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Cultured human leptomeningeal fibroblasts grafted into rat frontal cortex were localized to the implant pocket and to adjacent host leptomeninges. Immunohistochemical studies using a panel of human-specific and domain-specific APP antibodies revealed that all grafted cells expressed both APP and A β in situ. Remarkably, these antibodies also labeled rat pial and ependymal cells as well as reactive astrocytes adjacent to vessels. In addition, apical projections and cell bodies of many cortical pyramidal neurons contained human-specific APP immunoreactive material. Groups of subcortical neurons, particularly those of the amygdala, hippocampal formation and suprachiasmatic nuclei, were similarly labeled. The presence of human APP in host brains was confirmed by immunoblotting. Birefringent Congo Red staining was observed in the cortical neuropil and in leptomeningeal vessels. These data indicate that grafted leptomeningeal fibroblasts hyperexpress APP and A β which can diffuse into parenchyma and be taken up by specific rat cells.

Fetal basal ganglia astrocytes and C6 glioma cells were plated on the surface of 1.5 cm thick hydrated collagen I wafers. Both cell types migrated through the entire thickness of the wafer within 1 day after plating. The collagen in the wafer was digested and the fine collagen I fibrils were clumped into large strands. By 2-3 days, the collagen strands were digested from the wafers and replaced by a mass of fetal astrocytes or C6 cells joined by their processes. The collagen I digestion and cell migration suggested protease production. In a second series of experiments, cultured C6 cells and E14 fetal astrocytes were immunohistochemically stained for the presence of plasminogen activators as an index of protease production. Both tissue (tPA) and urokinase (uPA) types were observed. Fetal astrocytes and C6 cells were also positive for guanidinobenzoatase, a serine protease associated with migrating cells. These data demonstrate that rapid migration of the cells on and through collagen I fibrils is concomitant with expression of plasminogen activators and proteases which can either activate or function as collagenases and release the cells from the substrate.

The effects of spinal cord hemisection on protein labeling in the medulla, pons, midbrain, and cortex in the Cebus monkey were studied by measuring the uptake of [3H]lysine into the trichloroacetic acid (TCA) precipitable and non-precipitable fractions of brain. Animals were sacrificed 3, 6 or 13 days after hemisection. Two normal animals and a 3-day sham operate were utilized as controls. Differences included individual animals, operated vs. unoperated animals, brain areas, and variation of brain areas over all postoperative days. The overall trend in brain radioactivity over postoperative days indicates stress and non-stress related components of brain response to spinal injury. Left-right side differences (L greater than R) were noted in the uptake of [3H]lysine into specific areas of brain over postoperative time, including leg areas of motor cortex, occipital cortex, and superior colliculus. A biphasic action of leg sensory cortical areas over time was noted, where L greater than R for the first 6 postoperative days and R greater than L at day 13. The factors possibly mediating radiolabeled amino acid uptake into protein are discussed in relation to demonstrated anatomical and neurochemical regenerative processes.

The short term effects of spinal cord hemisection on protein synthesis in the Cebus monkey were studied by measuring the uptake of [ 3 H]lysine into the trichloroacetic acid (TCA) precipitable and non-precipitable fractions of spinal cord. Animals were sacrified 3, 6, or 13 days after hemisection. Two normal animals and a 3 day sham operate were utilized as controls. All data were pooled and subjected to analysis of variance. Significant group differences were resolved by a standardt-test over pooled operative group samples and a pairedt-test for left-right differences within groups. Compared to normal animals, significant increases in the mean uptake of precursor into TCA precipitable and non-precipitable fractions of spinal cord samples were noted in all postoperative groups. The observed increase in the uptake of [ 3 H]lysine into TCA soluble and insoluble fractions at 3 days after hemisection were attributed to stress, as were increases in the activity of the TCA insoluble fractions in the sham operate. By 6 days, the increases attributed to stress in the average protein activity seemed to subside, resulting in an overall decrease in the TCA precipitable protein activity with an increase in TCA soluble fraction activity. Between 6 and 13 days the activity of the TCA soluble and insoluble fractions returned to the 3 day levels with a sharp increase in the activity of both fractions in the area of the hemisection. This increase was attributed to increases in neuronal, neuroglial, and infiltrating cell metabolism possibly associated with regenerative changes.

