Neuroscience Vol. 101, No. 4, pp. 945–955, 2000
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䉷 2000 IBRO. Published by Elsevier Science Ltd
Printed in Great Britain. All rights reserved
0306-4522/00 $20.00+0.00
Stem cell grafts improve memory in aged rats
Pergamon
PII: S0306-4522(00)00408-5
www.elsevier.com/locate/neuroscience
CONDITIONALLY IMMORTAL NEUROEPITHELIAL STEM CELL GRAFTS
REVERSE AGE-ASSOCIATED MEMORY IMPAIRMENTS IN RATS
H. HODGES,*†‡ T. VEIZOVIC,† N. BRAY,* S. J. FRENCH,† T. P. RASHID,† A. CHADWICK,† S. PATEL†
and J. A. GRAY*†
*Department of Psychology and †ReNeuron, Institute of Psychiatry, King’s College London, De Crespigny Park,
Denmark Hill, London SE5 8AF, UK
Abstract—In order to investigate the effects of stem cell grafts on water maze deficits in aged (22-month-old) rats, three groups of
aged rats, assigned by pre-training latency scores to unimpaired, impaired control and impaired grafted groups, were compared with
young (five-month-old) controls, six to eight weeks after implantation of cells from the conditionally immortal Maudsley hippocampal stem cell line, clone 36 (MHP36 stem cell line), in the cortex, striatum and hippocampus. Grafted rats were substantially
superior to their matched impaired aged controls, and learned to find the platform as rapidly as unimpaired aged rats, although
young controls were more efficient than all aged groups in several measures of spatial search during training. On the probe trial,
however, aged rats with grafts showed significantly better recall of the precise position of the platform than any other group,
including young controls, possibly indicating some perseveration. A further comparison found that groups of unimpaired and
moderately impaired aged rats showed far less improvement from water maze pre-training to acquisition phases than young
controls, indicative of progressive deficits over time. Histological investigation showed that b-galactosidase-positive MHP36
cells migrated widely from the implantation sites to infiltrate the striatal matrix, all hippocampal fields and areas of the cortex.
Grafted cells showed both astrocytic and neuronal morphologies, with cells of pyramidal and granular appearance in appropriate
hippocampal strata.
Taken together, these results indicate that neuroepithelial stem cell grafts extensively colonize the aged rat brain and substantially reverse progressive cognitive decline associated with ageing. 䉷 2000 IBRO. Published by Elsevier Science Ltd. All rights
reserved.
Key words: MHP36 cell line, aged rats, intracerebral transplantation, water maze, spatial learning.
et al., 26 for example, showed that grafts of the Maudsley
hippocampal stem cell line, clone 36 (MHP36 stem cell
line), derived from the neuroepithelium of transgenic mice
expressing a mutant allele of SV40 large T antigen (H2KbtsA58), repopulated the area of hippocampal CA1 ischaemic
cell loss induced by four-vessel occlusion, expressed neuronal
and glial morphologies, and reversed deficits in water maze
learning. MHP36 cells have four key advantages for transplantation. They are: (i) conditionally immortalized by a
temperature-sensitive oncogene so that they expand in culture
but cease dividing when grafted; (ii) multipotent, expressing
neuronal, glial and oligodendrocyte phenotypes in vivo and in
vitro; (iii) multifunctional, improving performance in several
models of brain damage, in different strains of rat (Wistar and
Sprague–Dawley) and even across species in marmosets; (iv)
migratory and specifically attracted to damaged areas. 8,9,26,27
Migratory and multipotential properties are particularly relevant to repair of the aged brain, where it is not possible to
target diffuse cell loss surgically. Our recent findings, 9 that
impairments in spatial learning induced by cholinergic lesions
were reversed by MHP36 grafts, encouraged investigation of
their efficacy in improving deficits in impaired old rats.
Moderately impaired aged rats were also used to examine
the normal progression of deficits in ageing as a context for
graft effects, and to see whether decline was significant
enough to permit the use of repeated measures in all aged
rats to assess treatment effects in future studies, which
would avoid wastage of valuable aged animals.
A variety of changes occur in the aged brain, including
increased cortical GABAergic inhibitory tone, loss of cholinergic neurons and altered noradrenergic receptor sensitivity. 1,12,20 Age-associated memory impairments in rats have
been linked to these changes, particularly to progressive
degeneration of cholinergic neurons. 2,7,11,12 Transplant strategies to alleviate cognitive deficits in aged animals have
chiefly sought to enhance cholinergic system function, initially using cholinergic-rich primary fetal grafts to compensate
for transmitter loss. 5 More recently, genetically modified
fibroblasts, 4 encapsulated cells 28 or conditionally immortalized nerve growth factor (NGF)-releasing progenitor cells 15,16
have been investigated, since these types of graft circumvent
the ethical and practical problems associated with the use of
primary fetal tissue. These non-fetal grafts have been shown
to improve learning and restore cholinergic neurons in aged
rats. Martinez-Serrano and Bjorklund 17 also showed that NGFsecreting progenitors grafted in middle-aged (14- to 16-monthold) rats prevented the development of normal age-related
degeneration of cholinergic neurons and cognitive decline.
Grafts that target cholinergic neurons may not be able to
restore deficiencies in other neural systems contributing to
cognitive decline. 14 Conditionally immortal stem cell lines
which express different phenotypes in response to signals
from the adult brain permit more flexible repair. 13,24 Sinden
‡To whom correspondence should be addressed at: Department of
Psychology. Tel.: ⫹44-207-848-0252; fax: ⫹44-207-848-0987.
E-mail address: [email protected] (H. Hodges).
Abbreviations: CA, Cornu Ammonis; b-Gal, b-galactosidase; MHP36,
Maudsley hippocampal stem cell line, clone 36; NGF, nerve growth
factor.
EXPERIMENTAL PROCEDURES
Experiments were conducted in accordance with the UK Animals
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H. Hodges et al.
(Scientific Procedures) Act 1986, the Ethical Review Process of the
Institute of Psychiatry and the European Communities Council Directive 86/609/EEC. Care was taken to reduce stress and to increase
comfort by daily handling and attention to post-operative care.
