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Cerebellar diseases causing substantial cell loss often lead to severe functional deficits and restoration of cerebellar function is difficult. Neurotransplantation therapy could become a hopeful method, but there are still many limitations and unknown aspects. Studies in a variety of cerebellar mutant mice reflecting heterogeneity of human cerebellar degenerations show promising results as well as new problems and questions to be answered. The aim of this work was to compare the development of embryonic cerebellar grafts in adult B6CBA Lurcher and B6.BR pcd mutant mice and strain-matched healthy wild type mice. Performance in the rotarod test, graft survival, structure, and volume was examined 2 months after the transplantation or sham-operation. The grafts survived in most of the mice of all types. In both B6CBA and B6.BR wild type mice and in pcd mice, colonization of the host's cerebellum was a common finding, while in Lurcher mice, the grafts showed a low tendency to infiltrate the host's cerebellar tissue. There were no significant differences in graft volume between mutant and wild type mice. Nevertheless, B6CBA mice had smaller grafts than their B6.BR counterparts. The transplantation did not improve the performance in the rotarod test. The study showed marked differences in graft integration into the host's cerebellum in two types of cerebellar mutants, suggesting disease-specific factors influencing graft fate.

For many degenerative cerebellar diseases, currently, no effective treatment that would substantially restore cerebellar functions is available. Neurotransplantation could be a promising therapy for such cases. Nevertheless, there are still severe limitations for routine clinical use. The aim of the work was to assess volume and morphology and functional impact on motor skills of an embryonic cerebellar graft injected in the form of cell suspension in Lurcher mutant and wild-type mice of the B6CBA and C3H strains after a 6-month survival period. The grafts survived in the majority of the mice. In both B6CBA and C3H Lurcher mice, most of the grafts were strictly delimited with no tendency to invade the host cerebellum, while in wild-type mice, graft-derived Purkinje cells colonized the host's cerebellum. In C3H Lurcher mice, but not in B6CBA Lurchers, the grafts had smaller volume than in their wild-type counterparts. C3H wild-type mice had significantly larger grafts than B6CBA wild-type mice. No positive effect of the transplantation on performance in the rotarod test was observed. The findings suggest that the niche of the Lurcher mutant cerebellum has a negative impact on integration of grafted cells. This factor seems to be limiting for specific functional effects of the transplantation therapy in this mouse model of cerebellar degeneration.

Hereditary cerebellar ataxias are severe diseases for which therapy is currently not sufficiently effective. One of the possible therapeutic approaches could be neurotransplantation. Lurcher mutant mice are a natural model of olivocerebellar degeneration representing a tool to investigate its pathogenesis as well as experimental therapies for hereditary cerebellar ataxias. The effect of intracerebellar transplantation of embryonic cerebellar solid tissue or cell suspension on motor performance in adult Lurcher mutant and healthy wild-type mice was studied. Brain-derived neurotrophic factor level was measured in the graft and adult cerebellar tissue. Gait analysis and rotarod, horizontal wire, and wooden beam tests were carried out 2 or 6 months after the transplantation. Higher level of the brain-derived neurotrophic factor was found in the Lurcher cerebellum than in the embryonic and adult wild-type tissue. A mild improvement of gait parameters was found in graft-treated Lurcher mice. The effect was more marked in cell suspension grafts than in solid transplants and after the longer period than after the short one. Lurcher mice treated with cell suspension and examined 6 months later had a longer hind paw stride (4.11 vs. 3.73 mm, P < 0.05) and higher swing speed for both forepaws (52.46 vs. 32.79 cm/s, P < 0.01) and hind paws (63.46 vs. 43.67 cm/s, P < 0.001) than controls. On the other hand, classical motor tests were not capable of detecting clearly the change in the motor performance. No strong long-lasting negative effect of the transplantation was seen in wild-type mice, suggesting that the treatment has no harmful impact on the healthy cerebellum.

