(Stroke. 2001;32:1012.)
© 2001 American Heart Association, Inc.
Original Contributions |
Resolution of Stroke Deficits Following Contralateral Grafts of Conditionally Immortal Neuroepithelial Stem Cells
From ReNeuron Ltd (T.V., R.P.S., W.P.W., H.H.); Department of Psychology, Institute of Psychiatry, King’s College London (H.H.); and Department of Neurosurgery, Addenbrookes Hospital, Cambridge (J.S.B.), UK.
Correspondence to Helen Hodges, PhD, Department of Psychology, Institute of Psychiatry, King’s College London, Dr Crespigny Park, Denmark Hill, London SE5 8AF, UK. E-mail spjthmh{at}iop.kcl.ac.uk
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Abstract |
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Background and Purpose—Grafts of MHP36 cells have previously been shown to reduce dysfunction after global ischemia in rats. To test their efficacy after focal ischemia, MHP36 cells were grafted 2 to 3 weeks after transient intraluminal middle cerebral artery occlusion (tMCAO) in rats.
Methods—MHP36 cells were implanted into the hemisphere contralateral to the lesion, with 8 deposits of 3 µL of cell suspension (25 000 cells per microliter). Sham grafted rats received equivalent volumes of vehicle. Three groups, sham-operated controls (n=11), MCAO+sham grafts (n=10), and MCAO+MHP36 grafts (n=11), were compared in 3 behavioral tests.
Results—In the bilateral asymmetry test, MCAO+MHP36 grafted rats exhibited neglect before grafting but subsequently showed no significant dysfunction, whereas MCAO+sham grafted rats showed stable sensorimotor deficits over 18 weeks relative to controls. MCAO+sham grafted rats demonstrated spontaneous motor asymmetry and increased rotational bias after injection of dopamine agonists. MCAO+MHP36 and control groups exhibited no bias in either spontaneous or drug-induced rotation. In contrast to motor recovery, MCAO+MHP36 grafted rats showed no improvement relative to MCAO+sham grafted rats in spatial learning and memory in the water maze. MCAO produced large striatal and cortical cavitations in both occluded groups. Lesion volume was significantly reduced (P<0.05) in the MCAO+MHP36 grafted group. The majority of MHP36 cells were identified within the intact grafted hemisphere. However, MHP36 cells were also seen in the cortex, striatum, and corpus callosum of the lesioned hemisphere.
Conclusions—MHP36 cells may improve functional outcome after MCAO by assisting spontaneous reorganization in both the damaged and intact hemispheres.
Key Words: behavior, animal • stem cell transplantation • middle cerebral artery occlusion, transient • stroke, experimental • rats
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Introduction |
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Transient intraluminal occlusion of the middle cerebral artery (tMCAO) in rats provides a reliable model for the effects of transient embolic stroke, in which blockage of the middle cerebral artery (MCA) is implicated in 60% of cases.1 Typical therapies for stroke seek (1) to interrupt the cascade of events that lead to cell death2 and (2) to harness the substantial intracerebral reorganization after stroke to functional recovery by rehabilitation therapies.3 4 Most cerebroprotective drug treatments must be given within a short time frame, and although rehabilitation treatments require longer periods, residual disabilities may not undergo appreciable reduction after 3 to 6 months.5 Against this background, the possibility of promoting recovery from stroke damage by use of intracerebral transplants appears attractive, particularly for younger victims facing decades of disability.
Fetal tissue provides a possible donor source for grafts, and there is some evidence to suggest that fetal transplants are innervated by host neuronal networks, express features of normal host brain, and ameliorate stroke deficits in experimental animal models.6 7 8 Use of fetal grafts for stroke therapy, however, poses enormous practical and ethical difficulties, not the least of which is that homotypic replacement of damaged tissue might require both cortical and striatal dissections of different gestational ages. Use of laboratory-grown, immortalized cells may overcome these difficulties. Borlongan et al,9 using grafts of the NT2N cell line developed from human teratocarcinoma, showed improvements in passive avoidance and body swing tests after permanent MCAO in rats. Indeed, some functional recovery in stroke patients has recently been reported with NT2/D grafts.10 However, the provenance of these cells may pose difficulties for their development for large-scale clinical use. Our approach is to generate conditionally immortalized human stem cell lines by incorporating an immortalizing oncogene into fetal stem cells and creating clonal lines.