IgG-positive astrocytes have been reported in scrapie-induced and Alzheimer's cortical plaques, multiple sclerosis, and CNS tissue around abcesses, metastatic tumors and primary tumors of glial origin. The present experiments ascertain if this immunoglobulin positivity is specific for these cases or a function of astrocytes around any site of injury in the CNS. The spinal cords of 30, 300-g Sprague-Dawley male rats were lesioned by passing a 26 gauge needle through the cord at T 6 . After periods as long as 9 months, the spinal cords were processed for paraffin immunohistochemistry with antisera to IgM, IgG or double labeled for these immunoglobulins and GFAP (glial fibrillary acidic protein) a specific cytoplasmic marker for astrocytes. From 1 through 7 days after lesioning double labeled astrocytes in and around the site of injury are both IgM- and IgG-positive. From 14 days through 9 months postlesion, double labeled astrocytes surrounding the lesion are only positive for IgG. These data indicate a relationship between immunoglobulin availability, continued blood-brain barrier perturbation to immunoglobulins and the ability of reactive astrocytes to sequestor immunoglobulins. IgM is an early determinant for reactive astrocytes and IgG positivity is determinant for reactive astrocytes at any time period.

The fasciculus gracilis (FG) of the third cervical spinal cord segment (C3) of adult rats was aspirated and fresh whole pieces of rat 14-day gestation cervical spinal cord, prelabeled with Phaseolus vulguris leucoagglutinin, immediately placed in the pocket of one-half the operates. One to three months later there were no transplant derived rostro-caudal nerve fibers in the C1 or C2 segment of the aspiration-only or aspiration-graft FG. However, the neurons of the nucleus gracilis of the host medulla of the aspiration-only group were significantly atrophied whereas ther neurons of the aspiration-graft group were not statistically different from normal. There were normal numbers of neurons in all groups. Using immunohistochemical double labeling for prelabeled astrocytes with PHAL and GFAP, graft-derived astrocytes were found in the necleus gracilis of the host of the host medulla. These data indicate that the trophic action of spinal graft-derived astrocytes maintained neurons in the medulla of the host nucleus gracilis.

Axotomy of the olfactory sensory neurons in the adult primate squirrel monkey induces retrograde degeneration of the perikarya in the nasal neuroepithelium. The process of neuronal degeneration is rapid and by the 10th day the olfactory neuroepithelium is deprived of all mature neurons. Basal cells, supporting cells and the Bowman's glands are unaffected by the surgical procedure. The degeneration of the neurons is followed by intense mitotic activity of the basal cells of the neuroepithelium. At 30 days survival several young, mature neurons are present again in the neuroepithelium. At 60--90 survival days the neuroepithelium reacquires a population of neurons similar to controls. The persistence of neurogenesis and the replacement of experimentally degenerated neurons in an adult, non-human primate is briefly discussed.

Basal lamina is reconstructed over the lesioned surface of the spinal cord. The following experiment (90 rats) studies the ultrastructure of the formation of this membrane and the immunohistochemistry of laminin production (a major secreted component of basal lamina). After hemisection of the spinal cord at T6 animals were prepared for electron microscopy or antilaminin-biotin-avidin-peroxidase incubation. Three-5 days posthemisection, antilaminin reaction product was observed in astrocytes and their processes which faced the lesion, endothelia of blood vessels or pia. Ultrastructurally (3 days), basal lamina was polymerizing as small projections on the surface of astrocytic membranes facing the lesion, endothelia or pia. By 5 days the basal lamina was a single membrane, folded multiple sheets or in swirls. At 6-10 days the antilaminin reaction and the basal lamina (except for duplications) did not differ from normal. Reactive astrocytes secrete laminin for at least 3-5 days after hemisection and form basal lamina on the lesioned surface of the spinal cord after spinal cord hemisection.

