Transplanted precursors of nerve cells: their fate in the cerebellums of young rats.

The multiplying cells of the external granular layer in 7-day-old rats were labeled with [(3)H]thymidine. Slabs of the cerebellum were transplanted into the same region of uninjected hosts of the same age. The transplanted, undifferentiated cells of the donors migrated actively in the cerebellar cortex of the hosts and apparently differentiated there into basket and granule cells.

N-ethyl-N-nitrosourea-induced spinal tumors in an inbred strain of W albino rats.

Exposure to N-ethyl-N-nitrosourea (ENU) either transplacentally via the maternal bloodstream or postnatally by direct injection into the cerebellum or the cisterna magna resulted in a high incidence of spinal tumors in an inbred strain of W albino rats. After prenatal exposure to 60 mg ENU/kg maternal body weight, as many as 92% of the offspring developed 1 or more tumors in the spinal cord, whereas after postnatal exposure to 0.2 mg ENU/animal, 50% of the animals eventually developed spinal tumors. These tumors included relatively pure oligodendrogllomas, astrocytomas, and the usual mixed gllomas. Obvious clinical symptoms of paralysis of the limbs and weight loss accompanying the high incidence of tumors in the spinal cord make this system pertinent to the study of carcinogenesis in the central nervous system as well as to the study of related problems to the incidence of these tumors are discussed.

Neural transplantation: an historical perspective.

Literature on transplantation of neural and nonneural tissues into the brains of host animals is reviewed in the perspective of various issues. The two dominant issues determining this research were elucidation of embryological processes underlying the development of the nervous system and regeneration in the host brain. A comprehensive review of studies on regeneration in the central nervous system (CNS), using this technique of transplantation, indicates that regeneration of axonal fibers is small in magnitude and extent, and that it is more directly related to the trauma caused to the brain than to any other variable. This literature review attempts to provide a perspective to the contemporary research on neural transplantation and on regeneration in the CNS.

Experimental analysis of embryogenesis of cerebellum in rat. I. Subnormal growth following x-ray irradiation on day 15 of gestation.

Rat embryos of 15 days gestation were exposed in utero to 170 R of X-ray irradiation. Embryos collected 6 hours, 1, 2 and 3 days after irradiation, and animals of 2, 6, 15 and 30 days postnatal age were used for this study. Six hours after irradiation cells in the neuroepithelium and mantle layer along the roof of fourth ventricle were observed destroyed. Neuroepithelium showed only fragmentary regeneration during next two days, and it contributed to a small number of Purkinje cells. During postnatal development of cerebellum the external granular layer, the zone of proliferative cells that gives rise to neurons of postnatal origin, was found reduced. Other structures such as internal granular layer, molecular layer and medullary layer also were reduced. These multiple temporally sequenced developmental events resulted in a subnormal-sized cerebellum. Various quantitative measures helped establish that grossly the cerebellum in the X-ray irradiated animals was about half of that in the normal animals. Problems related to regeneration in the embryonic cerebellum, and to the factors determining the subnormal size of the adult cerebellum are discussed. Viewing this as an experimental approach to the study of neuroembryogenesis of cerebellum, the role of Purkinje cells in the regulation of development of cerebellum is brought out.

Experimental analysis of embryogenesis of cerebellum in rat. II. Morphogenetic malformations following x-ray irradiation on day 18 of gestation.

Rat embryos of 18 days gestation were exposed in utero to 170 R of X-ray irradiation. Embryos were collected six hours, 1, 2, and 3 days after irradiation, and animals of 2-, 6-, 15- and 30-day-old postnatal age were sacrificed. Six hours after irradiation pyknosis of cells was notices in the external granular layer along the posterior aspect of the cerebellum. Neuroblasts, destined to differentiate into Purkinje cells, were found arrested in their migratory path. During subsequent periods of embryogenesis the external granular layer was found recovered, and clustering of the neuroblasts were disorganized and fragmented. This abnormal clustering of neuroblasts was permanent, and the external granular layer followed the same abnormal pattern in its growth. During postnatal development the internal granular layer also was found to follow the abnormal pattern of Purkinje cell layer. These abnormal developmental events were seen to lead to malformed folia in the anterior regions of the cerebellum. In addition to it the cerebellum of X-ray irradiated animals appeared smaller than the normal. Issues having a bearing on the differential radiosensitivity of different cells, factors determining the small size of the cerebellum, and cellular events determining the morphogenetic malformations are discussed.