Animals
Fifty-six aged male Sprague–Dawley rats (22 months on arrival and
26 months at perfusion) and 21 young controls (three months on arrival
and seven months at completion) completed all phases of the experiments. All efforts were made to minimize the number of animals used.
Aged rats were caged in pairs and fed a restricted diet so as to
maintain their weight at a moderately stable mean of 709 ^ 97 g.
Young rats were housed five to a cage, fed ad libitum and weighed
422 ^ 49 g at the start of testing. All animals were maintained on a 12h/12-h light–dark schedule (lights on at 8.00 a.m.), and were handled
daily between arrival and the start of behavioural training.
allow diffusion away from the tip. Aureomycin powder was sprinkled
on the wound, which was closed with Michel clips, and the rats were
injected with Revivon (diprenorphine, 0.272 mg/ml, 0.01 ml/100 g).
Half of the aged impaired controls and young controls were sham
grafted with vehicle at the graft coordinates. This was intended as a
control for effects of surgery, and as the subsequent performance of
sham and unoperated animals was indistinguishable, the two aged
impaired groups were pooled into a single aged impaired control
group. After surgery, rats were housed individually until pre-operative
weight was regained, monitored daily, fed wet mash, which was given
by hand if necessary, until normal feeding and movement were
observed (one to two weeks). Grafted and control rats were injected
with cyclosporin A (Sandimmun, Sandoz, Basel, Switzerland; 10 mg/kg,
s.c.) mixed with Cremophor EL (a derivative of castor oil and ethylene
oxide; Sigma, UK; in a volume of 1:3) after surgery and three times a
week for two weeks.
Histology
Behavioural testing
Spatial learning and memory were assessed in the Morris water
maze, a 200-cm-diameter pool of black polythene. Wall height was
50 cm and the pool was filled to a depth of 25 cm with 24 ^ 2⬚C water
clouded with powdered milk. The rat’s task was to locate a 20-cm
platform submerged 2 cm below the surface of the water. Start points
were designated N, S, W and E, and the pool was conceptually divided
into four quadrants (1–4) and three annuli (A–C), with C outermost
and A innermost. Rats were trained for two trials/day with a 10-min
inter-trial interval. If they failed to find the platform within 60 s, they
were guided to it by the experimenter (who stood in an adjacent lobby
during the trial) approaching the pool and pointing down to the position. All animals rapidly learned to swim to the pointing hand, indicating that gross visual deficits or motivational differences were not
apparent in aged rats during training. Rats were left on the platform
for 10 s before being placed in the waiting box or towelled dry and
returned to the home cage. The swim path was recorded by an image
analysing system (HVS Image, Hampton, UK), which computed
latency to find the platform, distance swum, heading angle (a measure
of divergence from the direct path to the platform), and percentages of
time spent in the quadrants and annuli. After training, a probe trial was
given with the platform removed to assess memory for its location by
time spent in the training quadrant and in the vicinity of the platform
position (the counter area of twice the platform diameter), and by the
number of crossings of the platform position.
Aged rats and young controls were pre-trained in the water maze for
eight days, and old rats were divided into two groups above and below
the mean latency averaged over the last three days of pre-training (i.e.
both trials on days 6–8, when learning was maximal). Rats above the
mean, designated as “impaired”, were further divided into two groups
matched for latency and assigned to graft or control groups. The best
performers below the mean were assigned to the “unimpaired” control
group. These animals did not differ in pre-training latency from the
young controls. Six to seven weeks after transplantation, all the rats
were trained to find the platform in a different quadrant for 12 days
(two trials/day), followed by a probe trial on day 13 with the platform
removed. Rats were then perfused for histology.
Surgery
MHP36 cells (see Ref. 26 for their derivation) were taken from
frozen stock (passages 37–38). Two days before transplantation,
cells were pulsed with 0.5 mCi/ml [ 3H]thymidine for identification by autoradiography. For grafting, cells were suspended in 1 mM
N-acetyl-l-cysteine in Hank’s balanced salt solution at a density of
25,000 cells/ml. Initial viability averaged 92% and post-graft viability
was 85%, as counted by Trypan Blue exclusion in a haemocytometer.
Rats were pre-treated with 0.03 ml/100 g of Hypnovel (midazolam,
5 mg/ml) and after 5 min were anaesthetized with 0.01 ml/100 g of
Immobilon (etorphine hydrochloride, 0.074 mg/ml, and methotrimeprazine, 18 mg/ml). In grafted rats, six holes were drilled in the
skull and a 10-ml Hamilton syringe lowered under stereotaxic control
to deliver 0.3-ml infusions of cell suspension or vehicle bilaterally over
2 min in each of three regions defined by the following coordinates,
with bregma as the reference point, depth measured from dura and the
skull set in the flat position: frontal cortex, AP 3.2, L ^3.5, V ⫺3.0;
hippocampus, AP ⫺3.3, L ^1.5, V ⫺2.7; striatum, AP ⫺0.2, L ^3.2,
V ⫺5.2. The syringe was left in place for 2 min after each delivery to
At the end of behavioural testing, 10–11 weeks after grafting, rats
were perfused transcardially under terminal pentobarbital anaesthesia
with 4% paraformaldehyde in 0.1 M sodium phosphate buffer after
flushing with 0.9% saline. The brains were wax-embedded and 10-mm
coronal sections were stained with Cresyl Fast Violet. MHP36 cells
were identified using antibody to b-galactosidase (b-Gal), the protein
product of the LacZ reporter gene incorporated into the MHP36 cell
line, 26 and [ 3H]thymidine autoradiography. Some aged rat cells in the
hippocampus, most obviously in the CA1 field, appeared shrunken,
distorted and less dense than in young controls. As an indication of
whether CA1 cell damage might be associated with (i) cognitive deficits or (ii) with surgery under anaesthesia through possible ischaemic
damage via reduced cerebral blood flow or oxygen levels, cell counts
were undertaken in the dorsal CA1 field in Cresyl Fast Violet sections
at approximately 5.7 mm anterior to the inter-aural line in impaired rats
that underwent graft surgery, sham surgery or no surgery, in comparison with unoperated old unimpaired rats and sham-operated young
controls (n ˆ 5 per group). At this level, we have found the greatest
CA1 cell loss in animals subjected to global ischaemia (15 min of fourvessel occlusion), which correlated with deficits in spatial learning. 18
For counting, a frame of 0.0207 mm 2 was placed without bias over the
cell layer. Cells were counted within the frame, unless they overlapped
the top or right-hand side of the frame. Since the study was not
designed for full stereological assessment of hippocampal cells, 21
this limited count in a subset of the animals was used to provide an
estimate of cell numbers within the CA1 region that is most sensitive to
ischaemia.