Lurcher mutant mice represent a natural model of olivocerebellar degeneration. They serve as a tool to study pathogenesis, the functional impact of the degeneration as well as therapeutic approaches. Wild type littermates are used as healthy controls. Neurotransplantation may be a promising method of therapy for neurodegenerative diseases. The aim of this work was to compare the long-term survival rate of the solid embryonic cerebellar graft in adult Lurcher mutant and wild type mice of the B6CBA strain and to assess the fundamental structural features of the graft. The graft was obtained from 12-day-old GFP mouse embryos. The brains of host mice were examined histologically 6 months after the transplantation. The graft was identified according to its GFP fluorescence. The graft presence and structure was assessed. The graft survived in all 14 Lurcher mice and in 12 of the 14 wild type mice. Cell migration and fibre sprouting from the graft were poor. No marked differences in the graft morphology between Lurcher mutant and wild type mice were found. The graft survival and appearance were similar to those after a shorter period described in a previous study. This suggests that during the 6 months, no intensive or commonly occurring processes changing the graft had proceeded and that the Lurcher mutant cerebellum niche had no strong influence over the fate of the solid cerebellar graft., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Lurcher mutant mice represent a model of olivocerebellar degeneration. They suffer from complete loss of Purkinje cells and a reduction of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. The aim of the work was to compare solid embryonic cerebellar graft survival within a period of 9 weeks after their transplantation in adult Lurcher mutant and wild type mice of the B6CBA strain. The solid grafts were injected through a hole in the occipital bone. Host mice were sacrificed 3, 6, or 9 weeks after the transplantation and their cerebella and brain-stems were examined histologically to assess graft presence and structure. We did not find significant differences in graft survival rates between Lurcher mutant and wild type mice. The frequency of graft presence did not differ between mice examined 3, 6, and 9 weeks after the transplantation, neither in Lurchers nor in wild type mice. The grafts were of various sizes. In some cases, only small residua of the grafts were found. Nerve fiber sprouting and cell migration from the graft to the host tissue were observed more often in wild type mice than in Lurchers when examined 6 weeks after surgery. In the period 3-9 weeks after transplantation, massive dying out of the grafts was not observed despite regressive processes in some of the grafts. The degenerative changes in the Lurcher mutant cerebellum do not have strong impact on the fate of the solid cerebellar graft., ((c) 2009 Wiley-Liss, Inc.)

Possible effect of trophic factors from embryonic cerebellar graft transplanted in adult Lurcher mutant mice on LTP as electrophysiological marker of learning and memory process was studied. Also the combination of the transplantation and long-term forced motor training was investigated. An evaluation of LTP ability in four animal groups (transplanted, sham-operated, with and without forced motor activity) and comparison among them showed the highest LTP improvement in the group with combination of both influences (ie. transplantation and motor training).

Possible influence of embryonic cerebellar graft transplanted into the adult neurodegenerative brain in Lurcher mutant mice on long-term potentiation (LTP) in hippocampus was investigated. Evaluation of LTP ability and comparison with the tests of motor learning suggests similarities between magnitude of LTP and criteria of motor learning. Also interstrain differences were described. Our results support ideas about tight cooperation among brain structures which are involved in mechanisms of learning and memory.

Lurcher mutant mice suffer from complete loss of cerebellar Purkinje cells. The aim of the work was to compare the solid embryonic cerebellar graft survival in adult Lurcher mutant mice derived from strains C3H and C57Bl/7 and to assess the morphology of the grafts. Embryonic cerebellar tissue was obtained from 12-13 days mice embryos expressing green fluorescent protein (GFP). Embryonic cerebellum was injected with a glass microcapillary into the cerebellum of adult Lurcher mutant mouse. Host mice were sacrificed 2-12 weeks after the transplantation. Brainstems and cerebella were examined histologically. The graft and graft derived GFP-positive cells were detected according to their green fluorescence. To visualise the structure of the graft Nissl staining was used. Graft survival percentage was evaluated in groups of mice sacrificed during the first, second or third month after the transplantation. The graft was found in all C57Bl/7 mice and in 90.9% of C3H mice examined within one month after the transplantation. In the second month the graft was present in 83.3% of C57Bl/7 and 50.0% of C3H mice. Till the third month the graft survived in 68.2% of C57Bl/7 mice and 22.2% of C3H mice. In C57Bl/7 mice a cerebellar structure was developed in the graft and migration of graft derived-cells to the host tissue was observed more often than in C3H mice. C567Bl/7 mice seem to be more suitable for experiments testing functional consequences of transplantation into the cerebellum requiring good long-term graft survival.

