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Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

Transection of the hindlimb dorsal column fibers leads to a partial deafferentation of the neurons of the nucleus gracilis, the first relay of the ascending dorsal column-medial lemniscal (DC-ML) neural network. In response to the deafferentation, a synaptic renewal cycle is initiated and the neurons of the nucleus gracilis atrophy. The present study examines the molecular changes that occur in synaptic relays of the ascending DC-ML following a hindlimb dorsal column transection. Rats were sacrificed 0.5, 1, 24, or 72 hours postlesion. Steady-state levels of mRNA coding for c-fos were significantly elevated only at 24 hours postlesion in caudal dorsal medulla, which contains the nucleus gracilis. The increased c-fos is neuronal in origin since there is an increased level of c-fos immunoreactivity in both the cluster neurons and the interneurons of the nucleus gracilis. In the third relay of the DC-ML system, the somatomotor cortex, levels of c-fos mRNA were significantly decreased 72 hours postlesion. These data indicate that lesions of the hindlimb dorsal column fibers have transneuronal effects on gene expression that extend to at least the third synaptic relay in the DC-ML system.

Successful transplantation of the fetal brain into adult host brain has been accomplished. These studies explore the growth, differentiation, and viability of E11, E12, and E15 rat fetal cortex and fetal spinal cord implantation into the spinal cord of adult rats (donor and host, Sprague-Dawley). Under deep Chloropent anesthesia, 70 rats had 1-mm cubes of fetal cortex inserted with pressure or by stylus injection subpially between the dorsal horn and dorsal column (left side), or implantation of whole segments of fetal spinal cord. Animals were prepared for light microscopy 14 and 21 days and 1, 2, and 3 months later. Implants by both fetal tissues had a 69% survival rate. The younger the fetal implant the higher the success of the implant (E11 greater than E15). The diameter of fetal spinal cord implants reached the diameter of control postnatal animals after 30 days. The implants not only increased in mass (up to 7-fold in some cases) but differentiated and matured (apolar, unipolar, bipolar, and multipolar) neurons were observed one to three months postimplantation. By 30 days postimplantation, fetal neurons had large, often crenated nuclei, with a large single prominent nucleolus. The most successful implants were the young E11 fetal spinal cord into the adult host spinal cord. These implants represent an initial successful transplantation of fetal spinal cord into adult spinal cord.

The ependymal cells in rat host spinal cord were studied after transplantation of fetal cerebral cortex homografts. Adult Sprague-Dawley male rats had pieces of E14 fetal rat cortex pressure-injected into the spinal cord at T6. The tissue was prepared for light and electron microscopy and studied for over 2 months postimplantation. At 7 and 14 days postimplantation (DPI), there were dividing ventricular-ependymal cells lining cysts in the host spinal cord. After 21 days, cell division was no longer observed in these cells, and only mature ependymal cells lined these cysts. Mature ependymal cells were either: columnar, pseudocolumnar, cuboidal, squamous, or pseudomorphic; had cilia and villi on only one surface of the cell and shared tight junctions when contiguous. These data show that ependymal cells were mature cellular components of adult spinal cord after transplantation of fetal CNS grafts. In addition, ventricular-ependymal cells continued to divide within the parenchyma of the graft, indicating continued growth of transplanted fetal cerebral cortex in host spinal cord.

Cellular surface carbohydrates have been studied extensively in relation to cell interactions. However, the presence of intracellular carbohydrates not associated with cell surface membranes has not been well-defined histochemically. We describe a technique for the ultrastructural localization of intracytoplasmic carbohydrates using a wheat germ agglutinin peroxidase conjugate. The ultrastructural aspects of wheat germ agglutinin (WGA) binding to intracellular structures of rat somatomotor cortex layer V neurons and spinal cord motoneurons were examined using sonication to enhance penetration of the lectin–peroxidase conjugate. In addition to the membrane-associated structures previously described by others, WGA binding was observed on neuronal nuclear chromatin, nuclear pores, ribosomes, microtubules, vesicular organelles, and to synaptic complexes, primarily in the presynaptic bouton. The results support data indicating that complex carbohydrates may be important in neuronal molecular processing.