Statistical analysis
The behavioural data were derived from four groups of aged rats:
aged unimpaired (n ˆ 16), aged moderately impaired (n ˆ 16) with
scores below the mean pre-training latency, aged impaired rats with
MHP36 grafts (n ˆ 9) and aged impaired controls (n ˆ 15, including
five sham-operated and 10 unoperated animals) above the mean
latency, and two groups of young controls (one sham operated,
n ˆ 11, the other unoperated, n ˆ 10). The key comparisons (Experiment 1) were between the aged impaired grafted group, aged impaired
and unimpaired controls, and young sham-operated controls. These
four groups were compared for both pre-training and acquisition, in
order to see how grafting affected the performance of aged rats with
respect to the three control groups. A second set of comparisons
(Experiment 2) was made between aged unimpaired rats, aged moderately impaired rats and young non-operated controls, in pre-training
and acquisition. This was undertaken to look at natural changes over
time in old rats in comparison with young controls, as a context for the
effects of transplantation. Experiment 2 provided additional control for
the possibility that any apparent graft-induced improvement in Experiment 1 might reflect the effects of pre-graft learning on later performance. Experiment 2 also looked, in particular, for evidence that
changes in performance over time in all old rats might provide a
sufficiently clear baseline against which to measure treatment effects
in future studies, without the need to exclude non-significantly
impaired aged animals. This exclusion is standard practice in ageing
studies, but it is wasteful, since up to 50% of aged animals typically fall
into this category, and thus ethically questionable. In both experiments,
the data were subjected to ANOVA (Genstat V PC); orthogonal trends
of changes over days were extracted to show progression of learning by
Stem cell grafts improve memory in aged rats
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Fig. 1. Identification of grafted cells. MHP36 cells in the premotor cortex showing co-localization of [ 3H]thymidine autoradiography (grains) and immunoreactivity to b-Gal. Scale bar ˆ 20 mm.
significant linear trends and interactions between groups and the linear
trends of days. Groups were compared by the t ratio using the standard
error of the difference between means from the ANOVA.
Counts of cells in the dorsal CA1 field were compared between
groups by ANOVA, followed by comparisons of means by the t ratio.
Correlations across groups between the numbers of cells and the distance
swum to find the platform on day 8 of pre-training (before surgery), and
days 4 and 12 (i.e. early and late in training) of acquisition after surgery,
were made using Spearman’s rank order correlation coefficient. In all
cases, P ⬍ 0.05 was considered statistically significant.
RESULTS
Histology
Mean (^S.E.M.) counts of CA1 pyramidal cells/mm 2 at
5.7 mm anterior to the inter-aural line were 2950 ^ 95 in
young controls, 2873 ^ 236 in unimpaired aged rats and
2793 ^ 197 in impaired aged rats that did not undergo
surgery, and somewhat lower in the two aged groups
subjected to surgery: 2457 ^ 125 in impaired aged controls
and 2544 ^ 112 in impaired grafted rats. However, none of
the groups differed significantly. Moreover, no significant
correlations across animals were found between the number
of cells counted and distance swum to find the platform in pretraining (day 8), or early (day 4) or late (day 12) in training
(n ˆ 25, rs ˆ 0.03, ⫺0.25 and ⫺0.14, respectively, for the
three distance measures). Therefore, no associations were
detected between CA1 cell numbers at this level and spatial
learning ability, nor could reduced CA1 cell numbers be reliably associated with surgery in the animals examined.
Histology was undertaken to determine the presence and
distribution of grafted cells. Cells were injected at three sites
in the frontal cortex, striatum and the alveus above the hippocampus. Nissl-stained sections clearly revealed these injection sites. b-Gal-positive cells were seen in all grafted
animals, spreading out from the sites of injection. Double
staining of b-Gal and autoradiographic silver grains (Fig. 1)
confirmed that b-Gal reactivity labelled grafted cells. At the
most anterior site targeted to the frontal cortex (AP ⫹3.2), bGal-positive cells were seen in the secondary motor cortex, at
the injection site, from which they spread into the forceps
minor of the corpus callosum and into the primary motor
cortex (see Fig. 2A and B). At the second injection site, at
the level of the caudate–putamen (AP ⫺0.2), grafted cells
were evident in the needle tract, which went through the
primary and secondary motor cortices and into the striatum.
Cells migrated dorsolaterally through the cingulate cortex and
considerable distances along the entire corpus callosum.
Many b-Gal-positive cells within the caudate–putamen
nestled among host cells predominantly in the matrix
compartment of the striatum (see Fig. 2C and D). The third
injection site targeted the dorsal hippocampus, and b-Galpositive cells were seen in abundance in the alveus (the site
of injection) and in the underlying hippocampal layers (Fig.
2E–H). Density was relatively low in the pyramidal cell
layers of the CA1–CA3 fields, but cells were well integrated
(Fig. 2F). Cells were also seen in the strata oriens and radiatum of the CA3 region. The hippocampal fissure attracted rich
migration (Fig. 2G) and the dentate gyrus also contained wellintegrated cells in the granule layer, and many cells within
both the stratum moleculare and the hilus (Fig. 2H). In addition to dispersal within the hippocampus, cells colonized the
primary and secondary motor cortices in large numbers,
migrating up the needle tract, which penetrated these regions.
Cells migrated further through the white matter tracts [cingulum, corpus callosum (Fig. 2E) and the internal and external
capsule], and reached the caudate–putamen. b-Gal-positive
cells adopted several clearly differing morphologies: glial,
neuronal or possibly undifferentiated, which appeared to be
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H. Hodges et al.
Fig. 2.