The loss of afferent synaptic boutons is a prominent alteration induced by axotomy on adult central neurons. In this work we attempted to prove whether synapse loss could be reverted by reconnection with a new target. We severed the medial longitudinal fascicle of adult cats and then transplanted embryonic cerebellar primordia at the lesion site immediately after lesion. As previously shown, the transected axons from abducens internuclear neurons penetrate and reinnervate the graft [J Comp Neurol 444 (2002) 324]. By immunocytochemistry and electron microscopy we studied the synaptology of abducens internuclear neurons under three conditions: control, axotomy and transplant (2 months of survival time). Semithin sections of the abducens nucleus were immunostained against calretinin, to identify abducens internuclear neurons, and either synaptophysin (SF), to label synaptic terminals, or glial fibrillary acidic protein (GFAP) to detect the astrocytic reaction. Optical and linear density of SF and GFAP immunostaining were measured. Data revealed a significant decrease in the density of SF-labeled terminals with a parallel increase in GFAP-immunoreactive elements after axotomy. On the contrary, in the transplant group, the density of SF-labeled terminals was found similar to control, and the astrocytic reaction induced by lesion was significantly reduced. At the ultrastructural level, synaptic coverage and linear density of boutons were measured around the somata of abducens internuclear neurons. Whereas a significant reduction in both parameters was found after axotomy, cells of the transplant group received a normal density of synaptic endings. The ratio between F- and S-type boutons was found similar in the three groups. Therefore, these findings indicate that the grafting of a new target can prevent the loss of afferent synaptic boutons produced by the axotomy.

The ability of in vitro-expanded neural precursor cells or cell lines to differentiate following transplantation has significant implications for current research on central nervous system repair. Recently, interest has been focussed on grafts of such neural precursors implanted also into the eye or retina. Here, we demonstrate with a non-traumatizing subretinal transplantation method, that grafts of the two immortalized brain-derived cell lines C 17-2 (from postnatal mouse cerebellum) and RN33B (from the embryonic rat medullary raphe) survive for at least up to four weeks, after implantation into the adult normal rat retina. For both cell lines, implanted cells gradually integrate into all major retinal cell layers, including the retinal pigment epithelium, and judged by the morphology differentiate into both glial- and neuron-like cells, as shown by thymidine autoradiography, mouse-specific in situ hybridization, and using immunohistochemistry to detect the reporter gene LacZ. Our results suggest that these and other similar neural cell lines could be very useful in the continuous experiments in models of retinal disorders to further assess both the cell replacement and ex vivo gene therapy approaches., (Copyright 2001 Academic Press.)

Spinocerebellar ataxia type 1 (SCA-1) is one of several neurodegenerative diseases, including Huntington's disease, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and SCA-2, SCA-3, SCA-6, and SCA-7, each caused by an expanded number of CAG repeats in the coding region of their respective genes. The mechanism by which the resulting proteins are pathogenic is unknown. Clinical trials of neural transplants in Huntington's disease patients are under way. While initial reports are encouraging, definitive evidence of graft survival in patients despite the ongoing disease process is not possible with current imaging techniques. Transplants in primates have shown long-term survival of striatal grafts and recovery of function, but have used lesioning to model Huntington's phenotypically. Studies of striatal grafts in a transgenic mouse model of Huntington's have not yet shown a behavioral benefit. We describe a behavioral benefit of cerebellar grafts in a transgenic model of SCA-1 in which the ataxic phenotype results from expression of an expanded ataxin-1 protein. Mice were transplanted at an age when their ataxic phenotype is just becoming evident. Compared with sham-operated littermates, grafted mice showed better performance on multiple behavioral tests of cerebellar function. Differences persisted for 10 to 12 weeks posttransplant, after which there was a progressive decline in motor performance. At 20 weeks postsurgery, donor Purkinje cell survival was evident in 9 of 12 graft recipients. These results indicate that transplants can have behavioral benefits and grafts can survive long-term despite the ongoing pathological process in a brain actively expressing an expanded polyglutamine protein., (Copyright 1999 Academic Press.)

This study was designed to explore the efficacy of a human clone cell line as an alternative neural graft source and to validate the practice of cryopreservation and xenografting as logistical approaches toward conducting neural transplantation. We investigated the biological effects of transplanting cultured human neurons (NT2N cells) derived from a well-characterized embryonal carcinoma cell line into the brains of rats subjected to transient, focal cerebral ischemia induced by embolic occlusion of the middle cerebral artery. At 1 month and extending throughout the 6-month posttransplantation test period, ischemic animals that were transplanted with NT2N cells and treated with an immunosuppressive drug displayed a significant improvement in a passive avoidance task as well as a normalization of asymmetrical motor behavior compared to ischemic animals that received rat fetal cerebellar cell grafts or vehicle alone. Remarkably, cryopreserved NT2N cell grafts compared with fresh NT2N cell grafts, remained viable in the immunosuppressed rat brain and effective in producing behavioral recovery in immunosuppressed ischemic animals. The long-term viability of cryopreserved NT2N cell xenografts in vivo and their sustained effectiveness in promoting behavioral recovery suggest potential utilization of xenografting and cryopreservation as useful protocols for establishing clone cell lines as graft source in neural transplantation therapies for central nervous system disorders.

