Jerald J. Bernstein

Astrocytes secrete basal lamina after hemisection of rat spinal cord

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.

Axonal regeneration and formation of synapses proximal to the site of lesion following hemisection of the rat spinal cord

Although the central nervous system of the mammal is reported not to regenerate, axonal sprouting has recently been demonstrated in various regions of brain and spinal cord. The following study investigates the regenerative capacity of 72 rat spinal cords following left hemisection at T2. In addition to nine normals, rats were killed at 5, 7, 14, 30, 60, and 90 days after making the lesion. Three animals per groups were prepared for Golgi, Protargol-eosin-cresyl violet staining, and electron microscopy. Three additional animals had hemisections at C5 and the cord from C5 to T5 was stained by the Fink-Heimer method. Six animals had hemisections at T2 and the cords were subsequently rehemisected 90 days later at C5; three animals were prepared for the Fink-Heimer stain and three for electron microscopy. The dendrites of the reactive motor neurons proximal to the site of lesion became varicose from day 10 to day 60–90 after the lesion, until the entire dendrite was replete with irregularly shaped varicosities. At 15 days postoperatively, processes grew into the reactive neural zone and by day 30 could be identifified as axons (0.1–0.5 μ in diameter) and dendrites. The axons grew in fascicles, usually free of neuroglial cell processes. The amjority of the axons formed axodendritic synapses on the indentations of the dendritic varicosities by 60–90 days postoperatively. The Fink-Heimer stain reveled that a limited number of axons regenerated from long tracts into the reactive neural zone. Most regenerated axons appear to be axonal sprouts from the operated and unoporated portions of the spinal cord. The central nervous system of the rat does regenerate, but the regenerating axons do not grow past the reactive neural zone and thus do not reach the neuroglial scar.

Glioblastoma Cells Do Not Intravasate into Blood Vessels

ABSTRACT: GLIOBLASTOMA IS VERY rarely found outside the central nervous system. The ability of rat C6 glioblastoma cells to intravasate into central nervous system and pial blood vessels is tested using a rat homografting model and two in vitro models. In vivo, scanning electron microscopy demonstrates that upon grafting C6 cells into implantation pockets in rat cortex, blood vessels can be spared in large digestion cysts formed in host brain parenchyma. Immunocytochemistry of the grafted rat cortex reveals that the glioblastoma cells are upon the blood vessel basement membrane, surrounded by the extracellular matrix material, fibronectin. The endothelial cells of the blood vessel are inside the laminin and fibronectin, and there were areas of endothelial cell hyperplasia. C6 cells are not observed inside blood vessels. In vitro, C6 cell cultures seeded with blood vessels from fresh rat pia exhibit the same relationship of the C6 glioblastoma cells to the blood vessel as those in the other models. The C6 cells migrate upon the pial blood vessel basement membrane but do not intravasate into the blood vessel. To ascertain whether structure and components of the blood vessel basement membrane are important factors in glioblastoma cell exclusion from blood vessels, C6 cells are seeded upon artificial basement membrane hydrated gel wafers. C6 cells migrate into the artificial basement membrane gel wafer by 1 day after seeding. These data indicate that glioblastoma cells are confined to the central nervous system by an inability to pass through vital basement membrane.

C6 glioma cell invasion and migration of rat brain after neural homografting : ultrastructure

C6 tumor cells (10(6] were grafted as suspensions into freshly made implantation pockets in rat host cerebral cortex. Specimens were prepared for transmission and scanning electron microscopy 1 to 7 days postimplantation (DPI). By 3 DPI vacuolated C6 cells had migrated on or invaded the host brain. C6 cells were observed on the glia limitans on the surface of the brain, in the corpus callosum, subependymal space, and perivascular space and had invaded the cortex under the implantation pocket. In addition to the tumor mass that was observed under the implantation pocket, by 7 DPI individual C6 cells had migrated into the corpus callosum and internal capsule. Migrated C6 cells were observed in a perineuronal position in the hippocampus and other gray matter structures inferior to the corpus callosum. Micropockets were found around each C6 cell and the processes of these cells had replaced host parenchyma. The preferred routes of migration were on basal lamina and parallel and intersecting nerve fiber bundles. Invasion occurred through gray and white matter. The movement of homografted C6 cells in the brain suggests that these cells actively migrate as individual cells in addition to invading as a mass.

Neurogenesis of sensory neurons in the primate olfactory system after section of the fila olfactoria

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 Bowmans 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.