As "proof of principle," we have investigated the functional efficacy of the MHP36 conditionally immortalized murine stem cell line derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium, which constitutively expresses the temperature-sensitive tsA58 oncogene.11 MHP36 cells are capable of unlimited expansion to generate cells for grafting under permissive low (33°C) temperature in vitro, but they cease dividing and develop into mature neurons and glia on implantation into the higher temperature of the brain (37°C). They have been shown to improve outcome in several models of impairment, including hippocampal ischemia induced by 4-vessel occlusion, lesions to cholinergic forebrain projections, and old age.11 12 13 14 15 MHP36 grafts are well suited to repair the indiscriminate cell loss that occurs with MCAO because (1) they have the capacity to develop into neurons, glia, or oligodendrocytes in response to host signals and (2) they migrate to and engraft areas of damage in the host brain.13 14 15
The present experiment investigated the efficacy of MHP36 grafts placed within the intact cortex and striatum, contralateral to the lesion cavity, to alleviate behavioral deficits induced by 60 minutes of tMCAO. The intact side was chosen to avoid exposing the cells to the poorly vascularized, inflammatory environment of the developing ischemic lesion, in the process of forming a fluid-filled cyst.16 Rats were tested in tasks of sensorimotor (tape removal), motor (rotation), and cognitive (spatial learning) function to provide stable measures of several long-term deficits.17 18 19 Histological examination sought to provide preliminary evidence regarding whether migration to the areas of damage and/or enhancement of local reorganization in the intact hemisphere might contribute to functional effects.
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Materials and Methods |
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The time course of the experiment is described in Table 1
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Animals
Thirty-six male Wistar rats (Charles Rivers,
Maidstone Kent, UK) were used, weighing 280 to 320 g before
surgery and 560 to 630 g at the end of behavioral testing. They
were housed 4 to a cage, fed ad libitum, and maintained on a 12-hour
light/dark schedule (lights on 9
AM). Procedures accorded
with the UK Scientific Procedures Act of 1986 and the Ethical Review
Committee of the Institute of Psychiatry.
tMCAO Surgery
Twenty-one rats were subjected to left tMCAO under
halothane anesthesia (4% induction, 3%
maintenance in 70%/30%
NO2/O2). A 3.0-mm
polypropylene (Prolene) filament coated at the tip with silicon
(instant gasket, Halfords Ltd) was inserted 18 to 20 mm up the
exposed left MCA to the junction of the circle of Willis and tied in
place for 60 minutes. Anesthetic was discontinued, and the rat was
tested for neurological deficit (contralateral paw flexion and
circling) to establish the presence of ischemia. After 60
minutes the filament was retracted to the external carotid stump under
anesthetic, where it was left in place, the excess was trimmed off, and
the wound was sutured. Body temperature was maintained at 37±1°C
during surgery by rectal probe and heating pad. The rats were kept warm
by heating lamps for approximately 2 hours after surgery and then
placed in single cages in the postoperative room for recovery.
Neurological and health status was monitored for a week until normal
feeding was seen and postoperative weight was regained. Control rats
(n=11) were sham operated by exposure of the left internal carotid
artery only.
Transplant Surgery
Transplant and sham graft surgery was undertaken 2 to
3 weeks after occlusion or sham surgery, which (1) allowed for full
recovery; (2) enabled rats to be assigned to stroke-only and graft
groups on the basis of equivalent neurological deficit; and (3) enabled
pregraft baseline deficits in tape removal to be established. Rats were
anesthetized with Immobilon (etorphine hydrochloride, 0.074
mg/mL, and methotrimeprazine, 18 mg/mL; 0.01 mL/100 g IM) after
pretreatment with midazolam (Hypnovel; 0.03 mL/100 g IM) and placed in
a stereotaxic frame. Holes were drilled in the right side
of the skull to allow the penetration of a 10-µL Hamilton syringe at
the following coordinates20
(mm) derived from bregma, with the skull in the flat position
(-3.2 mm): anteroposterior, -0.3; lateral, -3.5; ventral,
-4.5, -6.0; lateral, -5.5; ventral, -4.0, -5.5;
anteroposterior, -1.3; lateral, -3.0; ventral, -5.0, -6.5;
lateral, -5.5; ventral, -5.0, -6.5.
