for treatment and care
of these children amount to a total of $11.5 billion per birth
year (2).
One of the major causes of perinatal brain damage is severe
fetal or neonatal asphyxia (3,4), resulting in cerebral hypoxic-
ischemic insults and hemorrhages. Experimental evidence has
shown that neuroprotective strategies using pharmacologic
agents or moderate cerebral hypothermia may ameliorate peri-
natal brain damage (5,6). However, limited clinical strategies
are available for functional regeneration of damaged nervous
structures in the perinatal period (7). Therefore, one of the
most urgent tasks for scientists and clinicians will be to
explore the enormous potential of cell therapies using stem
cells in general and hUCB-derived cells in particular to pro-
vide a therapeutic paradigm for perinatal neuronal repair.
For hUCB cell transplantation, a number of promising
experimental protocols have been established in sheep (8) and
rats (9). The study by Chen et al. (9) provides first experi-
mental evidence in adult rats that intravenous application of
umbilical cord blood cells results in the migration of these
cells toward brain regions affected by stroke. Furthermore,
behavioral studies on those animals revealed an at least partial
functional compensation.
The insult resulting from perinatal hypoxic-ischemic brain
damage in humans has been reproduced in a neonatal rat
model (Levine model) (10 –12). Here, we present a study
designed to investigate the potential of hUCB-derived mono-
nuclear cells to migrate to a hypoxic-ischemic brain lesion
from a distant transplantation site, and to examine their po-
tential to alleviate neurologic deficits in neonatal rats. Our
results demonstrate that intraperitoneal transplantation of
hUCB-mononuclear cells after a hypoxic-ischemic insult re-
sults both in the migration of these cells toward the lesion in
the neonatal rat brain and in the recovery from spastic paresis
as assessed by walking pattern analysis.
MATERIALS AND METHODS
hUCB-derived mononuclear cells. Blood from umbilical cord and pla-
centa was obtained from the Department of Gynecology and Obstetrics
(Ruhr-University Bochum, Germany), after receiving the mother’s informed
consent. The umbilical vein was punctured post partum, and the blood was
collected in umbilical cord blood collection bags containing citrate phosphate
dextrose as an anticoagulant (Maco Pharma, Langen, Germany) and stored at
room temperature up to 24 h until further processing.
Preparation of the mononuclear cell fraction was performed by Ficoll
Paque (Amersham, Freiburg, Germany) density gradient centrifugation ac-
Received March 17, 2005; accepted September 21, 2005.
Correspondence: Arne Jensen, M.D., Department of Gynecology and Obstetrics,
Ruhr-University Bochum, In der Schornau 23-25, D-44892 Bochum, Germany; e-mail:
Arne.Jensen@rub.de
Supported by grants of the Stem Cell Network North Rhine Westphalia (C.M., A.J.)
and by the Medical Faculty of Ruhr-University Bochum (FoRUM) (A.J.).
DOI: 10.1203/01.pdr.0000197309.08852.f5
Abbreviations: GFAP, glial fibrillary acidic protein; HLA-DR, human leu-
kocyte antigen type DR; hUCB, human umbilical cord blood
0031-3998/06/5902-0244
PEDIATRIC RESEARCH Vol. 59, No. 2, 2006
Copyright © 2006 International Pediatric Research Foundation, Inc. Printed in U.S.A.
244

cording to the manufacturer’s instructions. The mononuclear fraction of cells
was collected from the interphase, resuspended in 0.9% sodium chloride, and
the cell number was determined. Viability of resuspended mononuclear cells
was 97 62%.