Fetal CNS (cortex and spinal cord) transplants survive and grow in the spinal cord of adult rats. The present experiments study the initial stages (10 days) to determine the developmental pattern of early (E11) fetal cortex and spinal cord transplants implanted into the T6 spinal cord of adult rats (n = 50). Controls were age-matched fetuses or pups from the donor females and surgical and vehicle controls (n = 20). E11 fetal cortex (0.5 X 1.0 mm pieces of neuroepithelium) or spinal cord (1.0 mm) were dissected from caesarean delivered fetuses from time pregnant Sprague-Dawley rats. The fetal tissue was pressure-injected between the dorsal horn and dorsal column of the host. Hosts were prepared for light and electron microscopy 1, 3, 7 and 10 days postimplantation (DPI). At 1 DPI, ovoid, ventriculated multiple neuroepithelia formed in an implantation-induced cyst in the host spinal cord. The inner layer of cells lining the ventricle of the ovoid neuroepithelia was actively dividing. The nuclei of the cells differentiated as they reached the surface of the neuroepithelium. This pattern was repeated at 3-10 DPI. At 7-10 DPI, differentiated neurons and neuroglia surrounded actively dividing neuroepithelia. These cells comprised the bulk of the transplant in the cyst. Transplanted mesenchymal cells formed pockets of cartilage in the implanted spinal cord. Although transplanted CNS consisted of only neuroepithelial cells, they differentiated into neurons that were typical for cortex or spinal cord. These data indicate that the transplanted E11 fetal tissue formed neuroepithelia which established the pattern of growth for these transplants.

The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and homografted as a cell suspension into host thoracic spinal cord. Animals were sacrificed at 7, 14, 21 and 30 days postimplantation (DPI). Paraffin sections of cervical, thoracic and lumbar spinal cord were double-labeled for the presence of glial fibrillary acidic protein (GFAP) a specific marker for astrocytes, and PHAL, utilized as a marker for graft-derived cells. PHAL-GFAP positive cells were found throughout the thoracic spinal cord at all time periods, indicating that grafted astrocytes migrated along all 3 axes of the host spinal cord (rostral-caudal, dorsal-ventral and left-right). At 30 DPI, graft-derived astrocytes were found in host cervical and lumbar spinal cord. There appeared to be a minimal delay in the onset of migration.

Cell suspensions from 14-day gestation rat spinal cord can be successfully cultured on collagen gels containing laminin. After 7 and 14 days in culture the gels were removed from the dish and slices of gel with cultured cells were transplanted into the dorsal column of adult rats. Thirty days later there was no evidence of the gel. However, grafted neurons with well-developed organelles, axons, dendrites, axosomatic and axodendritic synapses were observed in the white matter of the host dorsal columns. Oligodendrocytes and astrocytes were also observed in the graft. This histocompatible substrate for culturing fetal CNS is a mechanism for transplanting cultured CNS cells as grafts of known maturity and synaptic organization into adult host CNS.

The extent of the projection of fasciculus gracilis from low thoracic (T12--13) spinal cord, to nucleus gracilis (NG) was studied by the Nauta--Gygax method in the rat in order to: (1) help define projection fields of fasciculus gracilis hindlimb and tail afferents along the longitudinal extent of NG; and (2) broaden the comparative data on the projection of the lower body to NG via fasciculus gracilis, already known in cat and monkey. Results showed that: (1) degenerated fasciculus gracilis axons are situated mainly dorsal to the corticospinal fibers, in the dorsal funiculus; (2) terminal projection fields were located at all levels of ipsilateral NG--caudal to the obex, terminal debris was confined to the medial two-thirds of NG--rostrad of the obex, in oral NG, degeneration was of finer caliber and projection fields more diffuse; and (3) ipsilateral degeneration was also seen at the level of a portion of Clarke's nucleus, and, in commissural and lateral cuneate nuclei and medial reticular formation in medulla.