This investigation explores the central nervous system protein-synthetic responses to hemisection of the spinal cord. Adult male rats hemisected at mid T2 were given intravenous injections of 14C-leucine at 0.7, 30, or 70 days postoperative. Four hours later the T1, T2, T3, and C6 segments of the spinal cord and right and left samples of somatomotor and occipital cortex were removed, weighed, and frozen. These samples were later homogenized and subjected to trichloroacetic acid (TCA) separations. Protein-synthetic activity was expressed as the ratio of the specific activity of the TCA precipitable proteins to the specific activity of the TCA free amino acid pool. In the spinal cord samples protein-synthetic activity was greater than in controls at 7 and 30 days postoperative in the T2 segment and at 30 days postoperative in the C6 and T3 segments. In the cortical samples protein-synthetic activity was greater than in controls in the right and left somatomotor cortex at 30 days postoperative, while it was less than in controls in the left somatomotor cortex at 70 days postoperative. Thus, T2 hemisection leads to initial increases and subsequent decreases in protein-synthetic activity in both spinal cord and somatomotor cortex. These results are discussed in terms of possible regenerative neural reactions occurring in the central nervous system following spinal cord injury.

Fourteen-day gestation fetal cerebral cortex homografts were transplanted into the thoracic (T6) spinal cord between the left dorsal column and dorsal horn of adult host rats. The transplants were soaked in 2.0 micrograms/ml of the lectin Phaseolus vulgaris leucoagglutinin (PHAL) prior to implantation. Transplanted host spinal cords were utilized at 7, 14, and 24 d and at 1 and 2 months postimplantation. Paraffin-sectioned spinal cords were double labeled for PHAL and glial fibrillary acidic protein (GFAP) by using FITC- and RITC-conjugated secondary antisera, respectively. Montages of FITC- and RITC-positive cells were analyzed for cells containing both fluorescences. Double-labeled cells (PHAL-GFAP) were transplant-derived astrocytes. Transplant-derived astrocytes were observed to initiate migration in the white matter columns of the host at approximately 14 d after transplantation. Double-labeled astrocytes were observed in cervical and lumbar spinal cord of the host (ca. 3.5 cm away from the center of the transplant) at 2 months postoperative. These astrocytes migrated at approximately 0.76 mm a day (after a 14-d delay). At 2 months, transplant-derived astrocytes composed as much as 50% of the astrocytes in the white matter of the host 2.0 mm from the transplant. The migrated astrocytes were hypertrophied and appeared reactive. Astrocytes in spinal gray matter only migrate about 1.0 mm from the graft-host interface. Transplant-derived astrocytes can migrate the entire length of the spinal cord white matter.

Fresh cells from two grade 3 human malignant astrocytomas were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then xenografted into freshly made implantation pockets in rat host cerebral cortex. Animals were sacrificed at 7, 14, 21 days, and 1 month postimplantation (DPI). Paraffin sections were double-labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes and differentiated astrocytoma cells, and PHAL, utilized as a marker for graft-derived cells. Grafted human astrocytoma cells were found on the glia limitans along the entire circumference of the brain, in the corpus callosum, internal capsule, entopeduncular nucleus, optic tract, and median eminence. In addition, astrocytoma cells were observed in the cingulum, habenula, arcuate, and supraoptic nucleus. Astrocytoma cells entered the spaces of Virchow-Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The corpus callosum was a major migration route for the astrocytoma cells. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes. The migration of the human astrocytoma xenografted cells in the rat brain followed the spread of human malignant astrocytomas in the human brain and is a valuable basic science tool in brain cancer research.

The thyroid hormones triiodothyronine (T3) and L-thyroxine appear to enhance regeneration in the peripheral and central nervous system (CNS). The following experiments examine possible metabolic substrates for the action of T3 on the adult rat CNS after spinal hemisection. The protein incorporation of (3H)lysine after a left spinal hemisection (T2) or control operations was examined 1, 3, 7, and 14 days postoperation. Triiodothyronine (1 microgram/kg body weight in a bicarbonate buffer) was injected daily for the postoperation or equivalent time period. One hour prior to decapitation, animals were given a subcutaneous injection of 200 microCi of (3H)lysine. Samples of brain and spinal cord were dissolved, and the radioactivity of acid-precipitable protein and acid-soluble fractions were determined by scintillation counting. T3 treatment influenced the general levels of incorporation of all treated groups over all days postoperation. Specific effects were observed in spinal hemisected T3-treated animals. A significant hemispheric (P less than 0.05) asymmetry was present at 3 days postoperation with the right somatomotor cortex higher in protein radioactivity than the left. In spinal cord, the area of the lesion and areas just caudal to the lesion were higher in (3H)lysine incorporation in T3-treated rats relative to controls. T3 effects appear to involve an increased sensitivity of the cells of the injured nervous system to the hormone.