Neurogenesis in the brain stem of the rabbit: an autoradiographic study.

With the aid of (H3)-thymidine autoradiography, neurogenesis was documented in the nuclear groups of the medulla oblongata, pons, and mid-brain, as well as in the brain stem reticular formation of the rabbit. Following single injections of (H3)-thymidine, counts were taken of intensely labeled neurons within the nuclei of the functional columns related to the cranial nerves, nuclei of several other functional classifications, and nuclei that did not fit into a functional category. In the brain stem as a whole, neurogenesis was found to occur between days 10.0 and 18.5 of gestation: however, the majority of nuclei studied contained intensely neurons only between days 12.0 and 15.0. Only in the pontine nucleus and the tectum were intensely labeled cells observed as late as day 18.5. Directional gradients of histogenesis were often observed within, as well as between, various nuclei. Within the nuclear columns related to the cranial nerves, a clear mediolateral spread of neurogenesis was observable such that nuclei of the motor columns reached a peak in neurogenesis before those in the sensory columns. Likewise, a mediolateral proliferation pattern was seen in the brain stem reticular formation. Other individual directional gradients were discernible; however, in the brain stem as a whole, distinct overall gradients were not observable. In many individual nuclei, gradients in neuron size were observed such that large neurons preferentially arose prior to smaller neurons. Information pertaining to gradients in neurogenesis, as well as to relationships among functionally related nuclei, are discussed.

Neurogenesis in the epithalamus, dorsal thalamus and ventral thalamus of the rat: an autoradiographic and cytological study.

Times of final mitotic division for neurons of the epithalamic, dorsal thalamic and subthalamic nuclei of the rat were determined with the aid of thymidine-H3 autoradiography. Intensely labelled neurons were observed in the brains of animals injected with radiochemical from days 13 to 19 of gestation. The pattern of distribution of the labelled neurons indicated that neurogenesis in the regions followed caudorostral, lateromedial and ventrodorsal neurogenetic gradients, all of which were found to operate simultaneously. Since neurogenesis in the epithalamus, subthalamus and caudolateral thalamic regions began on days 13 and 14 of gestation, the ventrodorsal and lateromedial proliferative gradients were clearly discerned only within the ventral and dorsal thalamus exclusive of the epithalamus. These directional neurogenetic gradients were apparent throughout the entire thalamus and within individual thalamic nuclei. No neurogenetic pattern based upon neuronal size was observed, i.e., large neurons were not preferentially formed earlier than smaller ones. Detailed information has also been provided on the cytological character of each thalamic nucleus.

Freezing of neural tissues and their transplantation in the brain of rats: technical details and histological observations.

Embryonic neocortical and brainstem tissues were frozen, stored for variable periods, thawed and transplanted into the cerebellum of neonatal host rats. Various conditions related to freezing, media for freezing, DMSO as the cryoprotectant, and thawing were analyzed. The findings indicated that the following conditions yielded best results for neocortical transplantation: freezing at a rate of 1 degrees C/min, using rat amniotic fluid as the medium for freezing, using 10% DMSO as the cryoprotectant, storing the frozen tissues at -90 degrees C, thawing the tissues fast just prior to transplantation, and transplanting them in the host brain with little or no delay. Other conditions having adverse effects on the neural tissues were considered. Issues pertaining to transplantability and retainability of the neural tissues inside the host brain, and effects of freezing and thawing on the long-term viability of the neural tissues and their growth are discussed.

Differentiation of Bergmann glia cells in the cerebellum: a golgi study.