Stem cell grafts improve memory in aged rats
949
double labelling is required to identify cell phenotypes.
MHP36 graft histology can be viewed at the ReNeuron
website (http//www.reneuron.com).
Experiment 1. Effects of MHP36 grafts in aged rats
Fig. 3. Experiment 1, pre-training: mean latency to find the platform in aged
rats and young controls. Aged rats were divided into impaired and unimpaired groups above and below the mean of the last three days of pretraining. Aged impaired rats assigned to control (n ˆ 15) and grafted
(n ˆ 9) groups were matched for latency. The aged unimpaired group
(n ˆ 16) was selected as showing the fastest latency below the mean.
Performance was comparable in the aged unimpaired and young control
(n ˆ 11) groups, which were both significantly superior (P ⬍ 0. 01) to the
impaired aged animals destined for control and grafted groups. Bar shows
twice the standard error for the difference in means (2 SED) for the
Groups × Days interaction.
related to their location. Cells around the injection sites were
large, rounded, lacked processes and may not have differentiated (Fig. 2C). Cells migrating along the corpus callosum
were predominantly linear and many appeared to be bipolar, a
morphology possibly influenced by the tightness of the
surrounding fibres (Fig. 2E). Cells within the hippocampus,
forebrain primary motor cortex and striatum appeared to
express several different morphologies, resembling classical
neuronal (pyramidal, granular) and astrocytic phenotypes (see
Fig. 2F–H for examples of grafted cell types within the hippocampus). The size of pyramidal-like cells was consistently
smaller than normal rat CA1–CA3 pyramidal cells, but
comparable to that of dentate granule cells. In summary,
grafted MHP36 cells dispersed widely, and colonized host
structures of the caudate–putamen and hippocampus in a
non-random way, since (i) they aligned within the cell body
layers of the hippocampus and the matrix compartment of the
striatum, and (ii) they adopted somewhat different morphologies according to the brain region occupied. However,
Assignment to groups: pre-training before transplantation.
Aged rats were divided on the basis of the mean latency to
locate the platform averaged over the last three days of pretraining, into two impaired groups of equivalent performance
above the mean; one subsequently received MHP36 grafts
and the other formed the impaired aged control group. The
best performers below the mean were designated as unimpaired. Aged rats were compared with young controls.
These groups differed significantly for latency (F3,47 ˆ 22.05,
P ⬍ 0.001; see Fig. 3). Aged unimpaired and young control
groups showed a more rapid rate of learning than the two
impaired groups (F3,329 ˆ 6.19, P ⬍ 0.001 for the interaction
of groups with the linear trend of days). In comparison of
means, overall time taken to find the platform was significantly reduced in aged unimpaired and young controls relative to the two aged impaired groups (P ⬍ 0.01), which did
not differ. Aged unimpaired and young control groups also
spent a higher percentage of time (F3,47 ˆ 10.38, P ⬍ 0.001)
searching appropriately in the platform quadrant than the two
impaired aged groups (P ⬍ 0.01 by comparison of means).
Thus, the division of aged rats at the mean latency late in
pre-training resulted in group assignments that (i) discriminated clearly between aged unimpaired and impaired groups,
(ii) provided two aged impaired groups that did not differ
in any respect, and (iii) confirmed that aged rats deemed
unimpaired were as efficient in spatial navigation as young
controls.
Acquisition after transplantation. Six to eight weeks after
transplant or sham transplant surgery, rats were trained in the
water maze for 12 days. The key issues were as follows. (1)
Did aged impaired rats with MHP36 grafts now perform
better than their matched impaired controls? (2) If so, did
they now perform as well as aged unimpaired animals? (3)
How well did all of the aged groups perform in comparison
with the young controls?
Latency. There was a substantial difference between groups
(F3,47 ˆ 43.58, P ⬍ 0.001), and between rates of learning
(F3,517 ˆ 12.79, P ⬍ 0.001 for the interaction of groups with
the linear trend of days). Young sham-grafted controls found
the platform more rapidly than all of the aged groups
(P ⬍ 0.01). However, the aged impaired group with MHP36
grafts, which before grafting had performed poorly, now
reached the platform as rapidly as the aged unimpaired rats
(see Fig. 4A), and both of these groups were substantially
superior to the aged impaired controls (P ⬍ 0.01).
Fig. 2. Distribution of grafted cells in the cortex, striatum and hippocampus. (A, B) Distribution of b-Gal-positive MHP36 cells (brown) in the frontal cortex:
migrating from the injection site (A, arrow), and at higher magnification showing both multipolar (B, arrowhead) and bipolar (B, arrow) morphologies. (C, D)
Distribution of b-Gal-positive MHP 36 cells (brown) in the striatum: near the injection site (C) and widely dispersed in the matrix compartment of the striatum
(D). At the injection site, cells were of a consistent rounded appearance, showing little differentiation. In the striatum, they showed both neuronal (arrows) and
astrocytic (arrowhead) morphologies. (E–H) Distribution of b-Gal-positive MHP36 cells (brown) in and above the hippocampus. In the corpus callosum
above the CA1 field (E), both rounded pyramidal-like (arrowhead) and elongated (arrow) cells are seen. In the CA1 field (F), pyramidal-like cells (arrows)
appear to be well integrated, but are smaller in size than the host CA1 cells (arrowhead). Many cells of both astrocytic and neuronal appearance are clustered in
the hippocampal fissure (G). In the dentate gyrus (H), MHP36 cells appear to be granular in the dentate granule layer (arrows), but of mixed interneuronal and
astrocytic appearance in the hilus. Scale bars ˆ 200 mm (A), 50 mm (B–F, H), 20 mm (G).
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H. Hodges et al.
the aged groups (F3,47 ˆ 13.30, P ⬍ 0.001; P ⬍ 0.01 in comparisons of young rats with the three aged groups). Thus, in
terms of path length, a measure of search accuracy that is not
confounded by motor effects, the young controls, aged
unimpaired and aged grafted groups were equivalent. The
three aged groups did not differ for swim speed, so that
motor effects cannot account for the difference between the
two impaired groups with and without grafts.