Neuronal Alteration and Reinnervation Following Axonal Regeneration and Sprouting in Mammalian Spinal Cord (Part 1 of 2)

Recent findings have shown that the spinalcord of mammals is capable of limited regeneration. The reparative process involves very limited regeneration of severed nerve fibers and growth of axonal spr

Migration of human malignant astrocytoma cells in the mammalian brain: Scherer revisited

Fresh suspensions of human glioblastoma multiforme were preincubated in the plant lectin Phaseolus vulgaris leucoagglutinin (PHAL) and implanted into cortical pockets in adult rat brain. Brains were investigated periodically over 30 postoperative days and the migration of the human glioblastoma cells was traced with anti‐PHAL immunofluorescence or the overexpression of human specific p185c‐neu a specific marker of a class of human malignant astrocytoma cells. The principal pathway of migration of the implanted human cells in the rat brain was ventrally through cortical gray matter and into the corpus callosum, with rapid lateral distribution in this and other parallel and intersecting white matter fascicles. Human glioblastoma cells also migrated on basement membrane lined blood vessels, pia‐glia membrane and spaces of Virchow‐Robin, as well as the subependymal space of the ventricles. These paths of migration of human glioblastoma cells in the rat brain are consistent with the pathways of spread of glioblastoma in the human brain as described by Scherer over 50 years ago, indicating that multifocal malignant astrocytomas have common migratory pathways in mature mammalian brain.

Ventral horn synaptology in the rat.

SummaryThe synaptology of the normal ventral horn of the rat was studied. Presynaptic boutons were classified as S (spherical vesicles), F (flattened vesicles), and G (predominance of 700–1200 Å granular vesicles). In addition, Cf, Cs, M, and T synaptic complexes were defined and quantitated. Synaptology was studied on α-motoneuron somata, α-motoneuron primary dendrites, peripheral dendrites and interneuron somata. In addition, organelles were quantified for the pre- and postsynaptic members of the synaptic complex. All counts were made on coded material and these data were analyzed statistically.Motoneuron somata had significantly more (P < 0.01) F (58%) than S (33%) boutons. This was also the case for the motoneuron primary dendrite (P < 0.01; F, 61%; S, 37%). The small dendrites had more (P < 0.05) S (56%) than F (44%) boutons. More Cf bulbs (P < 0.01) were found on motoneuron somata (9%) than on motoneuron primary dendrites (2%) or interneuron somata (3%). The C complex presynaptic bouton contained spherical (Cs) or flattened (Cf) synaptic vesicles which were attributed to the fixation employed. Cf bulbs were not observed on small dendrites. G bulbs were observed (< 1%) only on small dendrites. M bulbs were not observed on any postsynaptic structure.The boutons of the motoneuron primary dendrites (15% of total afferents) and peripheral dendrites (14% of total afferents) were frequently branched whereas there was significantly (P < 0.01) less branching of boutons on motoneuron and interneuron somata. Small postsynaptic subsurface cisterns were associated with boutons of both the S and F type on all structures. In addition, these cisterns were observed in motoneuron somata (4%) and interneuron somata (2%) without an accompanying bouton. C postsynaptic organelles were observed in motoneuron somata (3%) and primary dendrites (1%) with an overlying neuroglial cell process and no presynaptic bouton.The synaptology of the rat ventral horn is comparable to that in the cat and monkey. However, M (R) and P bulbs were not observed in the rat. This could be due to the sampling method which indicated that synapses with less than 1% occurrence fall at the level of statistical resolution in quantitative electron microscopy. The presence of postsynaptic specialization usually associated with presynaptic boutons with no presynaptic component may be a reflection of the dynamics of normal bouton renewal in the rat ventral horn.

ECM-MEDIATED GLIOMA CELL INVASION

Cell adhesion receptors of the integrin superfamily, CD44, and adhesion receptors of the immunoglobulin superfamily are expressed by high‐grade astrocytic tumors of the central nervous system. These receptors are critical for the invasion of these tumors in the nervous system. Glioma cells utilize these receptors to adhere to and migrate along the components of the extracellular matrix, which is uniquely distributed and regulated within the brain and the spinal cord. For this reason, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix. The receptor‐ECM component interaction is discussed, focusing on the role of cell adhesion molecules of the integrin family and CD44 in glioma cell adhesion and invasion. Microsc. Res. Tech. 43:250–257, 1998.

C6 glioma-astrocytoma cell and fetal astrocyte migration into artificial basement membrane: a permissive substrate for neural tumors but not fetal astrocytes.

Cortically homografted C6 glioma-astrocytoma cells both invade the rat host brain as a mass and migrate as individual cells. In contrast, fetal astrocytes derived from homografted whole pieces of fetal cortex migrate only as individual cells throughout the brain of the rat but are not capable of invasion. Our experiment explored the migratory capacity (over 7 days) of cultured purified fetal astrocytes and C6 cells after seeding 10(6) cells on a hydrated artificial basement membrane wafer (Matrigel). The artificial basement membrane wafer was not a suitable substrate for the growth of cultured fetal astrocytes. In contrast, C6 cells migrated as individual cells from the surface of the wafer into the substrate. Individual C6 cells migrated 1.8 mm in the first 4 days and then ceased migration. The C6 cells were observed at the base of a digestion tube that extended from and was open to the surface of the wafer. At 3 days, micropockets were observed to form around each C6 cell at the base of each tube. By 7 days, the majority of pockets observed were large and contained several C6 cells. These multiple cell groups appeared to be progenitors of tumor masses. These data indicate that C6 glioma-astrocytoma cells, which in vivo appear to be a model for glioblastoma multiforme, primarily migrate as individual cells through artificial basement membrane and secondarily form tumor masses. Progenitor tumor masses form by coalescence of individual C6 cell micropockets or the division of a single cell in an individual micropocket.