The migration of Bergmann glia cells from the external granular layer to the Purkinje cell layer and the differentiation of their processes were analyzed in Golgi-Cox preparations of the cerebellum of the rat. During their migration they showed numerous cytoplasmic excrescences and long thick processes, which suggested that they may follow ameboid form of movement for their migration. The differentiation of their processes was observed to involve a series of complex events related to retraction of the thick apical processes from the tips of which sheaves of filiform processes emerged, growth in the number and the thickness of the filiform processes, resorption of some of these filiform processes, cyclic changes in the appearance, resorption and reappearance of excrescences on the filiform processes, and changes in size and shape of the growth cones. These findings are discussed in the context of the role played by the Bergmann glia cells in the postnatal developmental events in the cerebellum, and the dynamic nature of differentiation of their processes.

Connectivity of neural transplants in adult rats: analysis of afferents and efferents of neocortical transplants in the cerebellar hemisphere.

Embryonic neocortical tissue, 3.5 mm3 in volume, obtained from 17-day-old Long-Evans rat embryos, was transplanted into the intact cerebellar hemisphere of normal adult rat hosts. Transplants examined 90-160 days later had grown to a final volume of 27.24 mm3, which reflected nearly an 8-fold increase in the initial volume of tissue transplanted. The transplants were all intraparenchymal, having replaced large parts of the cerebellar hemisphere and occasionally portions of the vermis and paramedian lobule during their course of growth and differentiation. They retained a cellular and cytoarchitectural identity characteristic of neocortical tissue. Anterograde degeneration studies and retrograde tracing methods on the light microscopic level revealed that transplants had received afferent connections from the ponto-, olivo- and spinocerebellar projection systems. In addition to these major connections, afferents from other nuclei such as the locus coeruleus and the lateral reticular nucleus were also observed with the HRP method. Efferent outgrowth as studied with degeneration methods revealed projections to the nearby host cerebellum and to the ipsilateral deep cerebellar nuclei. All transplants had developed massive intratransplant connections. Findings on the nature and magnitude of connections were analyzed in terms of different characteristics of the interface between the transplant and the host brain tissue. The surface of cortical transplants was found to consist of 7 distinct components, 5 of which were interface regions. Two types of interface regions, those between the cerebellar medullary and granular layers and transplants, were readily related to the magnitude of extrinsic afferent ingrowth, and hence were effective sprouting surfaces. Using two correlated estimates of the magnitude of afferent ingrowth to cortical transplants, volume of degeneration and surface area of degeneration of transplants resulting from lesions of host brain structures, the pontine system was found to provide more afferents to transplants than the olivary or spinal systems. Generally, extrinsic fibers were located nearer to transplant-host brain interface regions than deep within transplants. A positive correlation existed between the available effective sprouting surface area of transplants (an estimate of interactive host fibers with a suitable trajectory) and the magnitude of innervation of cortical transplants by extrinsic afferents.

Behavioral effects of CNS transplants in the rat.

Embryonic forebrain tissue from 17-day embryos was transplanted into the midline cerebellum of 10-day-old rat pups. The animals were allowed to survive for behavioral testing and were compared with animals receiving aspiration lesions of midline cerebellum and with normal controls. Subsequent histology indicated that the transplanted tissue had produced a compression lesion of the host cerebellum and had become fully integrated with the neuropil of the host animal. Behavioral results revealed no significant differences between transplant and control animals. Both of these groups were discriminably different from the lesion condition. It is suggested that the transplant may establish afferent and efferent connections similar to those present in the intact animal and thus may be anatomically well integrated with the host brain.

Neural transplantation: autoradiographic analysis of histogenesis in neocortical transplants.

Neurohistogenesis in neocortical transplants obtained from 15-, 16-, 17-, 18-, 19-, 20- and 21-day-old embryos, was studied employing [3H]thymidine autoradiography. The neural tissues were transplanted in the midvermis of cerebellum of the host animals. Following transplantation the host animals in different groups were injected with the radiochemical at 6 hr, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 day intervals, to label the neurons forming on different days in the developing transplants. Analysis of autoradiograms showed that all the neocortical transplants did undergo histogenesis in the host cerebellum, and that it was similar to that seen in a normally developing neocortex. Transplants from the 15-day embryos showed histogenesis lasting for 9 days, and at the other extreme transplants from the 21-day embryos showed histogenesis lasting only for 1 day. Histogenesis in other transplants fell between these two extremes in a graded fashion in relation to the age of the donor embryos. The magnitude of histogenesis in transplants from different donor embryos was closely related to the final size of the transplants. Transplants from 15-day embryos were the largest in size, and they were followed by those from 16-, 17-, 18-, 19-, 20- and 21-day donor embryos in a graded fashion. All transplants were intraparenchymal, and histologically appeared normal. They contained fully differentiated neurons, and were anatomically integrated with the host cerebellum without any glial scar tissue or necrotic tissue intervening between them.