Cerebral ischemia. The Levine model (10,13) was used to achieve repro-
ducible hypoxic-ischemic injury in neonatal rats. Seven-day-old Wistar rat
pups were deeply anesthetized by inhalation of 4% halothane and maintained
with 1.5% halothane in 50% N
2
O/50% O
2
. The left common carotid artery
was exposed, double-ligated with 6 –0 surgical silk, and severed. The duration
of anesthesia and surgery did not exceed 10 min. After surgery, the rat pups
were allowed to recover in their home cages for 1 h. To introduce systemic
hypoxia, the pups were subsequently placed in an incubator (Incubator 7510;
Dra¨ger, Lu¨beck, Germany) and exposed to a hypoxic gas mixture (8%
oxygen, 92% nitrogen) for 80 min. The environmental temperature was
strictly maintained at 36°C.
All surgical and experimental protocols were approved by the appropriate
institutional review committee (Bezirksregierung Arnsberg, Germany) and
met the guidelines of the German animal protection law.
Experimental protocol. Randomly selected animals were assigned to three
different experimental groups: controls (no lesion), lesion (without transplan-
tation), and lesion followed by transplantation of mononuclear cells.
Animals of the lesion group (lesion only, n511) were subjected to
ligation of the left common carotid artery, followed by systemic hypoxia.
Twenty-four hours after the insult, this group received an intraperitoneal
injection of 500
m
L of 0.9% sodium chloride (sham injection). In the
transplantation group (lesion followed by transplantation, n514), rats
received 1 310
7
hUCB-derived mononuclear cells (in a volume of 500
m
L
0.9% sodium chloride) by intraperitoneal injection 24 h after the hypoxic-
ischemic insult. The control group (no lesion, n511) comprised of animals
that did not undergo any treatment or surgery (n55), and of sham-operated
animals that were anesthetized, the carotid artery exposed but not ligated, and
surgery then terminated (n56). After initial analysis, these latter two groups
were indistinguishable in motor analysis, and therefore combined. Additional
control animals (no lesion, n510), which received intraperitoneal injection
of 1 310
7
hUCB-derived mononuclear cells, were included in motor
analysis. There was no use of immunosuppressants or analgesics in any
experimental group. Animals were included into subsequent analyses when
displaying a severe macroscopic lesion accompanied by cystic changes larger
than 4 mm upon hypoxic-ischemic damage without (11 out of 18 animals) or
with (14 out of 20 animals) transplantation of hUCB-derived cells, thereby
disregarding animals in which the Levine procedure resulted in minor lesions.
For the control group, animals were included when displaying an intact brain
without pigmentation or cystic changes (11 out of 13 animals).
Analysis of motor abilities. Footprint analysis was performed on postnatal
d 21 in a strictly blinded fashion. The paws were colored consecutively, and
the footprints were printed onto paper when the rat walked along a defined
gangway which was elevated by 35°. Footprints were then measured and each
imprint was analyzed regarding the distance between the first and fifth toe
(hind paws) or between the first and the fourth digit (forepaws) (14). In
addition, the step length was determined for each paw. A significant decrease
in footprint width and/or step length of the limbs contralateral to the lesion
compared with that of the ipsilateral site was considered to reflect spastic
paresis.
Macroscopic assessment. Rats were anesthetized by CO
2
exposition and
decapitated at postnatal d 21. Brains were dissected and brain injury was
assessed immediately after dissecting the brains (11). Neonatal rats were
considered to have suffered hypoxic-ischemic brain damage when displaying
a cystic infarction, similar to a unilateral internal hydrocephalus, on the brain
surface ipsilateral to the carotid artery occlusion.
Microscopic assessment. Brains were covered in tissue freezing medium
(Leica, Nussloch, Germany) and frozen in 8% methylcyclohexane in 2-methyl-
butan (vol/vol) (– 80°C). Histology and immunohistochemistry were per-
formed on cryosections of 14-
m
m thickness, which had been mounted onto
Superfrost Plus slides (BDH, London, UK) and heat dried for 1 h at 40°C
before fixation.
