Myelination of central nervous system axons in tissue culture by transplanted oligodendrocytes.

Unmyelinated mouse cerebellar cerebellar cultures in which oligodendrocyte differentiation had been suppressed by exposure to cytosine arabinoside developed axonal myelin after superimposition of kainic acid-treated cerebellar explants devoid of myelin-receptive axons. The latter explants contained differentiated oligodendrocytes. The operation of a diffusible myelin-stimulating factor was ruled out by the failure of myelination in cytosine arabinoside-exposed explants not in direct contact with oligodendrocyte-containing transplants.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. II. Ultrastructural studies.

Cytosine arabinoside-induced granuloprival cerebellar cultures lack both granule cells and differentiated glia and demonstrate marked synaptic reorganization. After kainic acid-exposed cerebellar explants, which contain granule cells and mature glia, were transplanted to the granuloprival cultures, the following ultrastructural features were noted: (1) parallel fibers formed normal synapses with Purkinje cell dendritic spines as well as with basket/stellate cell somata; (2) sprouted Purkinje cell recurrent axon collateral terminals were markedly reduced in number; (3) Purkinje cells matured and lost perisomatic spines; (4) astroglia formed sheaths around Purkinje cell somata and dendrites; and (5) axonal myelination occurred. The transplanted cultures demonstrated ultrastructural restitution toward normal after addition of missing elements.

Mature Purkinje cells in cerebellar tissue cultures: an ultrastructural study.

Mature Purkinje cells in mouse cerebellar tissue cultures were morphologically analyzed by electron microscopy. Explants maintained for 19 to 31 days in vitro contained Purkinje cells that were similar in most respects to those described in vivo except for incomplete arborization of the dendritic trees. Typical features included (1) absence of Purkinje cell perisomatic spines; (2) a paucity of naked Purkinje cell dendritic spines; (3) a 1:1 relationship of Purkinje cell dendritic spines to parallel fiber terminals; and (4) almost complete astroglial investment of Purkinje cell somata and dendrites. Minimal extracellular space was present in the neuropil of the explants and unusual synapses involving Purkinje cells were absent. Atypical features described by some investigators may be a function of retarded development in suboptimal culture conditions and do not represent the limit of tissue culture methodology.

Reorganization in granuloprival cerebellar cultures after transplantation of granule cells and glia. I. Light microscopic and electrophysiological studies.

Granuloprival cerebellar cultures were transplanted after 9 or 16 days in vitro with cerebellar explants that had been exposed to kainic acid. The latter contained granule cells and differentiated glia, elements lacking in granuloprival cultures. Changes induced by transplantation observed by light microscopy included interposition of granule cells among the large cortical neurons of host explants; a reduction of the excess neurites of the Purkinje cell axon collateral system that is characteristic of granuloprival explants; and the appearance of myelinated fibers in previously unmyelinated cultures. The most notable electrophysiologic consequence of transplantation was the disappearance of inhibition of cortical spontaneous activity in response to antidromic stimulation of Purkinje cell axons, correlating with the disappearance of excess neurites, and suggesting that Purkinje cell recurrent collateral inhibition was no longer the dominant mode of cortical inhibition. Restoration of missing elements in granuloprival cultures incited development of structural and functional characteristics resembling those of normal cerebellar explants.

Reorganization of organotypic cultures of mouse cerebellum exposed to cytosine arabinoside: a timed ultrastructural study.

This study was designed to examine the sequential changes in the developing granuloprival cerebellar culture. In this model of anomalous cerebellar development, organotypic cultures derived from newborn Swiss-Webster mice were exposed to the DNA synthesis inhibitor, cytosine arabinoside, at explantation and were fixed for electron microscopic examination on successive days in vitro. Similar developmental stages were compared in control explants. Granule cell destruction began early, and was widespread by 2 days in vitro, when oligodendrocyte destruction also began in treated cultures. A few granule cells survived, but no recognizable oligodendrocytes remained by 7 days in vitro, at a time when myelin was initially evident in control explants. Purkinje cell recurrent axon collateral sprouting began at 3 days in vitro in cultures exposed to cytosine arabinoside, and the sprouted terminals initially synapsed with Purkinje cell somata, somatic spines and dendritic shafts. Synapses with Purkinje cell dendritic spines developed later, at approximately the same time as parallel fiber-Purkinje cell dendritic spine synapses formed in control cultures. Astrocytic ensheathment of control Purkinje cells was well underway by 6 days in vitro and Purkinje cell somata were relatively rounded and almost completely ensheathed by 9 days in vitro. Glial ensheathment did not occur in cytosine arabinoside treated cultures, and Purkinje cell somata were scalloped at 7 days in vitro by excess impinging recurrent axon collateral terminals, and never developed the smooth contours characteristic of control Purkinje cells. Purkinje cell somatic spines persisted in treated explants, and reduction of excess extracellular space was delayed until 12 days in vitro, when most of the developmental changes had been completed. The earlier development of synapses by excess recurrent axon collateral terminals with Purkinje cell somata, somatic spines and dendritic shafts, followed by the later development of heterotypical synapses with dendritic spines, in parallel with synapse formation by normal presynaptic elements, suggests that the sequence of development of synapses is a function of the maturational state of the postsynaptic components.

