CONDITIONALLY IMMORTAL NEUROEPITHELIAL STEM CELL GRAFTS REVERSE AGE-ASSOCIATED MEMORY IMPAIRMENTS IN RATS

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1 Pergamon Stem cell grafts improve memory in aged rats Neuroscience Vol. 101, No. 4, pp , IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII: S (00) /00 $ 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 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: MHP36 cell line, aged rats, intracerebral transplantation, water maze, spatial learning. 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.: ; fax: address: spjthmh@iop.kcl.ac.uk (H. Hodges). Abbreviations: CA, Cornu Ammonis; b-gal, b-galactosidase; MHP36, Maudsley hippocampal stem cell line, clone 36; NGF, nerve growth factor. 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. EXPERIMENTAL PROCEDURES Experiments were conducted in accordance with the UK Animals 945

2 946 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 12- h/12-h light dark schedule (lights on at 8.00 a.m.), and were handled daily between arrival and the start of behavioural training. 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 [ 3 H]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, 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 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, 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 At the end of behavioural testing, 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 [ 3 H]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 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

3 Stem cell grafts improve memory in aged rats 947 Fig. 1. Identification of grafted cells. MHP36 cells in the premotor cortex showing co-localization of [ 3 H]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, r s 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), b- Gal-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

4 948 H. Hodges et al. Fig. 2.

5 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// 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, Experiment 1. Effects of MHP36 grafts in aged rats 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 (F 3,47 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 (F 3,329 ˆ 6.19, P 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 (F 3,47 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 (F 3,47 ˆ 43.58, P 0.001), and between rates of learning (F 3,517 ˆ 12.79, P 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).

6 950 H. Hodges et al. the aged groups (F 3,47 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 (F 3,47 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 (F 3,47 ˆ 7.26, P 0.001), with a slower decrease in path length over Days in aged impaired rats relative to the other groups (F 3,517 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 (F 3,47 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: F 3,47 ˆ 4.58, P 0.01) and at the perimeter (annulus C: F 3,47 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 (F 3,141 ˆ 5.96, P 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 (F 9,141 ˆ 2.29, P 0.025). Aged impaired rats with MHP36

7 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 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 (F 3,47 ˆ 8.72 and 7.64, P 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 (F 2,39 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 (F 2,39 ˆ 11.53, P 0.001), so that they covered more distance (F 2,39 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 (F 2,39 ˆ 3.56, P 0.05 for Quadrant 4, and F ˆ 7.04, P ˆ 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 (F 2,39 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 (F 2,39 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

8 952 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 (F 2,39 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 (F 2,39 ˆ 8.97, P 0.005) and search in the training quadrant (F 2,39 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 (F 3,117 ˆ 5.14, P ˆ 0.002), the Groups Quadrant interaction (F 6,117 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 (F 2,39 ˆ 11.18,

9 Stem cell grafts improve memory in aged rats 953 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 (F 1,624 ˆ , P 0.001; see Fig. 6). Although groups differed substantially (F 2,39 ˆ 57.82, P 0.001), there was a marked interaction between groups and phases (F 2,624 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 (F 1,624 ˆ , P 0.001), as did groups (F 2,39 ˆ 4.71, P ˆ 0.015), and there was a substantial interaction between groups and phases (F 2,624 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 (F 1,624 ˆ for annulus A and for annulus C, P in both cases) reflected this pattern. However, there were highly significant interactions between groups and phases (F 2,624 ˆ 11.73, P for annulus A and F ˆ 37.02, P 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 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 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 (F 2,39 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

10 954 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. Itis 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