Heading angle. Group differences in search accuracy were
further exemplified by measures of heading angle
(F3,47 ˆ 3.89, P ⬍ 0.02). Young controls showed the greatest
mean accuracy (39.6⬚) and the aged impaired non-grafted
control group was the least accurate (51.3⬚), with the aged
unimpaired (42.7⬚) and aged impaired group with MHP36
grafts (46.7⬚) intermediate. Both the young controls and the
aged unimpaired groups were significantly more accurate
than the aged impaired controls (P ⬍ 0.05), but the aged
impaired grafted group did not differ from either aged
impaired or unimpaired control groups.
Fig. 4. Experiment 1, acquisition: mean latency and distance swum to find
the platform in aged rats and young controls. Mean time taken (A) and
distance swum (B) to reach the platform in aged impaired controls (n ˆ 15),
aged impaired rats with MHP36 grafts (n ˆ 9), aged unimpaired rats
(n ˆ 16) and sham-operated young controls (n ˆ 11). The aged impaired
control animals showed substantial deficits (P ⬍ 0.01) relative to all other
groups on both measures. Aged unimpaired and aged impaired rats with
transplants took longer to find the platform than the young controls
(P ⬍ 0.01), but this reflected a slower swim speed, because they did not
differ from young controls in terms of distance swum. Bar shows twice the
standard error for the difference in means (2 SED) for the Groups × Days
interaction.
Distance. Groups differed significantly (F3,47 ˆ 7.26,
P ⬍ 0.001), with a slower decrease in path length over Days
in aged impaired rats relative to the other groups (F3,517 ˆ 8.79
for the interaction of groups with the linear trend of days; Fig.
4B). However, in contrast to latency, young controls did not
differ from aged unimpaired and grafted groups; thus, all three
groups were equally efficient in terms of path length to the
platform. These three groups swam shorter distances to locate
the platform than aged impaired controls (P ⬍ 0.01 in overall
mean comparisons). The discrepancy between the superiority
of the young controls over aged unimpaired and impaired
grafted groups in latency, but not in distance, resulted from
the fact that the young controls swam much faster than any of
Percentages of time in the pool sectors. Young controls
spent the highest percentage of time in the training quadrant
(Quadrant 1) relative to all three aged groups (F3,47 ˆ 7.76,
P ⬍ 0.001; P ⬍ 0.01 in overall comparisons of young rats
with the three aged groups). Within the three aged groups,
however, both the unimpaired controls (P ⬍ 0.01) and the
impaired grafted group (P ⬍ 0.05) spent more time in the
training quadrant than the aged impaired controls. The aged
unimpaired group was also marginally superior to the
impaired grafted group (P ⬍ 0.05). There were also significant differences between groups for time spent in the centre of
the pool (annulus A: F3,47 ˆ 4.58, P ⬍ 0.01) and at the perimeter (annulus C: F3,47 ˆ 3.66, P ⬍ 0.02), because the aged
impaired group ventured least into the centre and spent a
greater proportion of time circling the pool wall than all
other groups (P ⬍ 0.01), which did not differ in these
measures. All groups spent a comparable proportion of time
in annulus B containing the platform.
In summary, during acquisition, impaired aged rats with
MHP36 grafts were consistently superior to the aged impaired
controls on all measures apart from heading angle. They
performed as well as aged unimpaired rats in all respects
except for the percentage of time spent in the training quadrant. In comparison with young controls, both aged unimpaired and aged impaired rats with MHP36 grafts swam
equivalent distances to locate the platform, but young controls
showed greater accuracy than aged grafted animals in heading
angle. In addition, the percentage of time spent in the training
quadrant was greater for young controls than all the aged
animals, so that in these measures neither aged unimpaired
rats nor aged impaired rats with MHP36 grafts performed as
well as young controls.
The probe trial. This was used to see how far improvements
shown by aged rats with MHP36 grafts in learning the platform position resulted in improved memory for its location.
All rats spent longer in the former training Quadrant 1 than in
any other sector (F3,141 ˆ 5.96, P ⬍ 0.001 for the main effect
of Quadrant), indicating good recall of the general platform
position. However, groups differed in their distribution of
time, as shown by the Groups × Quadrants interaction
(F9,141 ˆ 2.29, P ⬍ 0.025). Aged impaired rats with MHP36
Stem cell grafts improve memory in aged rats
951
measures; see Fig. 5B) than any other group. Young controls
and aged unimpaired groups were superior to the aged
impaired controls in these measures of localized search, but
the differences were not reliable. Groups also differed in their
distribution of time in the annuli. Aged impaired rats spent a
lower (P ⬍ 0.05) percentage of time in annulus A than the
other groups, which did not differ, whilst spending a significantly greater proportion of time in annulus C (P ⬍ 0.05
in comparison with the other groups), thus maintaining the
inefficient search pattern evident during acquisition. All
groups spent a similar percentage of time in annulus B.
Results from the probe trial indicated that aged rats with
grafts remembered the precise platform position very well,
but the abnormal length of time spent there and large number
of crossings may indicate a degree of perseveration.
Experiment 2. Progression of deficits in aged rats
Since the pre-training and acquisition phases were spaced
10 weeks apart, there was an opportunity to examine the
effects of the passage of time in aged animals that initially
had no, or only mild, deficits relative to young controls.
Normal adult rats typically show marked improvement on
the second of two training periods in the same pool. This
analysis was undertaken to see whether such improvement
also occurs in aged rats. None of the animals had undergone
sham surgery. Two groups of aged rats were used, unimpaired
(n ˆ 16) drawn from the best performing rats above the mean
during pre-training, which also served as unimpaired controls
in Experiment 1, and the remaining “moderately impaired”
(n ˆ 16) animals above the mean. Aged rats were compared
with a group of young controls (n ˆ 10). Only data from the
first eight days of acquisition were used, to equate the number
of days across pre-training and acquisition phases.
Fig. 5. Experiment 1: probe trial performance of aged rats and young
controls. (A) Aged unimpaired rats (n ˆ 16), aged impaired rats with
MHP36 grafts (n ˆ 9) and young controls (n ˆ 11) showed a significant
preference for the training quadrant. This was not seen in the aged impaired
control group (n ˆ 15), which spent a lower percentage of time (P ⬍ 0.05)
in Quadrant 1 than the other groups. (B) Aged rats with transplants were
superior to all other groups (P ⬍ 0.01) in time spent in the platform (counter) area and number of crossings of the platform position. Bars show twice
the standard error for the difference in means (2 SED) between groups.