Posterior fossa subdural hematomas in neonates.

Posterior fossa subdural hematomas in the newborn infant are rare but potentially treatable. The infants are normal after birth, but within days, hydrocephalus hypotonia, and irregular respirations develop. Seizures and third nerve pareses are unusual. We report a neonate in whom this process was identified by computerized tomographic brain scan. We also discuss potential misinterpretations of the computerized tomographic brain scan in neonates.

Apneic spells in progressive dialysis encephalopathy.

A 53-year-old patient with progressive dialysis encephalopathy is discussed. In addition to the clinical features previously described in this syndrome, the patient had spells of sudden respiratory arrest in close association with the episodic EEG abnormalities characteristic of the syndrome.

'Glioblastoma'. Induction of a reproducible autochonous tumor in rats with murine sarcoma virus.

Inoculation of 0.02 ml of high-titer Kirsten strain Murine Sarcoma Virus into the brains of 10-day-old Wistar/Furth rats yields, with 100 percent incidence, a uniform glioblastoma-like tumor within 16 days. Light and electronmicroscopy confirmed the neuroectodermal origin of the parenchymal cells. The remarkable vascular component was studied with extracellular tracers. The permeability of the abnormal endothelium to constituents of the blood vascular compartment was confirmed. Accessory vascular channels, and blood channels devoid of endothelium entirely, were observed. This reporducible system should provide a useful model for further studies of the biology of brain tumors.

An ultrastructural study of cortical remodeling in cytosine arabinoside induced granuloprival cerebellum in tissue culture.

Mouse-derived cerebellar explants were exposed for 5 days to cytosine arabinoside, an inhibitor of deoxyribonucleic acid synthesis. They were then maintained in normal nutrient medium until fixation for electron microscopy at 15-20 days in vitro. The cerebellar cortex lacked granule cells, but Purkinje cells, Golgi neurons and a few basket and stellate cells survived. Astrocytes and oligodendrocytes were diminished in number and myelination was absent. Purkinje cell recurrent axon collaterals increased in number and formed synapses with the surviving cortical neurons and their processes. The ultrastructural alterations that occurred in the cytosine arabinoside-treated cultures were consistent with an interpretation of cortical remodeling in which Purkinje cell axon collaterals were the dominant inhibitory elements.

Climbing fibers in mouse cerebellum co-cultured with inferior olive.

Cerebellar explants derived from neonatal mice were co-cultured with medulla containing inferior olive. The explants exhibited extracellular climbing fiber-like responses to electrical stimulation. On ultrastructural examination, many terminals with a high packing density of round, clear vesicles in a dark filamentous cytoplasm were present. Similar terminals, characteristic of climbing fibers, had not been identified in previous studies with cerebellar explants incorporating dorsal pons, but excluding medulla. The climbing fiber terminals formed appropriate synapses with Purkinje cell dendritic spines. The findings suggest that when climbing fibers are introduced into cerebellar cultures, accurate functional and morphological units are established.

Choline acetyltransferase activity in mouse cerebellar cultures.

The finding of the acetylcholine synthetic enzyme, choline acetyltransferase, has been reported in mouse cerebellar cultures, and it has been used as an index of neuronal survival and maturation. These results are curious in light of immunocytochemical studies which show that this enzyme is localized within mossy fiber terminals in glomerular structures of the cerebellar cortex. Since most mossy fibers are of extracerebellar origin, a significant population of mossy fiber terminals would not be expected to be present in cerebellar cultures. The origin of this acetylcholine synthetic activity has been examined in mouse cerebellar cultures. Two groups of explants, one with and the other without incorporated dorsal pontine tissue, were cultivated. Only cultures that included pons showed well developed glomerular structures with mossy fiber rosettes. Homogenates of the cultures were assayed for their ability to synthesize acetylcholine, and the synthesis was shown to be due to choline acetyltransferase by use of the specific inhibitor, (naphthylvinyl)pyridine. Cultures lacking dorsal pontine tissue had only low levels of enzyme activity, whereas those which included pons had 20-60 times greater synthetic activity. These results indicate that the choline acetyltransferase activity arises from pontine tissue in cerebellar cultures and are consistent with mossy fibers being the source of this enzyme.

Toxic effects of kainic acid on mouse cerebellum in tissue culture.