Three microliters of suspension (25 000 cells per microliter) was infused over 2 minutes at each of the 4 sites (2 deposits/descent), and the cannula was left in place for an additional 2 minutes to allow diffusion from the tip. Controls received vehicle infusions to control for surgical and volume effects. Coordinates targeted somatosensory cortex (lateral sites) and striatum (medial sites) to ensure wide seeding of the area homologous to the region of stroke damage assessed in pilot animals. After transplantation, rats were injected with diprenorphine (Revivon; 0.272 mg/mL; 0.01 mL/100 g IM) and single housed in the recovery room until normal feeding, grooming, and weight gain were seen. Grafted rats received cyclosporin A (Sandimmun, Sandoz: 10 mg/kg IM mixed with Cremophor EL, Sigma, in a volume of 1:3) immediately after surgery and 3 times a week for 2 weeks, a regimen effective for MHP36 graft survival in rats with 4-vessel occlusion ischemia or lesions,11 12 13 14 which maintains stable blood cyclosporin A levels above the therapeutic minimum of 100 mg/dL.
Behavioral Tests
Bilateral Asymmetry Test
Strips of tape (1x5 cm) were wound around each
forepaw in random order. Animals were placed in an observation cage and
timed for latency to contact and to remove each tape. Random use of a
second observer established that interrater reliability was >90%.
Rats were tested before surgery and during the week before grafting to
establish preoperative and postoperative baselines. One session of 4
tests of 3 minutes was performed weekly for 12 weeks, commencing
approximately 6 weeks after transplantation, to assess long-term
recovery.
Water Maze Acquisition
Approximately 26 weeks after transplantation, rats
were trained to find a submerged platform (9 cm) in a large
(200-cm-diameter) pool filled to a depth of 25 cm with water maintained
at 24±2°C. Two trials of 60 seconds (10-minute intertrial interval)
were given daily for 16 days, followed by a probe trial (60 seconds)
with the platform removed. Rats that failed to find the platform within
60 seconds were guided to it by the experimenter. Animals remained on
the platform for 10 seconds before being removed and placed in a
holding cage or returned to the home cage. The swim path was
recorded by an image analysis system (HVS Image) that
computed path length, latency to mount the platform, and percentage of
time spent in each of 4 quadrants and 3 annuli into which the maze was
calibrated for analysis of swimming patterns and heading angle,
a measure of divergence from a straight path to the
platform.
Rotation
Spontaneous and drug-induced rotation were measured
approximately 38 weeks after transplantation in an 8-bowl rotameter
(TSE GmbH) in which rats were harnessed to swivels for 30 minutes at 30
minutes after injection. Swivels enabled turns to left or right to be
recorded. Rats were tested for response to saline (baseline). They
were then tested once a week, on alternate weeks, with either
amphetamine (2.5 mg/kg; Sigma) or apomorphine (0.5 mg/kg; Sigma) on 3
occasions over a period of 6 weeks. All injections were given in a
volume of 1.0 mL/kg IP.