Difference from young controls: *P ⬍ 0.05; **P ⬍ 0.01.
grafts spent longest in Quadrant 1 and the aged impaired
control group spent least time there, bracketing the aged
unimpaired and young control groups (see Fig. 5A). Young
controls, aged unimpaired controls and aged impaired grafted
rats did not differ in preference for the training quadrant, and
all three groups spent a significantly greater proportion of
time in Quadrant 1 than aged impaired controls (P ⬍ 0.05
for young and aged unimpaired controls, P ⬍ 0.01 for aged
impaired grafted rats). In terms of precise recall of the platform position, shown by time spent in the “counter” area of
twice the platform diameter and number of crossings of the
former platform position, the results showed a clear advantage for aged impaired rats with MHP36 grafts relative to all
other groups (F3,47 ˆ 8.72 and 7.64, P ⬍ 0.001 for time in
counter 1 and number of crossings, respectively). Aged
grafted rats spent significantly longer in counter 1 and crossed
the platform position more frequently (P ⬍ 0.01 for both
Pre-training. Comparison of latencies of the three groups
during pre-training (F2,39 ˆ 7.19, P ˆ 0.002) indicated that
moderately impaired aged rats took longer to find the platform
than aged unimpaired (P ⬍ 0.05) and young control groups
(P ⬍ 0.01), but aged unimpaired and young control groups did
not differ (see Fig. 6). However, young rats swam more
rapidly (F2,39 ˆ 11.53, P ⬍ 0.001), so that they covered more
distance (F2,39 ˆ 8.18, P ⬍ 0.001) at a faster rate than the aged
groups (P ⬍ 0.01 in all comparisons; see Fig. 7). The two aged
groups did not differ in swim speed. Groups also differed for
percentages of time in the pool sectors (F2,39 ˆ 3.56, P ⬍ 0.05
for Quadrant 4, and F ˆ 7.04, P ˆ 0.002 for annulus C),
because moderately impaired aged rats spent less time
(P ⬍ 0.05) in the training quadrant and more time circling
the perimeter (P ⬍ 0.01) than the aged unimpaired or young
control groups, which did not differ.
Acquisition. There was a clear separation in the performance of the three groups in time taken to find the platform
(F2,39 ˆ 67.39, P ⬍ 0.001). Young controls were markedly
superior (P ⬍ 0.001) to both of the aged groups, while the
aged unimpaired group found the platform more rapidly
(P ⬍ 0.001) than the moderately impaired aged rats (see
Fig. 6). Distance swum also differed markedly in the three
groups (F2,39 ˆ 17.12, P ⬍ 0.001). Path lengths for young
controls were also shorter than those for the moderately
(P ⬍ 0.001) and unimpaired (P ⬍ 0.05) aged groups (see
Fig. 7), whilst unimpaired rats swam shorter distances than
952
H. Hodges et al.
Fig. 6. Experiment 2: mean latency to find the platform in pre-training and
acquisition phases in aged unimpaired and moderately impaired rats, and
young controls. Young control rats (n ˆ 10) showed substantial improvement (P ⬍ 0.001) and aged unimpaired rats (n ˆ 16) showed modest
improvement (P ⬍ 0.05) over phases, but moderately impaired aged rats
(n ˆ 16) did not improve at all. Bar shows twice the standard error for the
difference in means (2 SED) for the Groups × Phases interaction.
moderately impaired aged rats (P ⬍ 0.01). Group differences
in speed (F2,39 ˆ 17.13, P ⬍ 0.001) arose because young
controls swam significantly (P ⬍ 0.001) faster than both of
the aged groups, which did not differ. However, motor effects
cannot account for their superiority because distance swum to
locate the platform was also significantly reduced. Parameters
of heading angle (F2,39 ˆ 8.97, P ⬍ 0.005) and search in the
training quadrant (F2,39 ˆ 38.77, P ⬍ 0.001) confirmed the
marked differences between groups in efficiency of spatial
navigation, with young controls performing significantly
better than unimpaired aged rats (P ⬍ 0.01), which in turn
performed significantly better than moderately impaired
aged animals (P ⬍ 0.01) in both of these measures. Moderately impaired aged rats also spent less time in the innermost
annulus A and more time circling the pool wall in annulus C
(P ⬍ 0.01 in all comparisons) relative to unimpaired aged
animals and young controls, which did not differ in annulus
measures. Results from acquisition indicated that although
Fig. 7. Experiment 2: mean distance swum to reach the platform in pretraining and acquisition phases in aged unimpaired and moderately
impaired rats, and young controls. Young controls (n ˆ 10) showed
substantial (P ⬍ 0.001) reduction in path length in acquisition relative to
pre-training, unimpaired aged rats (n ˆ 16) showed a reliable (P ⬍ 0.01)
reduction, but the moderately impaired aged group (n ˆ 16) showed only a
modest (P ⬍ 0.05) improvement. Bar shows twice the standard error for the
difference in means (2 SED) for the Groups × Phases interaction.
unimpaired old rats remained superior to the moderately
impaired animals, they were now significantly worse than
young controls in spatial navigation.
The probe trial. These studies (see Fig. 8) confirmed that
group differences seen in acquisition were reflected in accuracy of memory for the platform position. Although all groups
showed a preference for the training quadrant (F3,117 ˆ 5.14,
P ˆ 0.002), the Groups × Quadrant interaction (F6,117 ˆ 5.50,
P ˆ 0.001; Fig. 8A) indicated that young controls spent
significantly longer in the training quadrant than the aged
unimpaired (P ⬍ 0.05) or moderately impaired (P ⬍ 0.001)
groups; aged unimpaired rats in turn spent longer in the training quadrant than moderately impaired rats (P ⬍ 0.01).