Precise localization of the lesion areas was assured by histologic staining
according to Klu¨ver-Barrera, showing myelinated fiber tracts in light green
and cell bodies in blue. The lesion size was determined by measuring the
hemisphere areas in histologic sections. Measurements were performed in
nine histologic sections at three defined levels in the brain, and in three
animals per group (control, lesion, lesion plus transplantation). The extent of
brain damage was expressed as area of the left (lesioned) hemisphere as
percentage of the area of the right (nonlesioned) hemisphere.
For immunohistochemistry, adjacent cryosections were fixed in –20°C
cold ethanol, rinsed in PBS and preincubated in blocking buffer (BB, 10%
normal goat serum and 0.1% TritonX-100 in PBS) for 30 min. Primary
antibodies were diluted in BB and sections were incubated at 18°C overnight.
Samples were rinsed in PBS and incubated in 0.2% BSA in PBS. Incubation
with secondary antibodies was performed in BB at 18°C for 2 h. Sections
were mounted using the ProLong Antifade Kit (Molecular Probes, Leiden,
Netherlands). Fluorescence was documented using confocal imaging micros-
copy (Zeiss LSM 510 META). Data were collected using the single track
scanning module and exported as TIFF files into Adobe Photoshop 7.0
(Adobe Imaging Systems Inc., Mountain View, CA).
Primary antibodies (dilution and supplier in parentheses) were directed
against HLA-DR
a
-chain (1:50; DAKO, Hamburg, Germany), cleaved
caspase3 (1:100; New England Biolabs, Frankfurt, Germany), CD68 (ED1;
1:100; Serotec, Du¨sseldorf, Germany), glial fibrillary acidic protein (GFAP;
monoclonal: 1:100, Chemicon, Hofheim, Germany; polyclonal: 1:100, Sigma
Chemical Co., Taufkirchen, Germany). Primary antibodies used to determine
neuronal differentiation of HLA-DR-labeled hUCB-derived mononuclear
cells included the polyclonal GFAP antibody, neurofilament-68 (Chemicon),
and synaptophysin antibodies (Diagnostic Biosystems, Pleasanton, CA). Re-
activity of these antibodies with human cells had been confirmed in vitro (data
not shown).
Secondary antibodies were Alexa Fluor 488 or 546 conjugated goat-anti-
rabbit or goat-anti-mouse (1:3,000; Molecular Probes).
Statistical analysis. The Kolmogorov-Smirnov test was performed to
demonstrate that test distributions were normal. Further analysis of intra- and
intersample variability was done by two-way ANOVA (SuperANOVA; Aba-
cus Concepts Inc., Berkeley, CA), followed by the appropriate posthoc test
(Student-Newman-Keuls). Data are expressed as mean 6SEM. A value of p
,0.05 was considered statistically significant.
RESULTS
Hypoxic-ischemic brain damage in perinatal rats. Cere-
bral hypoxia-ischemia resulted in macroscopic changes, in-
cluding cystic lesions (n511). There was no evidence of any
brain damage in control rats (n512). The rostrocaudal extent
of the damage was determined and averaged 6.1 60.6 mm in
lesioned brains. Histopathological analysis revealed that hy-
poxic-ischemic brain damage affected cortex, hippocampus,
and periventricular areas (Fig. 1, A–C), and comprised inflam-
mation and neuronal cell death (see below).
The extent of inflammation was determined by CD68-
immunopositive activated microglia (15). Apoptotic neuronal
cell death was detected by cleaved-caspase3 immunoreactivity
(16). There were activated microglia (green) in a large region
of the lesioned hemisphere, whereas the apoptotic neuronal
cells were clustered within (red), as depicted in Figure 2, A
and B. Intact areas of the ipsilateral hemisphere as well as the
contralateral hemisphere were devoid of any immunoreactiv-
ity to CD68, indicating the absence of inflammation (Fig. 2, A
and C; green). However, weak cleaved-caspase3 immunore-
activity was detectable in intact hemispheres (Fig. 2, Aand C;
red), as described previously during brain development (17).