Histology
At the end of behavioral testing, rats were overdosed
with pentobarbital sodium (Sagatal, Rhone Merieux) and transcardially
perfused with heparinized saline followed by 4%
paraformaldehyde. Brains were placed in 30% sucrose
(Sigma) for 24 hours, blocked, and mounted on a cutting stage with
embedding compound. Brains were held at -22°C to -24°C in a
cryostat (Leica CM 1900), and 50-µm coronal sections were cut and
collected at 500-µm intervals from 6.3 to 3.7 mm before
bregma.20 Images of left and
right hemispheres were taken with a Leica stereo microscope. Datacell
image capture software (Pro-Plus) was used by an operator blind to the
rats’ identity for the automated estimation of lesion volume by region
of interest and slice interval using Simpson’s rule. Total
lesion volume was obtained by subtracting the volume of the lesioned
from the intact hemisphere. Intervening serial sections were processed
by immunocytochemistry for reactivity to ß-galactosidase (ß-gal),
the protein product of the
LacZ gene expressed by grafted
cells. ß-Gal immunocytochemistry was chosen in preference to in situ
hybridization or X-gal histochemistry to recognize
LacZ or ß-gal because (1)
pilot studies indicated that it yielded good bright-field and confocal
images; (2) in contrast to in situ hybridization, it permitted further
processing for histology; and (3) it was more stable and reliable than
X-gal.
Statistical Analysis
Sham grafted controls (n=11), ischemic rats
with sham grafts (MCAO+sham; n=10), and ischemic grafted rats
(MCAO+ MHP36; n=11) were compared in the water maze and bilateral
asymmetry tests by repeated-measures ANOVA, with groups as the
between-subjects factor and days or weeks as the within-subjects
factor. Weekly rotameter scores for baseline and drug-induced rotation
and lesion volumes were analyzed by 1-way ANOVA. Group means
were compared by Fisher’s least significant difference
test.
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Results |
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Bilateral Asymmetry Test
Before occlusion, rats did not show a mean difference in paw use as judged by the latency to contact and remove tapes from left and right paws (Figure 1
). After sham surgery the controls continued to show
no preference. However, rats subjected to tMCAO showed marked paw
disparity, with the right paw contacted and the tape removed
significantly more slowly than the left, indicative of contralateral
sensorimotor impairment (F1,64=19.72,
P<0.001;
Figure 1B). This robust and stable deficit persisted
throughout testing. In rats with MHP36 grafts, the stroke-induced
forepaw disparity was not significant by 8 weeks after transplantation,
and this improvement persisted through the 12 weeks of testing, so that
there was no difference between paws
(F1,64=1.15,
P=NS;
Figure 1C). Hence, grafted rats did not differ from controls
(compare
Figure 1A and 1C) since both groups showed paw
equivalence.
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Water Maze Acquisition
There was a substantial difference between groups in
latency to find the submerged platform
(F2,29=47.8,
P<0.0001) because the 2 stroke
groups, with and without transplants, were both impaired relative to
controls (P<0.001;
Figure 2). There was no difference between groups in speed
of swimming, minimizing the effects of a motor deficit on
performance. Ischemic groups were also impaired
relative to controls on all other measures, such as in the percentage
of time spent in the platform quadrant and annulus. During the probe
trial, in which the platform was removed, stroke rats exhibited
impaired recall of their precise position by spending less time in the
correct location and crossing the platform position less often than
controls. MHP36 grafts, therefore, did not ameliorate the marked
deficit in spatial learning and memory induced by
tMCAO.
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Spontaneous and Drug-Induced Rotation
Baseline (spontaneous) rotation was mildly asymmetric
in stroke rats without grafts
(Figure 3A), which turned more to the right than the left,
whereas control and grafted rats showed comparable turning in both
directions (F2,30=3.59,
P<0.05 for the difference
between groups). However, in response to amphetamine on weeks 2, 4, and
6 of testing, stroke rats without grafts showed marked leftward
turning, toward the lesioned side, indicative of increased dopamine
release on the intact side
(Figure 3B). Group differences were very substantial
(F2,30=5.71, 8.23, and 7.31,
P<0.01 for weeks 2, 4, and 6,
respectively), and the nongrafted group differed significantly
(P<0.01) from the grafted and
control groups, which did not differ in response to amphetamine. A
similar but less marked effect was seen with the postsynaptic dopamine
agonist apomorphine
(Figure 3C). Groups differed on weeks 3, 5, and 7
(F2,30=5.04, 3.32, and 3.66,
P<0.05), with the nongrafted
stroke group showing more marked leftward rotation than the grafted and
control groups (P<0.05), which
did not show a turning bias, apart from the grafted group on week 2. In
all groups the number of turns was lower in response to saline than to
the dopamine agonists. However, all groups showed similar activation,
so that drug-induced increases in bias in the nongrafted group were not
associated with differences in activity.
