Protective effects of fetal neocortical transplants on cognitive function and neuron size in rats with congenital micrencephaly

The rat with micrencephaly, produced by prenatal exposure to methylazoxymethanol, provides a useful model to study neurobehavioral abnormalities associated with congenital brain defects. The micrencephalic animals have a life-long learning impairment. As they age, their already impaired learning competence deteriorates further. To determine whether the aging-associated functional deterioration could be ameliorated by a neural transplant, micrencephalic rats bearing solid transplants of normal fetal neocortical tissue since infancy were evaluated on a visual pattern discrimination learning at 15 months and a spatial navigation test at 24 months of age. The transplant-bearing rats learned both tasks significantly better than the micrencephalic rats without transplants. Morphometric analyses revealed that cortical pyramidal neurons were larger in the transplant-bearing rats than in micrencephalic rats without transplants. The life-long presence of a transplant appeared to have protected the micrencephalic brain against aging-associated deterioration. This is the first demonstration that a neural transplant, placed in a congenitally defective infant brain, can ameliorate aging-associated cognitive deficits.     

Fetal frontal cortex transplant (14C) 2-deoxyglucose uptake and histology: survival in cavities of host rat brain motor cortex

Fetal frontal neocortex from 18-day-old rat embryonic brain was transplanted into cavities in 30-day-old host motor cortex. Sixty days after transplantation, 5 of 15 transplanted rats had surviving fetal transplants. The fetal cortex transplants were physically attached to the host brain, completely filled the original cavity, and had numerous surviving cells including pyramidal neurons. Cell lamination within the fetal transplant was abnormal. The (14C) 2-deoxyglucose uptake of all five of the fetal neocortex transplants was less than adjacent cortex and contralateral host motor-sensory cortex, but more than adjacent corpus callosum white matter. The results indicate that fetal frontal neocortex can be transplanted into damaged rat motor cortex. The metabolic rate of the transplants suggests they could be partially functional.     

Fetal brain grafts induce recovery of learning deficits and connectivity in rats with gustatory neocortex lesion

Three groups of rats showing disrupted taste aversion due to gustatory neocortex lesions, were studied. One group received a transplant of homotopic cortical tissue, another of heterotopic tectal tissue, obtained from 17-day-old fetuses. The third group remained without transplant as a lesioned control group. Comparisons of the taste aversion scores before and after graft, revealed that cortical grafted animals significantly improved the taste aversion, whereas those which received tectal grafts, and the cortical-lesioned controls did not. Moreover, results with horseradish peroxidase (HRP) histochemistry revealed that the homotopic, but not the heterotopic, brain transplants were able to re-establish connections with amygdala and with the ventromedial nucleus of the thalamus areas who normally kept connectivity with the gustatory neocortex. These results support the hypothesis that fetal brain transplants can reestablish cognitive functions, as well as connectivity with its host tissue.     

Integration of neocortical embryonal grafts with the neocortex of host rats examined by Leao's spreading cortical depression

Cortical spreading depression (SD) was used to assess the density and organization of neural elements in neocortical transplants and their connectivity with the host brain. Embryonal neocortex (E14) was transplanted into cavities in the frontoparietal cortex of 3-month-old rats. SD elicited in the cortex of anesthetized host rats (n = 12) 3 to 8 months after transplantation did not penetrate into the grafts. SD could be elicited in large transplants but did not propagate to the surrounding host neocortex. Spontaneous unit activity in the transplants was affected by SD elicited in the neocortex of the host rats anesthetized with urethane. Most units (n = 49) displayed excitatory-inhibitory (52%) or inhibitory (29%) reactions, whereas purely excitatory reactions were less frequent (8%). The results suggest that the packing density of neurons in the transplant can support SD but that the conditions at the graft-host boundary (glial scar, scarcity of neurons) stop SD propagation. High reactivity of the graft neurons to SD in the host neocortex indicates that afferentation from the host brain represents an important, predominantly excitatory contribution to the spontaneous activity of the transplant.     

