Elsevier

Behavioural Brain Research

Volume 143, Issue 2, 14 August 2003, Pages 177-191
Behavioural Brain Research

Research report
Transplantation of neurospheres after granule cell lesions in rats: cognitive improvements despite no long-term immunodetection of grafted cells

https://doi.org/10.1016/S0166-4328(03)00032-9Get rights and content

Abstract

EGF-responsive C17 murine-derived neural stem cells (neurospheres) were grafted into the dentate gyrus of adult male rats after dentate granule cells lesions produced by colchicine injections. Behavioural performance was evaluated over two post-grafting periods, using tests sensitive to hippocampal dysfunctions. The first period began 1 month after grafting and testing conducted in the water maze and the radial maze distinguished working- and reference-memory performance. The second period began 9 months after grafting and learning performance was also evaluated in a Hebb–Williams maze, in addition to both other tests. The lesions induced lasting deficits in all tests. During the first period, the grafts had no effect in either test. Conversely, during the second period, grafted rats showed a weak improvement in the water maze and a significant increase of reference memory performance in the radial maze. In the Hebb–Williams maze, performance of grafted rats was close to normal.

Strengthening the idea that dentate gyrus granule cells play an important role in the acquisition of new (perhaps more configural than only spatial) information, our results, moreover, suggest that neurosphere grafts may foster recovery after damage to point-to-point connection systems in the adult brain.

Introduction

Evidence suggests that fetal hippocampal grafts achieve considerable functional integration into a host brain. Field et al. [12] showed that such grafts placed in the hippocampus are contacted by host neurons in an appropriate laminar fashion, provided that cells are replaced homotypically within the lesion site. These observations demonstrate that fetal grafts may restore the information flow within the hippocampal formation in a relatively point-to-point manner. This data has been supported by the demonstration that grafts of dentate granule cells, but not of CA1 cells, restored long-term potentiation (a correlate of learning) in rats with dentate gyrus lesions [9]. In terms of cognitive effects, fetal grafts have also shown good tissue-specific potentials: CA1, but not CA3 or dentate granule cell grafts, improved water-maze performance in rats subjected to ischemic damage of CA1 neurons [17], [35], [36], [44]. Similarly, after colchicine lesions of the granule cells in the dentate gyrus, performance was restored only by grafts containing dentate granule cells [44], [63]. Thus, after a selective hippocampal cell field lesion, there is a high degree of specificity in the type of fetal tissue enabling restoration of spatial function in the water-maze test.

Such results suggest that fetal tissue from particular brain regions provide a possible donor source for intracerebral grafting. However, the limited availability of suitable tissue, along with the ethical [15], technical and safety issues surrounding the transplantation of human fetal tissue, pose a significant impediment to clinical progress. It may also be interesting, perhaps necessary, to elaborate other grafting techniques, based on the following remarks: the ideal source of tissue for neural transplantation would be cells that can be exponentially expanded in culture, banked, tested for the absence of adventitious agents, and cryopreserved. In addition, these cells should be capable of differentiating into appropriate phenotypes based upon environmental cues [13], [28], [30], [47], [48].

Neural stem cells (NSCs) appear to fulfil these requirements. Moreover, they can be isolated in vitro through the selective action of epidermal growth factor (EGF), and in response to EGF, individual NSCs proliferate to form clonally derived spherical colonies (neurospheres) floating in the culture medium. Neurospheres contain cells which, upon dissociation into single cells, give rise to new spheres colonies (self-renewal potential) and cells (progenitor cells) that can differentiate into neurons, astrocytes and oligodendrocytes (multilineage potential) [41]. Recently, it has been described that neurons derived from NSCs form functional synapses in vitro [52], [57] and in vivo [1]. According to these results and regarding the data which strongly imply a specificity of graft type-host connectivity in recovery after focal hippocampal damage, we investigated the functional efficiency of NSC-derived neurospheres (hereafter termed neurospheres) on cognitive deficits associated with experimental damage to the granule cells in the dentate gyrus of rats. Selective destruction of the granular cells was produced by injections of colchicine into the dentate gyrus as described previously [22]. Over two post-transplantation periods (1–4 months and 9–12 months), the rats were tested in tasks assessing cognitive function (water maze, radial maze and Hebb–Williams maze). In both the water maze and radial maze, the lesions used in the present study have induced robust working- and reference-memory deficits, as found in our previous study [22]. Histological and immunohistochemical examinations both enabled to verify the appropriateness of the lesions and tried to establish the survival of the grafted neurospheres.

Section snippets

Experimental procedures

All procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Council Directive #87848, 19 October 1987, Ministère de l’Agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animales; authorization #67-14 bis to H.J., #67-101 to S.S. and #6212 to J.C.C.) and international (NIH publication no. 86-23, revised 1985) laws and policies.

Histology and immunohistochemistry

After completion of all behavioural testing, each rat was given an overdose of sodium pentobarbital (100 mg/kg) and transcardially perfused with 60 ml of phosphate-buffered 4% paraformaldehyde (pH 7.4; 4 °C). The brain was then extracted. Ten rats (three Sham, three Col and four Graft rats) were used for histological verifications, the others were used for immunohistochemical evaluations.

For histological verifications, after extraction, the brain was post-fixed for 4 h and transferred into a 0.1 M

First series of test

The results obtained at the first delay will not be presented in detail here (results not illustrated but summarised in Table 2). Briefly, lesions were found to induce dramatic deficits in all tests and there was no effect of the grafts whatever test was considered.

Reference memory

Data are shown in Fig. 1. The ANOVA of the mean escape distances (Fig. 1A) showed significant effects of factors: Group (F(2,27)=10.6, P<0.001) and Day (F(4,108)=5.2, P<0.001) but no significant interaction. The ANOVA of the mean

Discussion

Based on a model of granule cell lesion in the dentate gyrus, this study aimed at investigating whether intrahippocampal grafts of neurospheres (1) survived in vivo after intrahippocampal transplantation, and (2) attenuated the cognitive impairments produced by intradentate administration of colchicine. Our present results enable discussion of each issue and can be summarised as follows.

During the first testing period (1–4 months after grafting), a clear impairment of reference and working

Conclusions

The present results confirm that rats with colchicine-induced lesions of granule cells in the dendate gyrus exhibited clear-cut cognitive deficits in spatial reference- and working-memory capabilities assessed in the water- and radial-maze tests. The data also demonstrate lesion-induced learning deficits in the Hebb–Williams maze, a test in which the spatial information processing is probably much less prominent than in both other ones. The most important finding was that in rats given

Acknowledgements

The authors wholeheartedly acknowledge Mr. O. Bildstein and Mr. R. Paul for their expert engagement in animal care.

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