Embryonic striatal precursors grafted into the lesioned adult host striatum show limited integration with little migration and restricted efferent projections. In the present study, the influence of an immature striatal environment on the integrative capacity of grafted neuroblasts was examined after transplantation of striatal progenitors into the striatum at different stages of postnatal development. Mouse progenitors, derived from embryonic day 13.5-14 lateral or medial ganglionic eminence or the cerebellar primordium, were transplanted as a single cell suspension into the developing postnatal day 1, 7 and 21 rat striatum. The grafted cells and their axonal projections were visualized using antibodies raised against the mouse-specific neural markers, M6 and M2. Cells from the lateral (but not the medial) ganglionic eminence showed a remarkable capacity to innervate selectively the striatal target structures, globus pallidus, entopeduncular nucleus and substantia nigra, reminiscent of endogenous striatal neurons, which is not observed after grafting into adult hosts. M6 and M2-immunopositive cellular profiles from both the lateral and medial ganglionic eminences were observed to have migrated extensively away from the injection site, in contrast to the cerebellar precursors which remained clustered at the implantation site. Cells from the lateral ganglionic eminence were largely confined within the striatal complex where they developed striatal characteristics, displaying expression of DARPP-32, the 32,000 mol. wt dopamine- and cyclic AMP-regulated phosphoprotein, whereas cells from the medial ganglionic eminence had migrated caudally along the internal capsule and were observed predominantly in the globus pallidus and thalamus, in addition to the striatum. The cells located outside the striatum were all DARPP-32 negative. The improved integration and increased projection capacity of the lateral ganglionic eminence precursors grafted into postnatal day 1 hosts gradually declined as the host advanced into later stages of development (postnatal day 7), and in postnatal day 21 hosts the grafted striatal precursors behaved similarly to grafts implanted into adult recipients. These results demonstrate the specific capacity of embryonic striatal progenitors to integrate into the developing basal ganglia circuitry during early postnatal development, and that the extent of neuronal and glial integration and graft host connectivity declines when the host has developed beyond the first postnatal week.

Activated Schwann cells such as those in the distal stump of a cut peripheral nerve, or those cultured in vitro, develop a molecular phenotype very different from that of quiescent Schwann cells, and express high levels of the transcription factor c-jun. We studied the expression of c-jun messenger RNA, by in situ hybridization, and Jun-like immunoreactivity of Schwann cells in segments of peripheral nerve, or in cell suspensions grafted into the adult rat brain. Schwann cells rapidly lost their Jun immuno-positivity, and down-regulated expression of c-jun messenger RNA once implanted into the brain, and only the Schwann cells contained in the portion of peripheral nerve which remained outside the brain maintained Jun-like immunopositivity. c-jun messenger RNA was also down-regulated in the grafts, but more slowly than the protein; however, a proximodistal gradient in the level of expression of c-jun messenger RNA along the graft, comparable to that found for Jun immunoreactivity, was not detected. Schwann cells transplanted into the lesioned central nervous system promote regeneration of some injured central nervous system axons, but this regenerative response is always much more limited than peripheral nervous system regeneration. We suggest a correlation between the limited regeneration of central nervous system axons into peripheral nerve grafts and the loss of c-jun expression in Schwann cells following exposure to the central nervous system environment.

The effects of neurotransplantation on development of audiogenic seizures (AS) were studied in Krushinskiĭ-Molodkina (KM) rats genetically predisposed to AS and in Wistar rats especially selected for AS. Within 24 weeks after bilateral combined transplantation of the newborn striatum and cerebellum into the parietal cortex, the AS didn't develop in Wistar rats in 74% of tests. In control Wistar rats with cortical grafts or saline injections into the parietal cortex there were no significant changes in the AS development. In the experimental KM rats the grafting led to the 4-5-times increase in the AS latency. In 50% of rats it decreased the AS severity by 1-2. In the control KM rats these parameters didn't change. It was concluded that it was possible to suppress or decrease the AS development by the transplantation of the newborn striatal and cerebellar tissue into the cortex of rats predisposed to this kind of seizures.