Grafts containing fetal hippocampal tissue reduce activity and improve passive avoidance in hippocampectomized or trimethyltin-exposed rats

Embryonic Day 16 or 17 rat tissue containing either hippocampus with some medial pallial anlage or cerebellar/alar plate anlage was transplanted to the site of the ablated hippocampus of otherwise normal adult rats or adult rats previously exposed to the neurotoxin trimethyltin. Ninety to one hundred five days later these rats were compared to control rats in acquisition of passive avoidance and in open field activity. Transplantation of both types of tissue produced behavioral recovery on both tasks in rats with hippocampal lesions that had not been exposed to trimethyltin. Only hippocampal transplants produced recovery of function in rats given trimethyltin. Although transplants of hippocampal tissue had an organotypic structure that was easily differentiated with cell and fiber stains from that of the cerebellar transplants, neither of these routine histological procedures nor immunocytochemical analysis revealed differences between transplants made into normal rats or toxicant-exposed rats. Either of two mechanisms may account for the ability of the transplants to produce behavioral recovery. These are reconstruction of damaged circuitry by the transplant and neurotrophic action of the developing transplant on the host brain. The second mechanism alone may be sufficient to restore function in brain-lesioned but otherwise normal rats. Therefore, either type of transplant is effective. Both mechanisms may be necessary for recovery in brain-lesioned, toxicant-exposed rats. Therefore, only transplants of tissue homotypic to the tissue removed from the brain are effective.     

Nerve growth factor increases the size of intracortical cholinergic transplants

The effects of continuous intracortical mouse Nerve Growth Factor on fetal rat basal forebrain transplants in denervated adult rat neocortex were investigated. Enzyme-linked immunoassay (ELISA) was used to measure the time course of endogenous NGF protein production in neocortex, hippocampus, and basal forebrain in a cohort of animals receiving unilateral ibotenic acid (IBO) lesions of the nucleus basalis magnocellularis (nBM). A second cohort of IBO-nBM lesioned animals received transplants of fetal basal forebrain followed by two to four weeks of continuous NGF or cytochrome-C infusion into the ipsilateral frontoparietal neocortex. To study the effects of abnormally high NGF doses on transplanted and host tissue, the cumulative dose of intracortical NGF was on the order of micrograms, compared with maximum picogram levels of neocortical NGF produced following IBO-nBM lesions. A four-fold increase in transplant size, and greater cell and fiber densities were observed in NGF-treated compared with NGF-untreated transplants. No adverse histological effects of long-term, high-dose NGF treatment were observed on transplanted basal forebrain or host neocortical tissue. These data indicate that cholinergic-rich mammalian brain tissue and intrinsic host tissue can be stimulated by high doses exogenous NGF without obvious deleterious effects.     

Fetal Neocortical Tissue Blocks Implanted in Brain Infarcts of Adult Rats Interconnect with the Host Brain

The purpose of the present study was to study if the connectivity of fetal neocortical tissue blocks placed in ischemic brain infarcts of adult rats would be enhanced in rats housed in an enriched environment. We also investigated whether the enriched housing conditions could enhance the postischemic and postgrafting functional outcome, in terms of motor behavior. This part of the study has been published recently. The middle cerebral artery was ligated on the right side in 37 inbred, adult male spontaneously hypertensive rats. The rats were placed at random either in an enriched environment (groups A and B) or in standard laboratory cages (group C). Three weeks after the artery occlusion, blocks of fetal sensorimotor cortex (Embryonic Day 17) were transplanted into the infarct cavity of rats from groups B and C. After 9 weeks all transplanted rats received an injection, into the graft, of a mixture containing the two tracers Fluoro-Gold and biotinylated Dextran amine. The transplants revealed a structured morphology with whorls and bands of cells reminiscent of normal neocortex. Tracing of efferent transplant to host fibers with biotinylated Dextran amine showed pronounced intrinsic transplant projections, as well as fibers, although significantly fewer, to the host ipsilateral sensorimotor cortex, striatum, and thalamus. Host to transplant projections were revealed by Fluoro-Gold-labeled cells found in the ipsilateral host sensorimotor cortex, the basal nucleus of Meynert, the thalamic ventrobasal, ventrolateral and posterior nuclei, and in the dorsal raphe nuclei. We conclude that fetal frontal neocortical block grafts placed in brain infarcts of adult rats develop a morphology reminiscent of normal neocortex and that both afferent and efferent neural connections, although sparse, are established with the host brain, whether the rats are reared under enriched housing conditions or not.     