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Histology
Lesion Volume
tMCAO resulted in severe cavitation, amounting to
approximately 26% of total brain volume, so that groups differed
significantly in brain tissue volume
(F2,23=9.68,
P<0.01;
Table 2). Ventricles on the lesion side were enlarged, so
that only a thin strip of striatal tissue separated the lateral
ventricle from the lesion. Ventricles were also enlarged to a lesser
extent on the intact side. Distortion, possibly via tissue loss, had
pushed the midline toward the lesion side
(Figure 4). In grafted animals lesion size was significantly
reduced to approximately 16% of total brain volume, and therefore the
area of degeneration was only 35.8%
(P<0.05) of that seen in
nongrafted stroke rats
(Table 2).
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Distribution of Grafted Cells
MHP36 cells, identified by ß-gal immunocytochemistry,
were seen at the injection site in the middle of the intact striatum
(Figure 5). Cells were also seen caudally and laterally
throughout the striatum and entering the parietal cortex. However,
approximately a third of ß-gal–positive cells had moved away from
the side of implantation and were seen in abundance in the corpus
callosum, straddling the midline, and within the lesioned hemisphere.
Some cells were seen in the residual strip of striatum, adjacent to the
lesion, and some had left the corpus callosum to enter somatosensory
cortex. Neuronal or glial phenotype of grafted cells cannot be
identified in the absence of double labeling, but cells showed bipolar
and multipolar morphologies of several types, including neuron-like
cells, of pyramidal and medium spiny neuron appearance, and
glial-like cells, suggesting a diverse pattern of
differentiation.
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Discussion |
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The experiment examined effects of grafts of conditionally immortal MHP36 cells on the promotion of functional recovery from stroke damage when placed in the intact hemisphere, contralateral to the lesion cavity. Previous studies have shown MHP36 cell survival, migration, differentiation, and efficacy in cognitive tasks after 4-vessel occlusion in the rat and hippocampal lesions in the marmoset,11 12 13 14 which resulted in circumscribed CA1 cell loss. In the present study we sought to determine whether MHP36 grafts also promoted recovery from MCAO, which affects several brain regions, many types of cells, and both motor and cognitive function, with assessments performed during an unusually long period of 10 months after transplantation.
Bilateral Asymmetry
The bilateral asymmetry test, as described by
Schallert et al,16
resolves within 1 month after stroke. Our modification of wrapping tape
around the forelimbs required both sensory cortex and striatal
integrity and revealed a robust behavioral deficit over 18 weeks in
sham-grafted stroke animals. Grafted animals showed a comparable
deficit before transplantation, but when testing resumed at 6 to 7
weeks after graft (approximately 8 weeks after tMCAO), grafted rats
showed no difference in the time to remove the tape from the affected
and unaffected paws throughout testing, indicating long-term
improvement in a test in which no spontaneous recovery
occurred.
Primary fetal grafts have been shown to improve motor performance after cortical infarction6 7 21 or striatal lesions,22 but this may only be significant when grafts have been paired with enriched environments or training, pointing to a role for experience-dependent neuronal plasticity in recovery.3 4 Indeed, Grabowski et al6 found that an enriched environment was as functionally effective as fetal neocortical grafts, with no additive effects apparent. The mechanisms are not well understood, although Mayer et al22 suggest that training enables animals to learn how to use their transplants. This evidently was not the case with the recovery from bilateral asymmetry, since animals showed immediate improvement when testing was resumed 6 weeks after implantation. Recovery from sensorimotor neglect may therefore be more dependent on graft-enhanced corticospinal pathway reorganization in the lesioned or intact side of the brain than on experiential factors.