Transplants of central nervous system to adult spinal cords are considered as potential aids in regeneration of the spinal cord and/or recovery of function after injury. The organization and development of the implant are important issues in seeking the potential for a transplant and host to become functionally integrated. This study uses embryonic cerebral cortex transplanted into the spinal cord of adult rats (T6) and examined the development and organization of the transplant with an antibody to vasoactive intestinal polypeptide (VIP). The cell bodies of VIP neurons are in the implants at 30 days postimplantation, but few of the somata have processes. By 45 days postimplantation, VIP neurons in the implant have dendrites and axons and are clearly recognizable as cortical bipolar cells which are not normally present in the thoracic spinal cord. These data show that neurons in embryonic cerebral cortical implants into the spinal cord elaborate the appropriate biochemical and morphological constituents in spite of the ectopic location. However, the cell processes develop at a slower than normal pace. Morphological interaction between the host spinal cord and the implant can be demonstrated possibly as early as 45 days postimplantation and clearly at 6 months following the implant. Thus, further examination of cerebral cortical implants as a potential aid in allevation of paraplegia subsequent to spinal cord injury is warranted.

The growth, differentiation and function of E11 fetal cortex was studied over 4 months after implantation into the degenerating nerve to the biceps femoris muscle of 70 rats. At 7 days postimplantation (DPI) the implant formed a neuroepithelium which contained mainly undifferentiated cells within a reconstituted perineurium. At 14–21 DPI the neurons contained differentiated, mature nuclei, poorly developed Nissl bodies and normal cytoplasmic organelles. Neuroglia were also observed with typical nuclei and a dense cytoplasmic ground. At 30 days the implants reached their greatest degree of differentiation. Neurons had mature nuclei and cytoplasmic organelles. There were few axosomatic synapses or neuronal cell processes. Neuroglia were prominent with many cell processes. The neuropil contained myelinated and unmyelinated nerve fibers, dendrites and many axodendritic synapses. At 40–50 DPI the neurons and neuronal cell processes were degenerating. At 60–120 DPI the implant contained only neuroglia. Stimulation of only the implant resulted in biceps femoris muscle contraction in 5 animals at 30 DPI and one animal at 40, 45 and 50 days. Injection of 10 μl of WGA-HRP into the biceps femoris muscle resulted in at least 20 neurons in 3 30-day implants that contained HRP reaction product. These data show that fetal cortical neurons can assume functions different from normal, functionally reinnervate adult muscle and can differentiate and form an ectopic nervous system for at least 30-days after implantation into a degenerating adult host peripheral nerve.

The present experiment examines astrocytes in fetal cerebral cortical homografts to adult rat spinal cords. The purpose of this study is to determine if astrocytes are structurally organized within the graft. Also, the presence of astrogliosis may be an indicator of the metabolic status of the graft. Embryonic cerebral cortex was taken at 14 days gestational age and transplanted into adult spinal cord at the level of the sixth thoracic vertebra. The homografts were examined at the light and electron microscopic levels from 7 days postimplantation (PI) to 6 months PI with glial fibrillary acidic protein antiserum which is specific immunohistochemical marker for astrocytes. At 7 days PI, immunoreactive astrocytes were present only at the periphery of the graft and appeared to be associated with blood vessels. By 30 days PI, normal protoplasmic astrocytes were present throughout the graft. No hypertrophied astrocytes are present at 30 days PI, but the numerical density of astrocytes is greater than that of normal cerebral cortical gray matter. Fibrous astrocytes are present in the periphery of the implant and many of these astrocytes extended their processes between the host and the graft. Occasional glial scarring is observed between the gray matter of the host and graft, but generally no glial scar occurred in the interface between the graft and the host gray matter. By 45 days PI, hypertrophies astrocytes can be seen in the graft, but are confined in this age group to perivascular regions. Gliosis progressed within the graft throughout the duration of the study, predominating within the graft by 5 months PI. The results of this study demonstrate that astrocytic processes can be shared by the host and graft and that the appearance of hypertrophied astrocytes possibly indicating a decrease in the functional capacity in the graft does not occur until after 30 days PI.

