Retinal afferent ingrowth to neocortical transplants in the adult rat superior colliculus is due to the regeneration of damaged axons.

Retinal afferent ingrowth to embryonic neural transplants in the adult rat superior colliculus may represent either sprouting of intact axons or the regeneration of transected axons. If ingrowth represents regeneration of damaged retinofugal axons, then lesions that axotomize more retinofugal axons at the transplantation site should induce greater retinal afferent ingrowth. Alternately, if ingrowth represents terminal or collateral sprouting of intact retinofugal axons at or near the transplant/host optic layer interface, then the magnitude of retinal afferent ingrowth should be directly related to the total area of this interface. To test between these two hypotheses surgical knife wounds were made either parallel (in the sagittal plane) or perpendicular (in the transverse plane) to the course of axons in the stratum opticum, embryonic neocortical tissue was transplanted at the coordinates of these tectal slits, and retinal afferent ingrowth visualized 1-90 days after surgery using anterogradely transported HRP. A zone of traumatic reaction (ztr) in the optic layers was seen in every case, characterized by hypertrophied axons and swollen terminal clubs at 1 day. Between 30 and 90 days the damaged retinofugal axons in the zone formed dense fascicles and neuroma-like tangles. Retinal afferent ingrowth occurred only across transplant interface regions with the ztr. The magnitude of ingrowth was directly related to the area of the ztr interface and not the total optic layer interface area. Retinal afferent ingrowth appears to reflect the intrinsic regenerative capacity of adult mammalian retinal ganglion cells and not sprouting of undamaged axons.

Diversity among reactive astrocytes: proximal reactive astrocytes in lacerated spinal cord preferentially react with monoclonal antibody J1-31.

An Astrocyte-specific antigen recognized by monoclonal antibody J1-31 is a more intense marker for proximal reactive astrocytes in lacerated rat spinal cord than is glial fibrillary acidic protein (GFAP). Thus, MAb J1-31 recognizes reactive astrocytes in the immediate vicinity of the lesion, whereas reactive astrocytes that are located at a distance from the lesion are not detected by immunofluorescent staining. These findings are relevant to the biochemical heterogeneity manifested respectively by reactive astrocytes located proximal and distal to a laceration-type injury of the spinal cord, and those that develop following axotomy with retrograde degeneration. Reactive astrocytes in the axotomy model are not stained with MAb J1-31, but are positive for GFAP.

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.

Spinal traumas: some postoperative complications in experimental animals.

Animals with severe spinal traumas show paraplegic syndrome and various somatic and autonomic dysfunctions. Of the various dysfunctions those related to hypothermia, bladder problems, and autophagia are of serious nature. The condition of animals with these complications deteriorates rapidly, and the animals are sacrificed for histological and pathological analyses. The findings show that the postoperative complications are related to the degree of severity of the trauma, and that 50-80% animals are lost due to these complications. Most of these animals are lost during the first two weeks after surgery, and the remaining at later stages. Transplantation of neural tissue at the site of lesion does not ameliorate these postoperative complications and improve the survival rate of the animals.

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.

Enhanced expression of a protein antigen (J1-31 antigen, 30 kilodaltons) by reactive astrocytes in lacerated spinal cord.

A mouse monoclonal antibody (MAb J1-31, isotype IgG 2b) was raised against an autopsy sample of cerebral white matter from a multiple sclerosis (MS) patient. MAb J1-31 recognizes a protein (J1-31 antigen) in human brain which has a molecular weight of approximately 30,000 daltons (30 kD) as determined by immunoprecipitation followed by SDS-gel electrophoresis (reducing conditions) and autoradiography (Singh et al.: Biosci Rep 6:73-79, 1986). By immunofluorescence microscopy, MAb J1-31 stains glial fibrillary acidic protein (GFAP)-positive cells, namely astrocytes, of both human and rat. Yet J1-31 antigen is distinct from GFAP (Predy et al.: Biosci Rep 7:491-502, 1987). In this paper we report that greatly enhanced staining for J1-31 antigen is exhibited by reactive astrocytes which arise following CNS injury. (Laceration-type surgical lesion of the rat spinal cord served as the experimental model). Enhanced expression of J1-31 antigen reveals some new aspect of the astrocyte response to injury, involving transformation to the reactive state. Consequently, MAb J1-31 may be a useful marker for studies on reactive astrocytes.

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 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.

Neocortical transplants in the rat brain: an ultrastructural study.

Electron microscopic analysis of neocortical transplants in the cerebellum of the host animals showed that the nerve cells, glial cells, and neuropil of the transplants were normal. These transplants showed anatomical integration with the host brain through various regions of interface. Neuropil interfaces were found to have a high density of synaptic profiles, and medullary interfaces had a very small number of synaptic profiles.

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.

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.

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.

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.

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.

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.