The mechanism of the behavioral improvement observed in parkinsonian primates that receive intrastriatal transplants of fetal dopamine neurons has not been firmly established. Dopamine production by grafted neurons may be the basis of the behavioral recovery. Alternatively, stimulation of the host dopamine system by the transplant procedure itself may be central to the outcome. The present study examined whether dopamine concentration was raised in the caudate nucleus of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated primate following grafting, and if so, whether the elevation was dependent on either (i) the introduction of the implantation cannula (sham), (ii) the brain region that was grafted, or (iii) the gestational age of fetal tissue that was grafted. Transplantation of early gestational age fetal ventral mesencephalon (embryonic days 40-50) was associated with significant elevation of caudate nucleus dopamine concentration to a mean of approximately 20% of control values in the vicinity (within 2 mm) of the graft, compared with more distant sites in the caudate nucleus. With early gestational age fetal ventral mesencephalon, the ratio of homovanillic acid/dopamine concentration near the graft site was normalized compared to the elevated value found in the caudate nucleus distant from the graft site. Grafts of later stage fetal ventral mesencephalon, or fetal cerebellum, or sham implantation did not increase dopamine concentration or lower homovanillic acid/dopamine ratio near the graft site. Biochemical and histochemical evidence suggests that host dopamine neurons terminating in the nucleus accumbens are not the source of the changes. Numerous tyrosine hydroxylase-positive neurons at the graft site were only observed in the MPTP-treated monkeys that received grafts of early gestational age fetal ventral mesencephalon. These data lend strong support to the hypothesis that dopamine derived from grafted dopamine neurons is the major basis for behavioral recovery observed following intrastriatal transplantation in our MPTP-treated monkeys.

Fetal grafts of normal cerebellar tissue were implanted into the cerebellum of Purkinje cell degeneration mutant mice (pcd/pcd), a model of adult-onset recessively inherited cerebello-olivary atrophy, in an attempt at correcting their cellular and motor impairment. Donor cerebellar cells engrafted in the appropriate sites, as evidenced by the pattern of expression of insulin-like growth factor-I (IGF-I) system genes. Bilateral cerebellar grafts led to an improvement of motor behaviors in balance rod tests and in the open field, providing evidence for functional integration into the atrophic mouse cerebellum and underscoring the potential of neural transplantation for counteracting the human cerebellar ataxias.

A synopsis is presented of the recent history of cerebellar tissue transplantation over the past 25 years. The properties of growth and differentiation of cerebellar grafts placed intraocularly or intracranially are reviewed, as well as the interaction of heterotopic and orthotopic grafts with the host brain. Particular emphasis is placed on the use of ataxic mouse mutants as recipients of donor cerebellar tissue for the correction of their structural deficits and the functional recovery of behavioural responses.

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.

Acrylamide (30 mg/kg body wt.) administered intraperitoneally daily to young adult male rats, five times a week for 3 consecutive weeks, affected the cerebellar functions, as exhibited by a significant reduction in rotarod performance, spontaneous locomotor activity, glutathione-S-transferase activity, and 3H-flunitrazepam binding in cerebellum. Transplantation of dissociated fetal cerebellar cells (E14) to cerebellum resulted in a significant recovery in behavioral and neurochemical parameters evaluated 9 weeks after transplantation. Light- and electron-microscopic studies confirmed the viability and specificity of cerebellar grafts.