“Homing” of human umbilical mononuclear cells. Trans-
plantation of human mononuclear cells (1 310
7
) derived
Figure 1. Histology of the hypoxic-ischemic brain lesion. (A) The level of
coronal sections shown in Band Cis indicated in a schematic drawing. (B)
Histologic staining (Klu¨ver-Barrera) reveals damage of the left brain hemi-
sphere, i.e. ipsilateral to the carotid artery occlusion. The lesion affects the
hippocampus (hc), parietal and temporal cortex (cx), periventricular areas
(pv), and results in an enlarged lateral ventricle (v). (C) Schematic represen-
tation of the histologic features observed in B. Boxed areas were analyzed
immunohistochemically, shown in Figures 2 and 3. Scale bar: 1 mm.
245UMBILICAL CELLS REDUCE SPASTIC PARESIS

from umbilical cord blood was performed by intraperitoneal
injection 24 h after the hypoxic-ischemic insult, i.e. in post-
natal d 8 rats (n514), in a sham-controlled fashion. Cells
were identified by immunohistochemical detection of human-
specific HLA-DR
a
-chain surface antigens (18). Thus, we
demonstrate that transplanted human mononuclear cells mi-
grate from the intraperitoneal cavity to the damaged brain
region (Fig. 3, Aand B; green). These cells were detectable in
the brain as early as 3 d after intraperitoneal transplantation
and were still present in the lesioned hemisphere 2 wk after
transplantation (Fig. 3, Aand B; green). Confocal microscopy
revealed that transplanted cells (green immunofluorescence in
Fig. 3B) were clearly incorporated into the astrocytic network
(red immunofluorescence in Fig. 3, B–D). In contrast, there
was no evidence for human cells outside the damaged brain
region (Fig. 3C), and there was no HLA immunoreactivity in
the lesioned areas of animals that had not received transplan-
tation of hUCB-derived mononuclear cells (Fig. 3D). Interest-
ingly, transplanted human cells found in the brain were strictly
confined to the area of activated microglia (compare Fig. 2A;
green).
Transplantation of hUCB cells did not change the severity
of morphologic damage. In control animals, left and right
hemisphere comprised approximately the same area. Upon
hypoxic-ischemic brain injury, the area of the lesioned hemi-
sphere was 34.99 64.7% of that of the intact hemisphere. In
lesioned animals that had received transplantation of hUCB-
derived mononuclear cells, the lesioned hemisphere area com-
prised 35.72 60.4% of the intact hemisphere area, which was
not significantly different from nontransplanted animals (p$
0.05). In addition, HLA-DR-immunopositive cells did not
display obvious signs of transdifferentiation in vivo, as as-
sessed by their phenotype and the absence of GFAP, an
intermediate filament expressed in neural progenitor cells, and
neuron-specific proteins like neurofilament-68 and synapto-
physin (Fig. 4), although expression of these markers was
observed upon cultivation of hUCB-derived mononuclear
cells in the presence of nerve growth factor and retinoic acid
in vitro (data not shown).
Perinatal brain damage causes spastic paresis. To exam-
ine whether transplantation of hUCB-derived mononuclear
cells after cerebral hypoxic-ischemic brain damage has bene-
ficial effects on locomotor behavior, we performed a strictly
blinded footprint and walking pattern analysis. Our analysis of
the toe distance 1 to 5 of the hind paw contralateral to the
cerebral damage at 3 wk of age revealed a highly significant
reduction (p,0.001) of this measure after cerebral damage
(0.99 60.04 cm; n510) compared with controls (1.15 6
0.04 cm; n512), which is characteristic for spastic paresis.
Figure 2. Activated microglia and apoptotic neuronal cells are located in the
vicinity of the hypoxic-ischemic lesion. (A) Schematic representation of the
lesion site as indicated by the presence of CD68-immunopositive cells (green)
and cleaved-caspase3 expressing cells (red). Immunohistochemistry is pre-
sented for areas Band C. (B) Immunohistochemistry for CD68 (green
fluorescence), showing activated microglia, and cleaved-caspase3 immunore-
activity (red fluorescence) indicating apoptosis in the lesioned hemisphere.