Tissue repair in the embryonic rat spinal cord following exposure to N-ethyl-N-nitrosourea.

The cytotoxic effects of N-ethyl-N-nitrosourea (ENU) and the potential for recovery from this damage in the developing rat spinal cord was investigated. Emphasis was placed on determining the severity and location of initial cell necrosis and the subsequent reorganizational changes in the damaged tissues. Pregnant rats were injected i.v. with a single dose of ENU (60 mg/kg) on one of days 12-16 of gestation. At 6, 12, 24 and 48 h post-injection one pregnant rat from each gestational stage was anesthetized, the embryos were removed, fixed and processed for embedding in paraplast or epon-araldite. Transverse sections from embryos killed at 6 h revealed extensive necrosis throughout the neuroepithelium in accordance with the temporal-spatial patterns of neurogenesis. At this dose level the post-mitotic neuroblasts appeared unaffected. Regeneration of the damaged neural tissue as defined by the restoration of the neuroepithelial cell layer and removal of necrotic debris proceeded quickly, and within 48 h a near-normal cytoarchitecture was observed. The embryonic age at time of ENU injection had no apparent influence on the actual sequence of tissue repair in the spinal cords although the events were slightly delayed within embryos exposed to ENU on days 12 or 13 of gestation.

Growth of neural transplants in rats: effects of initial volume, growth potential, and fresh vs frozen tissues.

Interactions between growth potential (as related to the age of donor embryos and type of tissue), initial volume, and fresh vs frozen conditions of neural transplants were studied in rats. Neural tissues with high growth potential (16-day gestation neocortical tissue) when used fresh yielded the best growth of the transplants, which was positively related to the initial volume of the tissue. At the other extreme, neural tissues with very low growth potential when used following their freezing and thawing yielded the poorest results. Changes in the initial volume of transplants did not seem to improve the final growth. Combination of these variables in between these two extremes yielded transplants of variable sizes.

Neural transplants: volumetric analysis of their growth and histopathological changes.

Neocortical transplants from 15-, 18- and 22-day rat embryos were transplanted into the cerebellum of the host animals. The necrotic changes and growth in the transplants, the blood in relation to them, and the space containing CSF were analyzed quantitatively in a developmental sequence. The histopathological changes were seen to last for 6-8 days. The neural tissues after initial regression showed recovery, and started to grow in size after 3-4 days. The transplants from 15-day donors showed the highest growth, and this was followed in sequence by those from 18- and 22-day embryos. Techniques involved in manipulation and dissection of the donor embryos, and in transplantation of the tissue, were found to play an important role in the necrotic and other changes in the transplants.

Gliogenesis and ependymogenesis during embryonic development of the rat. An autoradiographic study.

With the aid of [3H]thymidine autoradiography gliogenesis and ependymogenesis were studied in the brain of the rat during embryonic development. Gliogenesis was found to begin on day 17 of gestation in the caudal regions of the brain stem, and to spread rostrally. On days 20 and 21 of gestation gliogenesis reached a peak, and then declined. Ependymogenesis began earlier and showed the following pattern: day 14 of gestation in the 4th ventricle and cerebral aqueduct, day 15 in the 3rd ventricle, and day 17 in the lateral ventricles reaching a peak on different days in different sites. Both gliogenesis and ependymogensis continued up to the last day of gestation, day 22. Issues pertaining to gliogenesis and the formation of glioblasts, and the relationship between gliogenesis and ependymogenesis are discussed.