Chick/quail chimeras with partial cerebellar grafts have been performed to obtain further information about the origin and migratory movements of cerebellar cortical neurons. The grafts were performed by exchanging between these two species a precise, small portion of the E2 cerebellar primordium, as defined in Martinez and Alvarado-Mallart (Eur. J. Neurosci. 1:549-560, 1989). All grafts were done unilaterally. The chimeric cerebella, fixed at various developmental stages, were analyzed in serial Feulgen-stained preparations to map the distribution of donor and host cells in the ependymal layer (considered to be reminiscent of the primary germinative neuroepithelium) and in the various cortical layers. In some of the oldest cases, we also used antiquail immunostaining to recognize quail cells. In the ependymal layer, it has been possible to conclude that each hemicerebellar primordium undergoes a morphogenetic rotation that changes its rostrocaudal axis to a rostromedio-caudolateral direction. However, important individual variations were observed among the chimeric embryos with respect to the ependymal area expected to be formed by donor cells. These variations cannot be explained solely on the basis of microsurgical procedure; however, they suggest the existence of important reciprocal interaction between host and grafted neuroepithelia. Therefore, it was not possible to draw a precise fate map of the E2 cerebellar primordium. Nevertheless, the dispersion of grafted cells in the cerebellar cortex, when compared to the real extent of the ependymal grafted area in each particular case, provided important data: (1) The external granular layer (EGL), the secondary germinative epithelium, seems not to originate exclusively from the "germinative trigone," as is usually considered the case. It emerges from a larger but restricted portion of the primary cerebellar matrix extending about the caudal fourth or third of the ventricular epithelium, as defined after its morphogenetic rotation. (2) The Purkinje cells (PCs) develop from all areas of the cerebellar epithelium. Although the distribution of donor PCs parallels the grafted ventricular layer mediolaterally, donor PCs extend more in the rostrocaudal dimension. The PC layer is formed mainly by donor cells in the lobules underlain by the grafted ependymal layer. However, donor PCs are also observed in cortical lobules surmounting the host ventricular layer. In these lobules, the donor PCs form clusters of various widths interrupting the host PCs. Reciprocally, clusters of host PCs are also found in the lobules formed mainly by donor PCs. The alternate small clusters of donor or host PCs are surrounded by Bergmann fibers of the other species' origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Solid cerebellar grafts, taken from normal mouse embryos (gestational day 12-14), were transplanted into the cerebellum of adult Lurcher mice. The degree of Purkinje cell replacement was analysed one to three months after transplantation by means of immunocytochemistry (antibodies against calbindin, cGMP-dependent protein kinase and neurofilament proteins) and electron microscopy. Grafted Purkinje cells succeed in moving out of the graft and migrate into the host cerebellar cortex. They are present next to the graft in the granule cell and molecular layers, and far from the graft remnant, only in the molecular layer, indicating that, although both layers subserve Purkinje cell migration, the molecular layer is the ultimate target. In the host molecular layer, axons of transplanted Purkinje cells form thick bundles running in the frontal plane over long distances. Most of them terminate in the upper granule cell layer by enlarged bulbs resembling collapsed growth cones. Axons reaching their normal targets (the neurons of the deep cerebellar nuclei) are observed only in cases where the granule cell layer is disrupted and/or grafted Purkinje cells remain in the white matter. The projection is massive only from grafts lying in the close vicinity of the target neurons. Electron-microscopic analysis of grafted Purkinje cells populating the host cerebellar cortex reveals that their synaptic investment is abnormal. In the molecular layer, where the normal inputs are reduced, the compartmentation in proximal and distal dendritic segments is severely affected, climbing fibre synapses only form on a minority of grafted cells and "pinceau" formations are absent. In the granule cell layer, the synaptic investment is similar to that of Purkinje cells in agranular cerebellum, and even heterelogous synapses with mossy fibres have been observed. These results, compared to those previously obtained with grafting experiments in Purkinje cell degeneration mutant mouse, allow us to conclude that: (i) the Purkinje cell-deficient molecular layer of the host, despite its severe atrophy and reactive gliosis, still exerts a positive neurotropism specific for grafted Purkinje cells; (ii) the unlesioned host granule cell layer underlying the molecular layer containing grafted Purkinje cells, even if almost depleted of granule cells, remains an obstacle for the re-establishment of a corticonuclear projection; and (iii) the degree of synaptic integration of grafted Purkinje cells is directly related to the nearby presence of available host axon terminals. Hence, owing to the atrophy of the Lurcher cerebellum, the postgrafting restoration of the cerebellar cortical circuit is much less complete in this mutant.

Cerebellum of rat embryos at day 17 of gestation was transplanted into sensomotor cortex of adult syngenic rats. DNA content in granule and Purkinje cells of the transplant was determined cytophotometrically on day 30 after surgery. It is shown that granule cells are diploid; about 3% Purkinje cells contain hyperdiploid and tetraploid nuclei which corresponds to the content of such cells in the adult cerebellum. The conclusion is drawn that cell hyperploidy does not depend on the functional load but is essential property of the tissue.

To study the hypothesis that neural transplantations can alter seizure susceptibility in a genetic animal model of epilepsy, 93 pubescent genetically epilepsy-prone rats with stage 9 seizures received either bilateral inferior colliculi (N = 21) or lateral ventricle (N = 42) transplants or sham transplants (N = 30). The grafts consisted of embryonic locus ceruleus, neocortical, or cerebellar tissue. Starting 2 days after the transplantation the rats were subjected to audiogenic stimulations every other day for 61 days. Latency to the running and tonic phase, seizure severity score, and duration of the tonic and clonic phase were compared in the neural transplant and sham-operated controls. Rats that received transplants had a longer latency to the tonic phase and a shorter duration of the clonic phase than the controls. At age 110 days the rats had electrodes implanted bilaterally into the angular bundle and were kindled. No difference in kindling rate was found between the rats that received neural grafts and the sham-operated controls. Cerebrospinal fluid concentration of norepinephrine was not altered by the transplants. This study demonstrates that the anticonvulsant effects of neural transplants, using the genetically epilepsy-prone model of epilepsy, are mild.

Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.

Fragments of fetal rat cerebellar tissue were grafted into forebrain cortical lesion cavities of adult rats. After a survival ranging from 12-151 days, no graft was found to fill the cavity completely. Large neurons, occasionally grouped into nests, were identifiable from the 22nd day. Myelinated graft fibers, first seen at 32 days, failed to enter host brain. Although grafts demonstrated elements resembling cerebellar tissue, overall organization did not resemble that of the cerebellum.