Anatomical and behavioral sequelae of fetal brain transplants in rats with trimethyltin-induced neurodegeneration.

The effect of transplants of either fetal hippocampal or dorsal ventricular ridge (DVR) tissue into the brains of adult male rats exposed to TMT was determined for two behavioral tasks. Administration of TMT produced deficits in acquisition and performance of an operant differential reinforcement of low response rates (DRL) schedule and learning in the Morris water maze. The fetal transplants developed well within the TMT-damaged brains of the adult rats and numerous axons could be shown to cross the host-transplant interface. The transplants significantly reduced the DRL deficit produced by exposure to TMT. However, the TMT-induced deficit in water maze acquisition was made significantly worse by the hippocampal transplants. The improvement in DRL performance is attributed to the effect on the host brain of an unidentified trophic substance produced by the transplants. However, this positive effect may not protect the brain sufficiently to produce recovery in tasks demanding more complex neural computations than are required to withhold lever-press responses. The transplant-induced deficit observed in some aspects of water maze acquisition and performance may be attributable to either a tumor-like deleterious effect of the mass of the transplant or to abnormal neuronal activity transmitted from the transplant to the host brain. The results of the present study, and those from other similar studies, suggest that transplants of fetal tissue may be useful in producing changes in the brain of an animal exposed to an environmental neurotoxin, but that research should be focused upon development of transplant methodology that will minimize adverse effects of the grafts.     

Cultured embryonic retinae transplanted to rat brain: Differentiation and formation of projections to host superior colliculus

Retinae of rats on embryonic day 14 were placed in explant culture for 2–14 days prior to transplantation adjacent to the superior colliculus of newborn rats. In explant culture cell division and neuronal differentiation continued unabated. One month after transplantation host brains were examined for transplant survival, differentiation and formation of projections to the host brain. The culture retinal transplants survived and developed a morphology typical of mature retina, with normal cell and fiber laminae present. HRP injections into the host superior colliculus labeled neurons in the ganglion cell layer of the transplant which closely resembled ganglion cells in vivo. A small number of transplants received lesions. Degeneration material was traced into the superior colliculus and pretectal nuclei confirning that the cultured transplants had projections appropriate for retina entering the host brain. These results correlate closely with those seen after transplantation of embryonic rat retinae that had not been cultured^{26, 27}.     

Fetal cortical transplants in adult rats subjected to experimental brain injury.

Fetal cortical tissue was injected into injured adult rat brains following concussive fluid percussion (FP) brain injury. Rats subjected to moderate FP injury received E16 cortex transplant injections into lesioned motor cortex 2 days, 1 week, 2 weeks, and 4 weeks post injury. Histological assessment of transplant survival and integration was based upon Nissl staining, glial fibrillary acidic protein (GFAP) immunocytochemistry, and staining for acetylcholinesterase. In addition to histological analysis, the ability of the transplants to attenuate neurological motor deficits associated with concussive FP brain injury was also tested. Three subgroups of rats receiving transplant 1 week, 2 weeks, and 4 weeks post injury were chosen for evaluation of neurological motor function. Fetal cortical tissue injected into the injury site 4 weeks post injury failed to incorporate with injured host brain, did not affect glial scar formation, and exhibited extensive GFAP immunoreactivity. No improvement in neurological motor function was observed in animals receiving transplants 4 weeks post injury. Conversely, transplants injected 2 days, 1 week, or 2 weeks post injury survived, incorporated with host brain, exhibited little GFAP immunoreactivity, and successfully attenuated glial scarring. However, no significant improvement in motor function was observed at the one week or two week time points. The inability of the transplants to attenuate motor function may indicate inappropriate host/transplant interaction. Our results demonstrate that there exists a temporal window in which fetal cortical transplants can attenuate glial scarring as well as be successfully incorporated into host brains following FP injury.     

Motor deficits are produced by removing some cortical transplants grafted into injured sensorimotor cortex of neonatal rats.