Spatial Learning in the Morris Water
Maze
The failure of the MHP36 grafts to improve spatial
learning in the water maze in this MCAO model suggests that the grafts
(1) do not provide enough tissue or appropriate connectivity to promote
recovery and/or (2) may be more effective in sensorimotor than in
cognitive tasks. Other studies with fetal grafting after distal
MCAO21 also failed to show
improvements in this test. However, Aihara et
al8 found that striatal fetal
grafts improved spatial learning in the water maze, but not the radial
maze, in rats with MCAO, suggesting that grafts can in some
circumstances reduce spatial deficits after stroke. We have recently
found that intraventricular MHP36 grafts improved
water maze learning in rats subjected to 60 minutes of tMCAO, while
rats with intraparenchymal grafts showed no
recovery,23 as in the
present study. Thus, graft placement may influence connectivity or
transmitter release and facilitate specific behaviors, despite the
marked migratory capacity of MHP36 cells.
Rotation
Stroke rats displayed mild spontaneous rotation
to the right, possibly reflecting a stronger push by the unaffected
left paw,6 whereas dopamine
agonist drugs induced marked rotation to the left, consistent
with activation of dopamine receptors on the intact side of the brain.
Neither asymmetry was evident in grafted rats. Increased release of
dopamine from grafted cells, as occurs with fetal nigral grafts in rats
with unilateral nigrostriatal lesions, is not likely to
account for reduced bias because MHP36 cells rarely present as
tyrosine hydoxylase positive and do not reduce bias in rats with
unilateral nigrostriatal lesions (S.B. Dunnett, PhD,
unpublished data, 1999). Since the majority of grafted cells
remained on the intact side of the brain, asymmetry would have been
amplified had they adopted a dopaminergic phenotype.
Alternatively, the large volume of cells infused may have damaged the
intact side, so that the dopaminergic agonists were ineffective on both
sides of the brain. However, equally large infusions of vehicle in sham
grafted stroke animals did not eliminate bias, and dopamine agonists
increased activity comparably in all groups, indicating that grafted
rats were not subsensitive to their motor stimulant effects.
Normalization of rotation bias may therefore have involved either
enhanced thalamic function or recruitment of a normally silent
corticospinal pathway on the intact side of the brain, as discussed
below.
Lesion Volume
The reduction of lesion volumes in the brains of
the MHP36 grafted group seen 11 months after transplantation is
probably due to the reduction of secondary degeneration and atrophy,
distant to the original
lesions.16 It would not be
possible for infusions of 24 µL (600 000 cells) to fill out cavities
averaging 260 mm3, particularly since
only a third of the cells migrated to the lesion side. Moreover, cells
were scattered in the parenchyma and did not infiltrate the lesion, in
contrast to fetal grafts, which have been used to fill out cortical
cavities.6 7 21
Release of trophic factors from relatively few grafted cells might
conceivably have contributed to prevention of delayed secondary
degeneration, since neuroepithelial stem cells have been shown to
express a variety of cytokines and growth factor
receptors.24 Environmental
factors may also be implicated, since Mattson et
al21 found that fetal grafts
in cortical cavities reduced thalamic atrophy and improved behavior
only when animals were also exposed to an enriched environment.
Possibly our extensive behavioral testing offered comparable
stimulation. Reduced lesion volume appears to be a delayed effect
because we have not seen a reduction in rats examined 3 months after
transplantation of MHP36 grafts, which exerted positive effects on
bilateral asymmetry.23 A
time course study is therefore essential to compare the evolution of
lesion size in grafted and nongrafted animals after 60 minutes of MCAO
and to relate volume change to behavioral outcome and to expression of
trophic factors in MHP36 cells.
Cell Placement and Migration: Implications for
Graft Mechanisms
MHP36 cells were infused in the intact hemisphere
because stroke lesions might not have provided a sufficiently
well-vascularized matrix to support their survival. Hadani et
al7 found that fetal cortical
grafts survived in the penumbra but not in the lesion core. A key
finding was that MHP36 cells not only dispersed within the intact
cortex and striatum but also migrated to the damaged hemisphere,
probably via the densely populated corpus callosum, to colonize
somatosensory cortex and residual striatum. Approximately two thirds of
grafted cells remained within the side of implantation, while one third
migrated to the contralateral hemisphere. We have subsequently
confirmed this impression by detailed counts of grafted cells in
regions of interest and found that similar proportions (ie, 30% to
35%) of cells migrate to the contralateral hemisphere, whether
implanted in the lesioned or intact side of the
brain.25 Increased nerve
growth factor expression induced by MCAO both in perilesion regions and
in the homologous contralateral cortex, which has lost
innervation,26 may guide
implanted stem cells to both hemispheres after stroke.