Fragments of striatum or cerebellum from E 25 rabbit embryo were implanted into either the striatum or the mesencephalon of newborn mice. Implanted rabbit astrocytes were selectively identified by monoclonal antibodies to the GFAP which are unable to combine with mouse GFAP. Previous investigations had shown that xenogenic astrocytes have the capacity to migrate in host CNS. The purpose of this study was to compare the patterns of migration of transplant-derived astroglial cells according to the topographic origin of the transplant and location of the grafting site. We found that the migration pattern of the grafted cells from any of both selected sites of implantation was independent from the topographic origin of the transplant. The routes as well as the distances of migration were similar after homo- or heterotopic transplantation. We conclude that astroglial cells or their precursors do not express information which would direct them to move specifically toward a defined region in the host brain according to the region of origin in the donor.

Spontaneous hippocampal EEG activity and evoked field potentials were investigated in intact rats and in animals with fetal hippocampal grafts. Pieces of hippocampal grafts, derived from 15- to 16-day-old fetuses, were used to prepare cell suspensions and grafted directly into the intact hippocampus. Control animals received suspension grafts of the cerebellum derived from fetuses of identical age. Host hippocampal electrical patterns were monitored with chronic single electrodes or with a 16-microelectrode probe from 7 to 10 months after grafting. In contrast to previously reported high survival rates of fetal grafts in studies with damage to the host brain prior to grafting, survival of both hippocampal (60%) and cerebellar grafts (20%) was very poor in the intact hippocampus. In animals with cerebellar transplants or without surviving grafted neurons the electrical activity of the host hippocampus was indistinguishable from normal controls. In rats with hippocampal grafts short duration, large amplitude EEG spikes (up to 10 mV) were recorded, predominantly during immobility. When the EEG spikes (putative interictal spikes) were of large amplitude and contained population spikes, test evoked responses delivered to the perforant path were suppressed after the spontaneous events. In contrast, evoked responses were facilitated by interictal spikes without population spikes. The threshold of electrically induced afterdischarges did not differ significantly between groups of intact rats and animals with or without hippocampal grafts. However, in three rats with hippocampal grafts the evoked afterdischarges were associated with behavioral seizures. In two of these rats spontaneously occurring seizures were also observed. Synaptophysin-immunoreactivity demonstrated growth of the host mossy fibers into the graft.(ABSTRACT TRUNCATED AT 250 WORDS)

In mammalian cerebellum, granule cells are believed to migrate from the external germinal layer to the internal granular layer on radially oriented arrays of Bergmann glial fibers. It has been shown that similar arrays of Bergmann fibers are present in intact cerebellar transplants in rats. In the present study radial, parallel arrays have been observed in reaggregate and explant transplants in rats. These findings are consistent with the hypothesis that Bergmann fibers are involved in migration of granule cells and/or other aspects of formation of the trilaminar cortical pattern in cerebellar transplants.

Aggregates of cultured neonatal mouse cerebellar astrocytes were implanted into adult mouse sciatic nerves. Two different experimental models were used: aggregates were either placed between proximal and distal stumps of totally transected nerves, or were placed in gaps in partially transected nerves in direct apposition with the cut surface of the proximal stumps. In the model where aggregates were not placed in contact with the proximal stump, regrowing axons rarely entered the aggregates. Where aggregates were placed in contact with the proximal stumps, axons entered the astrocyte-rich environment. Experimental depression of the supply of Schwann cells available to comigrate with regenerating axons proved to be unnecessary: astrocytes provided an alternative substrate for axons. Some axons became myelinated by oligodendrocytes which differentiated within the aggregates; however, few axons remained, unmyelinated, in long-term association with the transplanted astrocytes.

It has been shown in a rat model that surgical injury to the right cerebellum with resultant ataxia can be corrected by implantation of embryonic cerebellar tissue into the injured cerebellum. Histological examinations of the cerebellar tissue revealed that mitoses of Purkinje cells of the implanted group were increased substantially over the control group's. The surgically induced ataxia resolved more rapidly in the cerebellar implant group than the control group. Based on this experimental data, a similar technique was applied in 6 patients with severe hereditary cerebellar degenerative ataxia. The preliminary results in these 6 surgically implanted patients with heredity degenerative cerebellar disease show 2 with marked improvement, 3 with moderate improvement and 1 with improvement for 2 months followed by mild deterioration but still better than presurgery. We also studied immunological markers in the blood and CSF in an attempt to determine whether rejection of implanted tissue occurs.