Neural transplantation in the spinal cord of adult rats. Conditions, survival, cytology and connectivity of the transplants.

Embryonic neural tissues of various types were transplanted into the intact, completely transected, and partially transected spinal cords of adult rats. The host animals were killed 4-6 months after the surgery, and the spinal cords and transplants examined. The best results were obtained when embryonic neocortical tissues obtained from 16-day rat embryos were used for transplantation into host animals that had been subjected to partial sectioning of the spinal cord. Use of other types of neural tissue, or transplantation of tissues into the intact or completely severed spinal cords was not successful. The successful neocortical transplants had survived, grown, differentiated, and established anatomical integration with the host spinal cords. The anatomical integration was established through an interface with the host spinal cord along the basal aspect. Along the lateral aspect glial scar tissue was present separating the transplants from the spinal cord parenchyma. The transplants contained well-differentiated and normal-looking neurons. They received afferents from the spinal cord only through the interface and not through the glial scar formations. The findings indicated that it is possible to transplant embryonic neocortical tissues into the spinal cords of the adult animals that become integrated with the spinal cord parenchyma. The axonal fibers in the adult spinal cord appear capable of regeneration and growing into the transplants only when an appropriate neural milieu, in the form of a healthy and viable interface, is available. In its absence the severed axons of the adult spinal cord do not grow into the neural transplants.

N-ethyl-N-nitrosourea-induced teratogenesis of brain in the rat. A cellular and cytoarchitectural analysis of the neocortex.

N-Ethyl-N-nitrosourea (ENU) was administered intravenously to pregnant Wistar-albino rats on days 14--21 of gestation in order to study the teratological effects of the carcinogen on the developing brain. Offspring were killed 60 days postnatally, and the brains examined histologically by cresyl violet staining and Golgi-Cox preparations. Macroscopic examination of the brains revealed a graded pattern of microcephaly. Injection of ENU on day 14 of gestation gave the most severe effects and injection on day 21 the least effects. Microscopic examination of the neocortex revealed a graded reduction in the lateral and sagittal lengths, thickness, and the number of cells in a sample slab of the cortex. In the Golgi-Cox preparations the pyramidal neurons of cortical layers III and V, as well as neurons of other layers, were seen to have fewer secondary and tertiary dendrites, and the length of their dendrites appeared stunted. These characteristics also were graded in relation to the day of injection of ENU. Altered cytology and cytoarchitecture of the neocortex and possible underlying mechanisms are discussed.

Perspectives in anatomy and pathology of paraplegia in experimental animals.

Three models of inducing spinal trauma in experimental animals--weight-dropping model, severance-by-knife model, and laceration-type-lesions model--are reviewed critically. Contributions by these models in understanding paraplegia in anatomical and pathological terms are brought out. Important distinctions between subthreshold traumas vs. threshold and suprathreshold traumas, transient and permanent paraplegic syndrome, and regeneration of served axonal fibers vs. prevention of development of permanent paraplegia, are stressed while evaluating each model of spinal trauma. Conceptual contributions by these three models and their bearing on the potential clinical applications are discussed.

Permanent alterations in the rat spinal cord following prenatal exposure to N-ethyl-N-nitrosourea.

Pregnant rats between gestational stages E14-E22 were given a single injection of N-ethyl-N-nitrosourea (ENU). Pups born of these females were sacrificed 60 days after birth and their spinal cords examined qualitatively and quantitatively. Quantitative analysis involved measurement of spinal cord length and volume, estimation of neuron number, and the measurement of individual cell dendritic number and length. Cytoarchitecturally spinal cords appeared normal in all animals regardless of the age when they were exposed to ENU. Animals exposed during the latter portion of neurogenesis in the spinal cord (E14-E16) had significantly (p less than 0.05) reduced volumes of gray matter and reduced cell counts. Cellular analysis showed that all animals exhibited some stunting of dendritic length, although the number of dendritic branches was significantly (p less than 0.01) higher than normal for neurons of the intermediate gray and the substantia gelatinosa. Increase in the number of dendrites per cell suggests a mechanism of structural compensation by the surviving neuronal cells following their exposure to the teratogen.