Groups differed substantially in time spent in the counter
area of the former platform position (F2,39 ˆ 11.18,
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Stem cell grafts improve memory in aged rats
Interaction between pre-training and acquisition phases
In order to clarify changes in performance over the two
phases of learning, pre-training results were compared with
those of the first eight days of acquisition for latency, distance
and percentages of time in the annuli. In these analyses,
improved acquisition is reflected by a difference between
phases. A more marked improvement in young than aged
rats would yield interactions between groups and phases.
Latency was significantly faster in acquisition than pretraining (F1,624 ˆ 146.14, P ⬍ 0.001; see Fig. 6). Although
groups differed substantially (F2,39 ˆ 57.82, P ⬍ 0.001),
there was a marked interaction between groups and phases
(F2,624 ˆ 39.17, P ⬍ 0.001), because improvement in acquisition was far greater in the young controls (P ⬍ 0.001) than in
the aged unimpaired group (P ⬍ 0.05), whilst moderately
impaired aged rats did not show any improvement over
phases. Results for distance (see Fig. 7) were similar to
those for latency. Phases differed (F1,624 ˆ 277.91, P ⬍ 0.001),
as did groups (F2,39 ˆ 4.71, P ˆ 0.015), and there was a
substantial interaction between groups and phases (F2,624 ˆ
41.89, P ⬍ 0.001). This occurred because distance was
substantially decreased in young controls (P ⬍ 0.001), reliably decreased in aged unimpaired rats (P ⬍ 0.01), but only
modestly (P ⬍ 0.05) decreased in moderately impaired aged
rats during acquisition relative to pre-training. As shown in
Experiment 1, a good search strategy is reflected by high
percentages of time crossing the middle of the pool, and
low percentages spent swimming around the pool wall, so
that time in annulus A would be expected to increase and
time in annulus C to decrease during acquisition relative to
pre-training. The substantial differences between phases
(F1,624 ˆ 325.80 for annulus A and 369.3 for annulus C,
P ⬍ 0.001 in both cases) reflected this pattern. However,
there were highly significant interactions between groups
and phases (F2,624 ˆ 11.73, P ⬍ 0.001 for annulus A and
F ˆ 37.02, P ⬍ 0.001 for annulus C) because the percentage
time increase in annulus A and decrease in annulus C were
more marked in young controls and aged unimpaired rats
(P ⬍ 0.01) than in moderately impaired aged rats (P ⬍ 0.05).
Fig. 8. Experiment 2: probe trial performance of aged unimpaired and
moderately impaired rats, and young controls. Moderately impaired aged
rats (n ˆ 16) showed no preference for the training quadrant (A), spent a
minimal time in the counter area and rarely crossed the platform position
(B) relative to the aged unimpaired (n ˆ 16) and young control (n ˆ 10)
groups (P ⬍ 0.001 in comparison with young controls and P ⬍ 0.05 relative
to aged unimpaired rats). The unimpaired aged rats showed reduced preference for the training quadrant (P ⬍ 0.05), reduced time in the counter area
(P ⬍ 0.01) relative to the young control group. Bars show twice the standard error for the difference in means (2 SED) between groups. Difference
from young controls: *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001.
P ⬍ 0.001; see Fig. 8B), young controls spending more time
in counter 1 than unimpaired (P ⬍ 0.01) or moderately
impaired (P ⬍ 0.001) aged rats. A similar but less marked
pattern was seen for the number of platform position crossings
(F2,39 ˆ 4.97, P ⬍ 0.015). In this measure, the only reliable
difference lay between young controls and moderately
impaired aged animals (P ⬍ 0.01). As during acquisition,
moderately impaired aged rats spent less time in annulus A
than aged unimpaired (P ⬍ 0.05) and young control
(P ⬍ 0.01) groups, but more time in annulus C (P ⬍ 0.05 for
both comparisons), whilst young controls were more accurate
in heading angle (P ⬍ 0.05) than both of the aged groups.
DISCUSSION
Results from Experiment 1 showed that impaired rats with
MHP36 grafts in the hippocampus, frontal cortex and striatum
improved to a level that was substantially superior to that of
non-grafted impaired aged rats, and comparable to the performance of unimpaired aged rats in measures of latency, path
length and appropriate exploration. However, both unimpaired
and impaired grafted rats were less accurate than young
controls in heading angle, search in the training quadrant
and latency, although lower latency in young rats did not
necessarily reflect more rapid spatial learning, since they
also swam faster. Unimpaired and impaired grafted aged
groups were just as efficient as the young controls in distance
swum, and all three groups were substantially superior to the
aged impaired controls in most measures. In the probe trial,
grafted aged rats showed as strong a preference for the training quadrant as the unimpaired aged group and young
controls, and were significantly more accurate than any
other group in recall of the precise platform position, as
shown by counter and crossings measures. These results
provided good evidence for the accuracy of spatial memory
954
H. Hodges et al.
in grafted rats, but the surprisingly high scores may indicate
that grafted animals showed a degree of perseveration. Young
controls and unimpaired old rats searched more readily in
other areas of the pool after failing to find the platform in
the expected location. This persistence may reflect cognitive
rigidity thought to characterize the performance of animals
with hippocampal dysfunction, 6 suggesting that some subtle
deficits were not attenuated and may even have been
unmasked by grafts. Improved spatial navigation in the grafted
rats is likely to reflect a cognitive change, rather than, for
example, improved motor function, because the swimming
speed of the three aged groups did not differ. Thus, MHP36
grafts appear to ameliorate spatial learning deficits associated
with widespread degenerative changes in the aged brain 12 just as
effectively as they do those induced by discrete CA1 damage
following global ischaemia 26 or cholinergic lesions. 8,9
Findings from Experiment 2 indicated that moderately
impaired aged rats did not show improved spatial learning
during a second exposure to the water maze in measures of
latency, distance or heading angle, whilst unimpaired aged
rats showed only modest improvements, in comparison with
the substantial gains shown by young controls. These results
indicate a marked deterioration in moderately impaired rats
over a period of only 10 weeks, since initially small differences from young controls in pre-training were substantial at
acquisition. Given this cognitive decline in aged rats, the
effects of MHP36 grafts appear to be all the more remarkable,
suggesting that grafts did not merely halt the deficits in spatial
learning and memory, but substantially reversed them. This
evidence for progressive deficits suggests that it would be
possible to design repeated measures experiments in which
treatment efficacy is measured by comparative changes in
cognitive function over time rather than by group improvements relative to normal or impaired aged controls at a single
time-point. Typically, only the 25–35% of aged rats with and
without deficits are selected, and up to 50% of rats that perform
at intermediate levels may be discarded in behavioural experiments on ageing. Changes in performance over time relative to
controls are routinely used in clinical efficacy trials, and the
present results indicate that this approach with aged rats
would ensure optimum use of these valuable and informative
experimental animals, without loss of statistical power.