The cerebral cortices from 14-day gestation rat embryos were prelabeled with Phaseolus vulgaris leucoagglutin (PHAL) and then homografted into freshly made implantation pockets in the host cerebral cortex. Animals were sacrificed at 30 and 60 days postimplantation (DPI). Paraffin sections were double labeled for the presence of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes, and PHAL, utilized as a marker for transplant-derived cells. Transplant-derived astrocytes were found on the glia limitans along the entire circumference of the brain, in the hippocampal commissure, corpus callosum, internal capsule, entopeduncular nucleus, habenular commissure, brachium of the superior colliculus, optic tract, optic chiasm, and sensory root of the trigeminal nerve. Transplanted astrocytes entered the spaces of Virchow-Robin, and migrated along parenchymal blood vessels and between the ependymal and subependymal layers of the third and lateral ventricles. The presence of basal lamina or parallel nerve fiber bundles was a common factor for these migration routes.

The ultrastructure of cortex and spinal cord from 11-, 12-, and 15-day-old fetuses implanted into the spinal cord of adult rats was studied over 3 months. Under deep Chloropent anesthesia, a 0.5 × 1.0-mm square of fetal cortex or a 1.0-mm segment of fetal spinal cord was implanted subpially between the left dorsal column and the dorsal horn of 70 adult rats. Implants grew toward gray matter, usually interfacing with the host at the isthmus between the horns of the spinal cord. However, implants were observed that occupied the entire left dorsal and ventral horns of the left half of the host spinal cord. Implants had concentric zones: A central zone with basal lamina lined joined channels and subjacent neuroglia; a zone of differentiating implant nervous system; a zone with basal lamina lined implant with overlying pial cells on the dorsal and lateral surfaces of the implant; a zone that interfaced with the host with overlapping neuropil on the lateral and ventral surfaces of the implant. Neuron types were typical for cortical or spinal implants. Implants survived for 3 months and reached stages of neuronal and neuroglial maturation similar to controls. Both fetal spinal cord and brain were successful as implants, had delayed differentiation, and formed complex neuropils. The zone of overlapping interface of the donor and host is an anatomical indication of physiological and functional integration.

The relative amounts of incorporation of [3H] lysine into various brain areas and segments of the spinal cord in adult Cebus and Rhesus monkeys were studied. One hour after subcutaneous injection of [3H] lysine, animals were sacrificed and the brain and spinal cord samples taken and processed for [3H] lysine incorporation into protein (Lowry et al., 1951; Wells and Bernstein, 1976). Analysis revealed marked differences in metabolism among brain areas within each species of monkey as well as across species. Cortical structures were generally higher in radioactive uptake than subcortical structures in both species. The Cebus monkey had the greatest variation among brain areas, particularyly between cortical regions, whereas the Rhesus monkey did not show cortical variation in uptake. The ratio of incorporated to nonincorporated lysine in the brain and spinal cord was higher in the Rhesus than the Cebus monkey. Both species showed a rostrocaudal gradient of radioactivity following [3H] lysine incorporation into the spinal cord. Considering weight, size, and availability differences, the metabolism of [3H] lysine appears to differ by brain areas and spinal cord segments between species.

The effects of ischemia (removal of oxygen and glucose for 4 h) and anoxia (removal of oxygen alone) on astrocytes were studied in dissociated cultures of E14 spinal cord containing both neurons and astrocytes. In addition, a group of cultures was treated with a low Na+, low Ca2+, and high K+ medium during the 4-h ischemic period (ischemia-protected group), a process that protects neurons from ischemic damage under identical conditions. Astrocytes were examined immunohistochemically using glial fibrillary acidic protein (GFAI) antiserum 24 h after insult. Densitometry and statistical analysis (1-way analysis of variance [ANOVA], a priori; 2-tailed Tukey-t, a posteriori) of the digitized images of the somata and processes of astrocytes in the anti-GFAP reacted cultures showed significant differences between the groups; a significant increase (P less than 0.01) in the GFAP-positive reaction in the somata of ischemic astrocytes and a significant decrease (P less than 0.01) in the GFAP-positive reaction in the processes of ischemic, ischemia-protected, and anoxic astrocytes. There were no significant differences in the GFAP immunoreactivity of somata between control, ischemia-protected, and anoxic astrocytes or of processes from ischemic, ischemia-protected, and anoxic astrocytes. These data show that following ischemia cultured astrocytes increase somatic GFAP immunoreactivity compared to all other groups tested whereas the staining intensity for GFAP was decreased in the processes of all three experimental groups compared to controls. Ischemia protection resulted in the absence of the enhancement of somatic GFAP immunoreactivity. The relationship of the astrocytic response and the type of cellular stress is discussed.