Fetal frontal cortex was transplanted into cavities formed in the right motor cortex of neonatal rats. As adults, the animals were trained to press two levers in rapid succession with their left forelimb to receive food rewards. Once they had reached an optimal level of performance, the effect of removing their transplants was assessed. Surgical removal of transplants significantly impaired the performance of 2 of 4 subjects. Placing a cross-strain skin graft to induce the immunological rejection of the transplants produced a behavioral deficit in 1 of 2 subjects with complete transplant removal. Skin grafts produced no behavioral effects in four subjects that had surviving transplants. Since the motor deficits produced by transplant removal resembled those observed following the removal of normal motor cortex, we propose that these three transplants functioned within the host brain. Histology showed that the procedures used to remove cortical grafts did not injure any host brains. Therefore, host brain damage is unlikely to account for the behavioral deterioration that followed transplant removals.     

Electrophysiological properties of embryonic neocortex transplants replacing the primary visual cortex of adult rats

Solid pieces of the occipital neocortex derived from 17-day rat fetuses were placed in a cavity formed by complete unilateral aspiration of the primary visual cortex in adult rats. Vital labeling of the brain with bisbenzimide was used to differentiate grafts from the host brain tissue. 2 to 10 months after operation electrophysiological experiments were performed in which neuronal activity and field potentials in transplants were recorded in response to sensory and electrical stimulation of the host brain. This study shows that in a large portion of the transplants (14 out of 25): (1) the majority of neurons (183/270) are controlled by visual stimuli and many of them respond to electrical stimulation of the lateral geniculate body (53/62) and the homotopic sites of the contralateral neocortex (28/62); latencies of these responses are within the ranges typical of the normal visual cortex; (2) there is a topical representation of the visual field on the transplants; (3) receptive field sizes, the preference to stationary flashes or to moving visual stimuli and the temporal response pattern of the grafted neurons are similar to those of the primary visual cortex. However, the field potentials evoked visually were recorded only in part of the transplants (8/14) which revealed clear neuronal visual responses, and field potential depth profile differed from that in visual cortex in situ. The functional organization of the transplants remained unchanged throughout the long-time testing. Taken together, these results suggest that after primary visual cortex removal, fetal neocortex transplants may be able to replace functionally the damaged neural circuitries of the host brain.     

Regeneration of adult dorsal root axons into transplants of fetal spinal cord and brain: a comparison of growth and synapse formation in appropriate and inappropriate targets

Cut dorsal root axons regenerate into transplants of embryonic spinal cord and form synapses that resemble those found in the dorsal horn of normal spinal cord. One aim of the present study was to determine whether these axons also regenerate into and establish synapses within transplants of embryonic brain. A second aim was to compare the patterns of growth in embryonic brain and spinal cord transplants. Embryonic spinal cord or brain was transplanted into the lumbar enlargement of adult Sprague-Dawley rats, the L4 or L5 dorsal root was cut, and the cut root was juxtaposed to the transplant. The transplants included whole pieces or dissociated cell suspensions of embryonic day 14 (E14) spinal cord, or whole pieces of E14 neocortex, E18 occipital cortex, E15 cerebellum, or E18 hippocampus. One month later the regenerated dorsal root axons were labeled by immunocytochemical methods to demonstrate calcitonin gene-related peptide (CGRP). CGRP-immunoreactive axons regenerated into all the transplants examined and formed synapses in the neocortex and cerebellum transplants in which they were sought. Synapses were far rarer in neocortex and cerebellum than we had observed previously in transplanted spinal cord, and the patterns of growth differed in transplants of spinal cord and brain. In solid transplants of spinal cord, regenerated axons remained relatively close to the interface with the dorsal root, branched, and formed bundles. Areas of dense ingrowth were separated by regions with few labeled axons. In transplants of brain regions, the regenerated axons were few, unbranched, and appeared as individual fibers rather than in bundles, but they were distributed widely in neocortex transplants. The results of quantitative studies confirmed these observations. The area fraction occupied by regenerated axons in solid spinal cord transplants was significantly larger than in occipital cortex or cerebellum transplants. Distribution histograms of the area occupied in transplants demonstrated that regenerated axons were distributed sparsely but homogeneously in transplants of brain, whereas spinal cord transplants were heterogeneous for regenerated axons and contained areas in which growth was dense or sparse. In contrast, several measurements of axon distribution, including area, longest axis, and length of lateral extension, indicated that CGRP-labeled axons spread more widely in occipital cortex transplants than in solid transplants of spinal cord or cerebellum. The results indicate that embryonic CNS tissues that are not normal targets support or enhance the growth of severed dorsal roots and suggest that the conditions that constitute a permissive environment for regenerating axons are relatively nonspecific.(ABSTRACT TRUNCATED AT 400 WORDS)     