(C) The nonlesioned hemisphere is devoid of activated microglia (green
fluorescence) and shows few weakly labeled caspase3-positive cells as char-
acteristic for neonatal rat brain. Scale bar: 100
m
m.
Figure 3. Intraperitoneal transplantation of mononuclear hUCB-derived
cells resulted in a specific “homing” of these cells into the CNS and
incorporation around the lesioned area. (A) Schematic representation of the
distribution of human cells in the lesioned hemisphere. HLA-DR-positive
cells (green) are detected in the area of the hypoxic-ischemic lesion. Con-
tralateral hemispheres were devoid of human cells. Immunohistochemistry is
demonstrated for boxed areas in B(lesioned hemisphere) and C(nonlesioned
hemisphere). (B) HLA-DR-positive mononuclear cells (green) are located
within a scaffold of GFAP-positive astrocytes (red) in the area of the
hypoxic-ischemic lesion. (C) Nonlesioned parts of the brain, as demonstrated
by the absence of inflammatory and apoptotic events (compare Fig. 2), are
devoid of human cells (absence of green fluorescence). (D) Lesioned areas of
the brain of animals that did not receive transplantation of human cells are
devoid of HLA-immunostaining. Note absence of green immunofluorescence
(HLA-staining) within the red scaffold of GFAP-positive rat astrocytes. Scale
bar in B, C: 100
m
m; in D: 20
m
m.
246 MEIER ET AL.

As animals after hypoxic-ischemic lesion were smaller (43.2
63.2 g) than control animals (50.3 63.1 g) of the same age,
an intraindividual comparison of footprint width was per-
formed. In control animals (without transplantation), there
was no significant difference in toe distance between left (1.16
60.03 cm) and right (1.15 60.04 cm; p$0.05; Fig. 5A) hind
paws. In nonlesioned control animals, transplantation of
hUCB cells did not result in significant differences in the toe
distance of left (1.19 60.01 cm) and right (1.21 60.01 cm;
p$0.05) hind paws. However, in lesioned animals (without
transplantation), the toe distance of the ipsilateral hind paw
(1.12 60.03 cm) differed significantly from that of the
contralateral hind paw (0.99 60.04 cm; p,0.05; Fig. 5A),
and this difference is independent of the animal’s weight.
Furthermore, in animals with hypoxic-ischemic lesion,
there was evidence for a reduction of the step length of the
right hind paw (contralateral to the lesion; 7.57 60.07 cm)
compared with the left hind paw controls (8.26 60.33 cm; p
,0.05; Fig. 5B), which is also characteristic of spastic paresis
(19).
Intraperitoneal transplantation of umbilical mononuclear
cells reduces spastic paresis. Most importantly, our results
show that these neurologic deficits were eliminated following
transplantation of hUCB mononuclear cells. There was a
Figure 4. Transplanted HLA-immunopositive human UCB-derived mononu-
clear cells do not reveal signs of transdifferentiation upon “homing” to the
hypoxic-ischemic lesion. (A, B) GFAP (red), characteristic for astrocytes as
well as neuroglial progenitor cells, is located in close vicinity to human
HLA-DR-positive cells (green), however, there is no obvious overlap be-
tween GFAP- and HLA-DR-immunofluorescence. The neuron-specific pro-
teins NF-68 (C, D) and synaptophysin (E, F) are expressed in the brain tissue
(red fluorescence), however, HLA-positive cells (green fluorescence) are
devoid of NF-68 and synaptophysin immunostaining. Scale bar in A, C, E: 20
m
m; B, D, F: 50
m
m.