Histological findings showed that although atrophy
occurred in some aged brains, notably in pyramidal cells of
the hippocampal CA1 field, there was no evidence for group
differences on the basis of cell counts in a selected CA1
region. There were no correlations between water maze
distance scores and these cell numbers, and no evidence
that surgery affected cell counts. This is in agreement with
recent stereological findings that no cell loss occurs in the
principal neurons of the hippocampus in both aged Long–
Evans 21 and Wistar 22 rats, relative to young controls, despite
impairments in spatial learning. Possibly, a more sensitive
procedure to quantify cell abnormality might suggest a relationship between hippocampal cell viablity and behavioural
performance that is not captured by cell counts. However,
Markowska et al. 14 found that evidence for relationships
between a wide range of behavioural, neurochemical and
receptor markers in aged Sprague–Dawley rats was surprisingly sparse, so that structural and neurochemical indices of
changes in the aged brain may not necessarily be easy to relate
to behavioural performance. It may be more relevant to look
for subtle relationships between behaviour and functional
parameters such as second messenger 19 or electrophysiological 23 activity in hippocamapal neurons.
Grafted cells, identified by autoradiography and b-Gal
immunoreactivity, were widely dispersed in the brains of
aged rats. This pattern of migration differed from that seen
in rats with acute ischaemic CA1 cell loss (15 min of fourvessel occlusion), where MHP36 cells placed in the alveus
migrated selectively to the damaged CA1 field. 8,26 Clearly,
some differences would result from the fact that cells were
also grafted to the cortex and striatum in the aged rats.
However, examination of the hippocampus alone, where the
sites of grafting were the same in aged and ischaemic rats,
showed that in old rats MHP36 cells migrated to all pyramidal
cell fields, including dendritic and cell body layers, and to
both the hilar and dentate granule sectors of the dentate
gyrus. In ischaemic rats, MHP36 cells migrated to additional
regions of cell loss in the CA3 and dentate gyrus fields 10 only
when damage was increased by extending the duration of
four-vessel occlusion from 15 to 30 min. Taken together,
these results suggest that migration of MHP36 cells is related
to the extent of damage, with cell loss distal to the injection
site capable of attracting extensive migration.
Although many grafted cells expressed a glial morphology,
a proportion of cells showed site-specific differentiation,
some cells in the striatum resembling medium-sized spiny
neurons and those in the dentate gyrus presenting a granular
appearance. These findings suggest that MHP36 cells adopted
different and appropriate local phenotypes. MHP36 cells
made extensive use of white matter tracts for migration,
presenting a distinctive elongated appearance en route. It is
not known if cells leaving these tracts further change their
phenotype. If so, this would indicate considerable developmental flexibility. Since grafts placed in three brain regions
colonized wide areas of the cortex, striatum and hippocampus, it is not possible to determine which area was critically important for their effects on spatial learning. However,
rats with MHP36 grafts did not swim faster than the other
aged groups, so that motor effects are not likely to have
played a major role in recovery of spatial learning. Effects
of fetal and NGF-releasing grafts in improving water maze
performance of aged rats 5,15,16 have suggested that cholinergic
system atrophy contributes to ageing deficits in spatial navigation, and we have shown that both fetal cholinergic-rich and
MHP36 grafts improve spatial learning in rats with lesion
damage to the cholinergic projections. 9,25 However, the efficacy of fetal CA1 and MHP36 grafts in ischaemic rats 9,26
suggests that grafts may also ameliorate spatial deficits by
repairing intrahippocampal circuitry. The present findings
are potentially consistent with either mechanism. If MHP36
cells achieve their functional effects in aged rats by improving
transmission within the hippocampus and/or cortex, this
action could compensate either for local circuit damage or
for attenuation of cholinergic input to these regions.
Astrocytes may also play an important part in the functional recovery induced by MHP36 grafts, since grafted
cells readily adopt this phenotype. For example, we have
found that approximately 18% of MHP36 cells pre-labelled
with the fluorescent marker PKH26 and grafted in the ischaemic CA1 field adopted a glial phenotye, whilst 38% adopt a
neuronal phenotype, according to double labelling of PKH26
with either glial (glial fibrillary acidic protein) or neuronal
(NeuN) markers. 10 Bradbury et al. 3 found that astrocyte grafts
promote recovery from deficits induced by cholinergic lesions
Stem cell grafts improve memory in aged rats
as effectively as primary fetal tissue. Glial contributions
might include: (i) provision of a matrix to support the survival
and integration of the neuronal population of grafted cells; (ii)
release of trophic factors that sustain damaged host or grafted
neurons; or (iii) release of transmitter substances to supplement the activity of host cells.
CONCLUSION
The present findings indicate that spatial learning of
impaired aged rats was improved to the level of unimpaired
aged controls, and in some respects to the level of young adult
rats, following grafts of conditionally immortal MHP36 cells.
Since aged rats with moderate performance levels failed to
show improvements from pre-training to acquisition, this
achievement in grafted animals suggests that cognitive
955
decline was reversed rather than retarded. From three injection
sites in the cortex, striatum and hippocampus, b-Gal-positive
grafted cells migrated widely along white matter tracts to
occupy areas of the sensorimotor cortex, the matrix of the
striatum and the hippocampus, where they adopted a laminar
distribution. Grafted cells displayed neuronal and astrocytic
morphologies, but double labelling with specific markers is
required to establish their identity. These findings demonstrate the substantial capacity of a migratory stem cell line
to repair diffuse damage in the aged brain, and offer encouragement for the development of human clonal stem cell lines
with similar potential.
Acknowledgements—This work was supported by ReNeuron Ltd.
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(Accepted 1 September 2000)