The effect of spinal dorsal funicular lesions (T 12) upon the frequency of boutons on, and diameters of the somata of pyramidal cells in layer VB of hindlimb cortex was studied. Adult rats sustained bilateral damage to either the dorsal column (DC, n = 10) alone or DC combined with the corticospinal tract (CS) (DC + CS, n = 34) and were utilized 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperatively (DPO). Neurons randomly sampled from 44 lesioned and 13 unoperated cases were analyzed for the number of silver-impregnated boutons (Rasmussen method) on the circumference of the soma as well as diameters of the soma, nucleus, and nucleolus. Analyses of variance comparing across lesioned and normal groups were significant for bouton counts on the soma (P less than 0.01), and diameters (long axis) of somata (P less than 0.01) and their nuclei (P less than 0.05). Both lesioned groups exhibited significant decreases from normal for these latter three parameters. With respect to survival time for the DC + CS-lesioned animals we noted the following: (1) Bouton counts on the soma significantly decreased below normal between 1 and 60 DPO; this decrease was most dramatic during the first three days postlesion. (2) Somal diameter (long axis) significantly decreased below normal between 2 and 120 DPO (except at 14 and 90 DPO). (3) Nuclear diameter (long axis) significantly decreased below normal only at 90 DPO. (4) Bouton counts on somata of neurons in layers VB and IV [Ganchrow and Bernstein, 1981] of hindlimb cortex correlated negatively and significantly across 120 postlesion days. The rapid shrinkage and reduced afferentation of layer VB somata during the first week following DC + CS lesions suggest initial, retrograde reactions to CS axotomy. Since bouton counts on layer VB somata were significantly less (P less than 0.05) in DC- than DC + CS-lesioned rats, it is hypothesized that CS axotomy regulated a set-point for increased afferentation which was maintained on the shrunken somata between 7 and 120 DPO.

The feasibility for growth, maturation, and differentiation of fetal nervous system implanted into adult mammalian peripheral nervous system was studied. Thirty-five adult rats had the epineurium of the sciatic nerve crushed, perineurium minced, and fetal rat cortex or spinal cord implanted. Rats were utilized 7, 14, and 21 days, and 1, 2, 3, and 4 months later. A 1-mm cube of cortex or a 1-mm segment of spinal cord of 11-, 12-, or 15-day gestation fetuses was placed into the epineurium. Age-matched controls (7 DPI (days postimplantation) control for E15 implant was a 1-day pup, 21 days' gestation) were utilized for comparison (two per time group). Five animals had sciatic crush and perineurial mince only, and the gait and toe-spreading response were observed over 4 months. All implanted animals walked normally at 30 days. All implants were successful and survived the duration of the experiment. Cortical implants produced prominent bulges in the epineurium (21 days-4 months). Maximal neuronal and neurolgial cell division was observed at 7-21 days, decreased at 30 days, and stopped by 60 days. Most implanted cortical neurons had mature nuclei and immature dendritic patterns (apolar), and rarely had mature dendritic patterns. Neuroglia were abundant. The younger the cortical implant, the larger the cell mass produced (E11 greater than E15). The spinal cord implants survived, were viable, contained mainly neuroglia, and grew minimally. Host Schwann cells and nerve fibers were found in and around the implants. These data show that adult peripheral nervous system can act as an environment for growth and viability of fetal CNS implants.

The transneuronal effect of bilateral, dorsal funicular lesions (T 12) on the frequency of boutons on cells in layer IV of hindlimb cortex was studied. Adult rats were utilized 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperative (DPO), and tissue was processed for the light microscopic visualization of silver-impregnated boutons (Rasmussen method). Bouton counts were taken on soma, or along 5- and 10-micrometers segments or proximal dendrite branching from soma. The soma diameter also was measured on those neurons chosen for bouton counts on the circumference of the soma. Statistically significant, increased afferentation on soma and proximal dendrite occurred during the first postlesion week relative to longer survival times; bouton counts on the proximal dendrite showed a trend (not statistically significant) toward increases above normal. These data mirror similar, consistent increases in bouton counts reported in thalamic ventroposterolateral nucleus of these same cases. At 14 DPO, bouton counts on the soma decreased below normal (P less than 0.005) and, except at 60 DPO, remained so through 120 DPO (P less than 0.025). Bouton counts on the proximal dendrite also decreased below normal at 14 DPO (P less than 0.005), thereafter exhibiting either periodic (along 5-micrometers) or extended (along 10-micrometers) periods in significant decreases from normal. Correlations in lesioned cases between the number of boutons on the soma and either bouton counts on proximal dendrite or soma diameter were positive and statistically significant (P less than 0.005 in all correlations). Possible anterograde (via the dorsal column-medial lemniscal system) and/or retrograde (via the corticospinal tract) transneuronal mediation of these effects in hindlimb cortex is discussed.