Figure 5. Transplantation of hUCB-derived mononuclear cells reduces spas-
tic paresis as assessed by footprint analysis of 3-wk-old animals. (A) Hypoxic-
ischemic brain damage results in spastic paresis of the distal limb muscles,
causing a significant reduction of footprint width (toe distance 1 to 5) of the
right hind paw (contralateral to the insult; black columns) compared with the
left (ipsilateral; gray columns) hind paw. Intraperitoneal transplantation of
hUCB-derived mononuclear cells after hypoxic-ischemic brain damage re-
duced spastic paresis. In these animals, differences between ipsi- and con-
tralateral hind paws were no longer detectable. Photographs of footprints
(right hind paws) illustrate the footprint widths (arrows) of control animals
without (left) and with (center left) transplantation, upon hypoxic-ischemic
lesion without (center right) and with (right) transplantation of hUCB-derived
mononuclear cells. (B) In control animals with and without transplantation,
the step length of left and right hind paws is equal. In contrast, hypoxic-
ischemic lesion resulted in a significantly reduced step length of the right hind
paw (black columns) compared with the left hind paw (gray columns). This
reduction in step length of the hind paw contralateral to the lesion, also
indicative of spastic paresis, was largely alleviated upon transplantation of
hUCB-derived mononuclear cells. Data are presented as mean 6SEM; *p,
0.05; ***p,0.001.
247UMBILICAL CELLS REDUCE SPASTIC PARESIS

dramatic alleviation of the contralateral spastic paresis for all
parameters back to normal (Fig. 5). Upon transplantation,
there was no significant difference detected between toe dis-
tances 1–5 of the ipsilateral (1.20 60.03 cm) and contralateral
(1.21 60.04 cm) hind paws of animals after hypoxic-
ischemic lesion and transplantation (p$0.05). The step
length of the ipsilateral (8.46 60.4 cm) hind paws also
equaled that measured on the contralateral side (8.40 60.32
cm; p$0.05). Interestingly, the weight of these animals (51.4
61.4 g) was similar to that of control animals, however, it
differed significantly from that of lesioned animals that had
not received hUCB-cell transplantation (p,0.05).
DISCUSSION
In this study, we used a model of perinatal hypoxic-
ischemic brain damage in rats. The insult was morphologically
characterized by hemorrhage, neuronal death, and inflamma-
tion, and functionally by spastic paresis of contralateral fore-
and hind limbs. Intraperitoneal transplantation of human
UCB-derived mononuclear cells resulted in a) migration of
cells from the peritoneal cavity to the CNS, b) incorporation of
cells around the cerebral lesion (“homing”), and c) an allevi-
ation of spastic paresis.
Unlike other studies, we used the intraperitoneal cavity for
transplantation. The migration of transplanted cells from the
intraperitoneal cavity to the damaged region of the brain
suggests the presence of very specific and powerful chemoat-
tractant signals. One component of the complex mechanism
seems to be the expression of cytokines/chemokines in the
brain in response to hypoxic-ischemic brain injury (20). The
chemoattractant effect of adult ischemic brain tissue had pre-
viously been demonstrated by cell migration assays, in which
hUCB-derived cells were shown to migrate towards tissue
extract derived from lesioned brain hemispheres (9). In our
paradigm, invasion of mononuclear cells was very focused in
that it was confined to the hypoxic-ischemic region of the
brain. More general patterns of brain damage as observed in
human genetic metabolic disorders like Krabbe leukodystro-
phy lead to a more widespread distribution of damaged neu-
rons, and hence result in a ubiquitous distribution of trans-
planted human hUCB cells (21,22).
The specificity of “homing” within the hypoxic-ischemic
brain may both be linked to specific chemoattractants (20) and
to the lack of a functioning blood-brain-barrier in the damaged
brain regions (13,23) leading to a facilitated intrusion of
human mononuclear cells into damaged regions and to a
preferential regional incorporation of these cells. Furthermore,
successful “homing” might be related to the early transplan-
tation after the insult, because then concentrations of chemoat-
tractants are likely to be maximal, and, secondly, there is a
known optimal point in time for transplantation in rodents
within the first 2 wk after the insult (24).