Cultures of mouse cerebellum were exposed for various intervals after explantation to kainic acid, a glutamic acid analog. Purkinje cells and intracerebellar nucleus neurons were destroyed and cortical laminar formation was inhibited by exposure to kainic acid, while granule cells were relatively spared. Prolonged kainate treatment also reduced the granule cell population. The destructive effects of kainic acid were evident upon exposure of Purkinje cells prior to the development of parallel fiber-Purkinje cell synapses, the neurotransmitter for which is believed to be glutamic acid. Glutamate application to intracerebellar nucleus neurons in vitro did not evoke extracellularly recorded excitatory effects, suggesting that these kainate-sensitive neurons do not have significant numbers of glutamate receptors. The combination of these observations suggests that neuronal toxic effects of kainic acid are not exclusively mediated by action on glutamate receptors, but involve other, less specific mechanisms as well.

Myelination of mouse cerebellar explants by rat cultured oligodendrocytes.

It has been shown that transplanted central nervous system tissue containing oligodendrocytes will myelinate neuronal processes in vitro and in situ. In this study we propose to show that cultured rat oligodendrocytes have the capacity to myelinate mouse cerebellar neuronal processes in vitro. Cultured rat oligodendrocytes were transplanted to cytosine arabinoside-treated mouse cerebellar explant cultures, then observed for myelination. Ultrastructural examination showed myelin and myelin-like figures in co-cultures. Control cytosine arabinoside-treated cultures and cultured oligodendroglia were without compact myelin.

Phenytoin neurotoxicity in developing mouse cerebellum in tissue culture.

Phenytoin applied to developing neonatal mouse cerebellar cultures at concentrations of 9-46 micrograms/ml of nutrient medium from the day of explantation to 16 days in vitro induced cerebellar cortical degeneration. The degree of neurotoxicity correlated with drug concentration. Purkinje cells were the most susceptible of the cerebellar elements, and intracerebellar nucleus neurons were the most resistant. In contrast, mature mouse cerebellar explants were resistant to chronic exposure to high concentrations of phenytoin.

Effect of gentamicin and dexamethasone on the natural history of the rat Escherichia coli brain abscess model with histopathological correlation.

In a rodent model of Escherichia coli brain abscess, the natural history of the infection was studied and the influence of a glucocorticoid (dexamethasone) and an antibiotic (gentamicin) on the development of brain abscess and the survival of abscess-bearing animals was evaluated. The administration of steroids using three different dosage schedules suppressed the macrophage and glial response, decreased collagen formation, increased the number of pathologically evident bacteria, and decreased host survival. The administration of antibiotics by parenteral routes decreased the number of viable bacteria in the abscess. The simultaneous administration of systemic gentamicin and dexamethasone resulted in increased host survival to a level that was intermediate between that of animals treated with dexamethasone alone and that of those treated with gentamicin alone. Thus, some of the adverse effect of corticosteroids on host survival could be mitigated by the simultaneous administration of antibiotics. Finally, it was observed that the abscess in this model tends to expand along white matter tracks. This path of least resistance may be responsible for the observation that brain abscesses tend to rupture into the ventricle rather than into the subarachnoid space via the cortex.

Surgical resection of a pineal tumor containing elements of germinoma and astrocytoma.

A 15-year-old boy presented with hydrocephalus due to a pineal tumor presumed to be a germinoma. After a shunting procedure, gross total tumor resection was carried out via an occipital transtentorial approach. Histopathological examination of the tissue demonstrated that, in addition to the expected germinoma, the tumor also contained areas of astrocytoma and areas that, taken in isolation as in a needle biopsy, could have been diagnosed as pineocytoma. The diagnosis and therapy of a pineal tumor of mixed histological types such as this lesion would be seriously compromised by "conventional" empiric radiation and even a stereotactic needle biopsy may not have provided an adequate tissue sample.

Cerebrovascular permeability and delivery of gentamicin to normal brain and experimental brain abscess in rats.

Antibiotics vary widely in their ability to penetrate the blood-brain barrier. In studies of 70 rats, the permeability of the normal blood-brain barrier to gentamicin was shown to be poor. In experimental brain abscesses, during the cerebritic stage of development, the penetration of intravenous antibiotics was increased compared to normal brain but was very inconsistent. Antibiotic delivery to brain abscess was not significantly altered with the administration of high-dose steroids, but the macrophage and glial response was markedly decreased with high-dose steroid therapy. Reversible osmotic blood-brain barrier modification with mannitol increased the delivery of gentamicin both to brain abscess and to the surrounding brain. It also resulted in more consistent tissue drug levels. The clinical implications of these studies suggest that, because of the inconsistent delivery of gentamicin to brain abscess, the therapeutic efficacy of medical management alone may be quite variable. This mode of therapy could possibly increase the efficacy of medical management of brain abscesses, especially in patients with multiple or surgically inaccessible brain abscesses.