Implantation of fetal dopamine-containing tissue into preformed cavities in the caudate nucleus of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian monkeys leads to behavioral recovery. Recovery may be related to two sources of dopamine: the grafted cells and/or the sprouted fibers from host dopaminergic neurons. The authors undertook this study to determine whether behavioral recovery requires release of dopamine by the implanted tissue, and to establish if nondopaminergic fetal central nervous system implants can induce sprouting of dopamine fibers in the primate brain and cause behavioral recovery. Rhesus monkeys with MPTP-induced hemiparkinsonism or full parkinsonism and a stable neurological deficit were used for this study. Cavities were created in the caudate nuclei anterior to the foramen of Monro via an open microsurgical approach. Fetal cerebellum or spinal cord was implanted into the preformed cavities of three monkeys. Control parkinsonian monkeys showed no recovery. However, implant-induced improvement was stable for up to 6 months after implantation. Sprouted dopaminergic fibers oriented from the ventral striatum and nucleus accumbens were found in the area of the tissue implant in the animals that received fetal grafts but were not present in the control monkeys. It is concluded that brain implants do not need to contain dopamine to induce functional recovery in MPTP-induced parkinsonian primates. Implant-induced and trophic factor-mediated dopaminergic sprouting by the host brain plays a role in the behavioral recovery and may well be responsible for the clinical improvement seen in parkinsonian patients after brain implants.

A prominent feature of the mammalian cerebellum is its organization into parasagittal compartments. One marker of such compartments is the zebrin I molecule that is expressed by bands of Purkinje cells (PC). In order to understand better the basis for the development of this organization, we have transplanted dissociated rat cerebellar anlage, taken during the period of proliferation of PC precursors, into kainic acid lesioned adult rat cerebellum. As previously observed, the resultant grafts exhibited trilaminar structures reminiscent of the normal cerebellum. In every case, the PC in the resultant grafts were organized into zebrin I+ and - compartments. In one case, most of the grafted PC were integrated into a region of PC deficient host molecular layer that was induced by pretreatment with kainic acid. Clear bands defined by zebrin I reactivity were seen where groups of the grafted PC had entered the host molecular layer. These bands did not correlate in distribution or size with host bands. Hypotheses compatible with these findings that involve specific and non-specific aggregation of PC are discussed.

Improvements in MPTP-induced deficits were only found in subjects that received fetal substantia nigra transplants into the caudate nucleus. The MPTP-induced deficits were assessed using an object retrieval task that examined cognitive and subtle motor performance and by behavioral observation to determine the overall status of the subjects. Subjects that were also moderately or severely impaired by MPTP administration but that received inappropriate donor cells or implant sites (cerebellum to CN or SN to cortex) did not show any evidence of behavioral recovery. These subjects could not respond on the task in the months after grafting and were sacrificed, showing no improvements in parkinsonian signs or healthy behavior signs, up to 5-6 months after surgery. Grafting of SN cells into the striatum of non-MPTP lesioned subjects failed to modify normal behavior or induce abnormal behavior determined by our 2 behavioral assessment methods. In those monkeys that received the appropriate transplants, TH immunohistochemistry revealed that cells of the fetal substantia nigra grafted into the caudate nucleus survived and extended neurites into the host striatum. Indeed, grafted dopamine neurons were often associated with appreciable innervation of the caudate nucleus and appeared to be well incorporated into the host brain. In contrast, examination of the striatum of subjects in the inappropriate-graft group (e.g., cerebellar cells grafted into the caudate) showed no evidence of TH staining within the graft or host caudate nucleus. This indicated that there was no evidence of dopamine neurons present in the grafted tissue and that the mere presence of a fetal graft did not appear to induce sprouting in these MPTP-treated subjects. Although behavioral recovery occurred in only those monkeys that received appropriate transplants (fetal SN to host CN) and not in those that received inappropriate grafts (fetal cerebellum to CN or fetal SN to cortex), the CSF HVA levels did not distinguish those monkeys with improved parkinsonism from those that remained severely parkinsonian. The finding that in some SN-CN grafted subjects reported here, there was evidence of increased dopamine and lowered HVA/dopamine ratio in the vicinity of the SN grafts (cf. Elsworth et al., 1990b) is consistent with the hypothesis that graft-derived or graft-induced dopamine production is responsible for behavioral recovery. In addition, the finding that CSF HVA levels in non-MPTP lesioned subjects were unchanged by fetal SN grafts further indicates that CSF HVA levels may not be sufficiently sensitive to changes in central dopamine production to reflect release of dopamine from relatively small grafts that may, in lesioned subjects, modify behavior.(ABSTRACT TRUNCATED AT 400 WORDS)