Behavioral and morphological consequences of primary astrocytes transplanted into the rat cortex immediately after nucleus basalis ibotenic lesion

Adult male rats received transplants of dissociated 30-day old cultured cortical astrocytes into the ipsilateral frontal and parietal cortex immediately after unilateral ibotenic acid lesion of the NBM or after sham injury. We hypothesized that transplants of astrocytes into the acetylcholine-deprived cortex might provide trophic support to terminals arising from damaged NBM neurons. Twenty four hours after transplantation and every other day for 11 days post surgery, the animals were tested for locomotion and habituation in an open field. NBM lesion reduced vertical movements only as compared to no lesion and no transplant counterparts. Nine days after surgery rats with NBM lesion and astrocyte-transplants into the cortex were as impaired in the acquisition of a passive avoidance (PA) task as untreated counterparts. Animals with no lesions and transplants into the cortex also had significant PA acquisition deficits. All rats with ibotenic lesion were significantly impaired on PA retention as compared to rats with no lesions. Astrocyte-transplants survived up to 2 months after cortical implantation but these transplants produced severe laminar disruption and gliosis. This effect was greater in rats with NBM lesion than in intact animals with transplants into the cortex. These data show that astrocyte-transplants do not promote functional recovery after NBM lesion and suggest an immune rejection of the astrocyte transplants by the host brain.     

Intraparenchymal fetal striatal transplants and recovery in kainic acid lesioned rats

Striatal kainic acid (KA) lesions induce behavioral and biochemical deficits which resemble symptoms encountered in patients suffering from Huntington's disease. In rats with KA lesions, fetal striatal transplants have shown to reverse the pervasive nocturnal hyperactivity induced by the lesion. In the present study 4.6 mm3 of fetal striatal tissue were delivered bilaterally into the anterodorsal portion of the lesioned caudate nucleus. Care was taken to deliver the transplant within the host parenchyma and away from the lateral ventricles. Locomotor behavior analyzed using the Digiscan animal activity monitors before and after the transplants demonstrated a reversal of the hyperactivity following transplants in 70% of lesioned animals. Microinjections of horseradish peroxidase delivered into the globus pallidus and substantia nigra of a small group of functionally recovered transplanted animals, did not reveal evidence for reinnervation between host nigra or pallidum and the transplant at 10 weeks post-transplantation. Other laboratories have reported anatomical connections by 6 months post-transplantation. Ventricular/brain ratios demonstrated that intraparenchymal transplants significantly reduced the ventricular dilation following KA lesion. These results suggest that functional recovery can be obtained when the transplant is immersed into the host's striatal parenchyma regardless of the existence of long-range anatomical connections.     

Cerebrovascular permeability in intracerebral implants of foetal basal forebrain cell suspensions

Blood-brain barrier permeability was measured using [14C]-labelled a-aminoisobutyric acid (AIB) quantitative auto-radiography in rats which had previously received unilateral ibotenate-induced lesions of the nucleus basalis followed by intracortical implantation of foetal basal forebrain cell suspensions. The permeability characteristics of intracortical transplants were found to be dependent upon the site of implantation. Superficial transplants were invariably associated with AIB transfer constants (Ki) 3- to 4-fold higher than those in corresponding contralateral host cortex. In transplants sited deep in host neocortex, Ki values were not significantly different from those measured in surrounding host brain tissue.     