In addition to location, the identity of grafted cells is crucial to understanding the mechanisms of their effects. This preliminary study examined only cell distribution and lesion volume. Subsequently we have shown that up to 40% of grafted cells were neuronal by double labeling of PKH26, the fluorescent marker incorporated into cells before grafting, and the neuronal marker NeuN. Most of the remaining grafted cells were astrocytes (PKH26 and glial fibrillary acidic protein [GFAP] positive). There were more grafted neurons in the intact striatum, which harbored more cells than the lesioned striatum, but the proportions of neurons and glia were similar whether grafts were initially placed in the intact or lesioned hemisphere or in the ventricles. Grafted cells were found to adopt striatal phenotypes (positive for somatostatin, parvalbumin, calretinin, and choline acetyltransferase), but work is ongoing to quantify cell types in regions of interest (M. Modo, MSc, unpublished data, 2000) and to look for relationships with behavior.
Functional recovery after implantation distal to stroke damage found in this study provides some pointers to the possible mechanisms involved. Two possibilities are (1) that grafted cells migrated to the area of damage and reconstituted local circuits that were sufficient to sustain some functions and (2) that grafts augmented spontaneous reorganization on the intact side sufficient to undertake, or compensate for, some lost contralateral functions. Evidence from imaging studies suggests that both possibilities are reasonable. The contralateral hemisphere undergoes substantial plastic changes after stroke,27 28 suggesting the "unmasking" of ipsilateral corticospinal projections. Thus, Cramer et al27 found both enhanced activation in the intact side of the brain and foci of activation on the rim of the infarct in response to finger tapping in patients recovered from hemiparesis, while Dettmers et al28 found bilateral representation of movement in cortical motor association areas of stroke patients. The present results indicate that the majority of grafted cells dispersed within the side of implantation, and therefore a major contribution to recovery may have occurred though reinforcement of normally suppressed host ipsilateral corticospinal pathways. We subsequently showed25 that cells migrate to the opposite side whether placed ipsilaterally or contralaterally to stroke damage. Unexpectedly, apolipoprotein E (apoE) was upregulated in the intact striatum of rats with both ipsilateral and contralateral grafts, but not in sham-grafted controls, where it was confined to the lesion borders and ipsilateral thalamus (K. Hopkins, BSc, unpublished data, 2000). ApoE, a lipid transporter, is associated with both clearance of cell debris and recovery of neurons after ischemic brain damage.29 It was coexpressed in both neuronal (NeuN positive) and glial (GFAP positive) grafted cells prelabeled with PKH26, as well as in host neurons and glia. This unique expression of apoE, in association with host and grafted neurons in the intact striatum of stroke rats, suggests that it may provide a marker for graft-associated neuronal remodeling. Thus, grafts may stimulate a silent into a functionally active pathway, a suggestion that requires further investigation. Taken together, these results suggest that several mechanisms may be involved in recovery after stem cell grafts in rats with stroke damage, including interactions with site of grafting and with host tissue undergoing both degeneration and reorganization.
Conclusions
The results suggest that MHP36 grafts
contralateral to site of stroke damage exert positive functional
effects and reduce lesion volume. These findings provoke an
investigation of novel graft mechanisms, which will increase our
knowledge of how grafts may be used to harness and augment brain
plasticity in response to stroke damage. Stem cell grafts may serve to
augment constitutive and inducible mechanisms of plasticity rather than
to "fill the hole." These results may increase the flexibility of
transplant surgery, so that grafts may be placed at a distance from
stroke damage in brain regions that are more favorable to their
survival and
integration.
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Acknowledgments |
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This work was supported by ReNeuron Ltd. We wish to thank Peter Sowinski, MvetSc, for care of the animals to ensure their long-term survival.
Received July 20, 2000; revision received December 18, 2000; accepted December 27, 2000.
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