Successful “homing” may be also related to the use of
crude, rather than predifferentiated, hUCB-derived mononu-
clear cells for transplantation in this study, resulting in a
minimal manipulation of cells, which was limited to density
gradient centrifugation and resuspension in sodium chloride
only. Thus, the full potential of cell (sub-) populations and that
of cellular responsiveness to sense signals important to entrain
migration to the damaged brain region may have been pre-
served.
Transplantation of human mononuclear cells into neonatal
rats also warrants some considerations concerning graft– host
interactions. Our results suggest some degree of immune
tolerance toward cord blood cells, possibly mediated by di-
minished generation of cytotoxic responses of the host (25)
through IL-10 release by these cells (26).
Neurologic deficits like contralateral spastic paresis and
reduced step length are characteristic of pyramidal tract im-
pairment in human newborns. Thus, the most important issue
in any therapeutic approach, i.e. functional recovery, was
achieved in our model. Footprint analysis represents one of the
most sensitive parameters to detect pyramidal motor dysfunc-
tion in early neonatal life (19).
Irrespective of the striking “therapeutic” effect of mononu-
clear cell transplantation that also included the normalization
of the weight, the underlying mechanisms remain largely
unknown. Interestingly, we produced no evidence for neural
differentiation of these transplanted human cells in the rat
brain, even though hUCB mononuclear cells in our hands
showed expression of a variety of neural markers in vitro
using differentiation media (data not shown). This appears in
part at variance with observations by Zigova et al. (27).
However, this study is not directly comparable due to the
methods used by those authors, i.e. predifferentiation of hUCB
cells, intracisternal injection of cells, and the use of P1 rats. In
our study, there was no change in the phenotype of HLA-
positive mononuclear cells or in the expression of glial or
neuronal marker proteins even though there was clear struc-
tural integration of the human cells into the three-dimensional
astrocytic scaffold of the rat brain. None of the neural markers
used yielded positive immunohistochemical staining of HLA-
positive transplanted human cells. Although we were able to
demonstrate a strong therapeutic effect of the transplantation
by largely alleviating the neurologic deficits observed in rats
after cerebral hypoxic ischemia without transplantation, the
beneficial functional outcome appears to be unrelated to true
cell replacement by differentiation of transplanted human
cells. Thus, a permissive effect is suggested, as observed in the
lesioned heart (28) and brain (29). Hence, in our model of
hypoxic-ischemic brain damage, we propose secondary mech-
anisms, which may reduce adverse effects resulting from
neural damage per se. Tentative candidates for these second-
ary mechanisms are increased vascularization, reduced edema,
detoxification, prevention of gliosis, and others. Also, “by-
stander effects” mediated by other trophic factors, including
glial cell line– derived neurotrophic factor, brain-derived neu-
rotrophic factor, and nerve growth factor, may contribute to
functional recovery caused e.g. by extensive host axonal
growth and increased neuronal survival (30). Furthermore,
recruitment of endogenous neural stem cells, e.g. elicited
through cytokine and growth factor release, ought to be
considered.
In summary, we show in a neonatal cerebral hypoxia-
ischemia model that hUCB-derived mononuclear cells trans-
248 MEIER ET AL.

planted intraperitoneally enter the rat brain and incorporate
specifically around the lesion (“homing”) in large numbers.
Transplantation results in substantial alleviation of spastic
paresis as assessed by footprint recordings of the hind paw
contralateral to the damaged brain hemisphere and walking
track analysis. This study provides important information for
the development of therapeutic strategies using hUCB stem
cells after perinatal brain damage to reduce potential sensori-
motor deficits.
Acknowledgments. The authors thank Nicole-Christiane
Kozik, Hans-Werner Habbes, Janet Moers, and Kerstin
Schmitz for excellent technical assistance, and Petra Paraken-
ings and Helga Schulze for expertly done photographic work.
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249UMBILICAL CELLS REDUCE SPASTIC PARESIS