The incorporation of [3H]lysine into the trichloracetic acid (TCA) precipitable protein and soluble fractions of brain and spinal cord of rats subjected to immediate transection distal and crush proximal or transection distal, two week interval, and crush proximal of the left sciatic nerve has been studied at 5, 11, and 18 days postoperative. In addition, the regeneration of the nerve fibers was measured. A greater rate of (P 18 day group > 5 day group. However, no significant differences in uptake into protein in spinal cord and brain were seen between the simultaneous lesion sciatic animals over postoperative days. In brain, differences between the [3H]lysine incorporation into protein of the left (L) and right (R) cortical hemispheres was noted. The incorporation into somatomotor cortex showed a L > R difference which was most pronounced after simultaneous sciatic nerve lesion. Interestingly, the largest R > L difference was in occipital cortex after interval lesions of sciatic nerve. In spinal cord, the protein radioactivity of the L5–6 and S1–4 segments which include most of the affected neurons was greater on the right (unlesioned) side than on the left (sciatic crush) side over all lesioned animals. This effect was most pronounced after interval sciatic lesion. The alteration of central nervous system incorporation of lysine after peripheral nerve (sciatic) lesion and regeneration are discussed.

The intra-hippocampal organization of theta rhythm was examined in chronically implanted rats. Small lesions were placed in the posterior aspects of the hippocampal formation and the subsequent effect of the lesion ascertained electrophysiologically. Lesions of the subiculum significantly diminished the amount of theta rhythm present and the effect appeared to be permanent. Lesions of the lateral entorhinal region produced a large, but transient, increase in the amount of theta rhythm present. The electrophysiological consequences of these lesions were organized in ‘lamellae’ as previously suggested. In addition, the effects of the discrete lesions were expressed ipsilaterally only. The fiber connections underlying these effects were reviewed and the subiculum was postulated to be a critical region for the maintenance of normal ‘on-going’ hippocampal electrical activity. The increase in theta rhythm following lateral entorhinal lesions was postulated to be result of the loss of high-frequency influences from other neural regions. It was concluded that, although paced by the septal regions, hippocampal theta rhythm is dependent upon intra-hippocampal formation maintenance and modulation. In particular, the present data indicate the importance of the subiculum in this maintenance. Furthermore, the data suggest an important role of the hippocampal formation in the organization of theta rhythm.

Allografts of mature rat superior cervical ganglia (SCG) survived for up to 120 days following transplantation to regenerating and degenerating peripheral nerves of adult rats. Transplants contained the constituents of normal superior cervical ganglia and there was evidence of outgrowth of catecholamine containing fibers from the transplants. However, fibers did not attain the length necessary to enter peripheral muscles and no muscle response could be elicited by electrical stimulation of the implanted nerve where the transplanted mature SCG was the only source of nerve fibers.

This study investigated the regenerative capacity of the long tracts in the goldfish spinal cord. The spinal cord was transected anterior to the dorsal fin and the regenerative capacity of the descending fiber tracts (tectospinal, cerebellospinal, ventral tract group) assessed. Degenerating axons were stained 9 and 60 days later. Seven animals in the 60-day group underwent a subsequent cord retransection, one segment rostral to the original site of lesion and stained 9 days later. The degenerative patterns of the descending and ascending tracts were determined for a spinal cord segment 2 cm caudal to the original site of lesion. Following spinal cord transection all fish were paralyzed but normal swimming returned by day 25. Nine days postoperatively, the descending tracts were completely degenerated, with no evidence of degeneration in the ascending tracts. Sixty days postoperatively regenerated nerve fibers in the descending tracts were approximately 35–49% of the usual complement of axons. Approximately 94% of the regenerated nerve fibers were 4–8 times of normal diameter. Retransection of the spinal cord one segment rostral to the first lesion resulted in degeneration of the large diameter regenerated axons but not the normal diameter component. The relationship of the return of function to the quality and quantity of regenerated nerve fibers is discussed.