Status marmoratus in fetal cortical transplants

Adult rats received unilateral transplants of either embryonic day-17 fetal neocortex or striatum. Sixty-two percent of the fetal neocortical transplants grafted into the striatum, and 25% of these neocortical transplants placed in host neocortex had regions of hypermyelination, whereas none of the striatal transplants showed such an effect. There are similarities between these neuropathologic findings and status marmoratus in humans and animals with perinatal hypoxic injuries.     

Transplantation of central nervous tissue

Studies of post-lesional reorganization of central nervous connections have shown that central nerve fibers respond to nearby denervation by sprouting and formation of new terminals. The connections in the central nervous system (CNS) are accordingly much more plastic than was thought for a long time. This has revived the interest in transplantation of central nervous tissue. In this study we present some historical data on CNS transplantation supplemented by recent results obtained in our laboratory. Pieces of hippocampal tissue from embryonic or early postnatal rats were transplanted to different parts of the brain of littermates or adult rats. About two-thirds of the transplants were recovered after survival times ranging from 4 d to 2 years, and their cytological organization and intrinsic connections were monitored by cell and fiber stains and histochemical methods (AChE staining and Timm sulphide silver method). Comparison with both a normal and a lesioned control material revealed that in most transplants the tissue had developed as it does when left in situ in the donor brain, but deprived of its major afferent connections. In several instances we found evidence of a major exchange of connections between the transplants and host brains. The conditions needed for this to occur appeared to involve growth stimulation of host brain fibers by transection (host to transplant) and denervation of host neuropil (transplant to host). In cases where these conditions are met, the use of transplants may have future implications in attempts to repair lesions in the central nervous systems.     

Testosterone-induced enhancement of male medial preoptic tissue transplant volume in female recipients: a possible neuronotrophic action

To characterize further the action of gonadal hormones on the development of the brain, this study was designed to test the ability of testosterone treatment to modify the volume of male brain tissue transplants in female recipients. One-day-old females, in addition to having either medial preoptic area (MPOA) or caudate nucleus (CN) tissue from neonatal males bilaterally implanted into their own MPOA, also received a subcutaneous injection of either 200 micrograms testosterone propionate (TP) or oil concurrently, and on the following 4 days. All recipients were sacrificed at 30 days of age postnatally. An analysis of male transplant volumes indicated that MPOA transplants in oil-treated recipients were substantially reduced in size (0.06 +/- 0.01 mm3) compared with the initial transplant volume of 0.19 mm3. However, MPOA transplants in recipients treated with TP showed a 79% increase above the initial transplant volume (to 0.34 +/- 0.05 mm3). The resulting 5- to 6-fold difference in MPOA transplant volume between oil and TP treated animals was highly significant. In sharp contrast, the same TP treatment to recipients receiving male CN transplants resulted in no enhancement of transplant volume. Therefore, transplants involving a brain area known to concentrate 3H-labeled testosterone neonatally (i.e. the MPOA) responded to TP treatment with an enhancement of volume which was not observed for transplants consisting of brain tissue not known to concentrate 3H-labeled testosterone (i.e. the CN). The above results suggest that testosterone is a 'neuronotrophic' agent during development that acts specifically on cells within steroid-sensitive brain areas, perhaps to prevent neuronal death within these areas.     

A preliminary study of homotopic fetal cortical and spinal cotransplants in adult rats

Neocortical and spinal tissue from a given E16-17 rat fetus were homotopically transplanted into lesion sites of adult rats which had undergone combined cortical and complete lower thoracic spinal cord lesions. Spinal cord transplants were placed either directly into the gap in host spinal cord or embedded in a collagen matrix. Animals were killed from 4 days to 8 months and tissues were processed for light microscopy. All cortical transplants survived and integrated with host brain. Many axons appeared to grow between the cortical transplant and subjacent host parenchyma. Only collagen-embedded spinal transplants survived. At 8 months, two animals underwent spinal cord transection and HRP implantation two vertebral segments rostral to the spinal cord transplantation site. Both animals revealed HRP-labeled neurons in the cortical transplants. It was concluded that 1) homotopically transplanted fetal cortical tissue can survive and may be capable of extending axons to midthoracic levels, and 2) a collagen matrix may enhance the survival of fetal tissues transplanted into a complete gap in host spinal cord.