Search PubMed⌕ Search

Biomedical subjects

H D Webster

Publications and source records attributed to H D Webster.

At least 73 records · Page 4Linked to original sources
PubMed1982

Distribution of papovavirus, myelin-associated glycoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions.

To study how viruses interact with oligodendroglia and produce demyelination, we immunostained paraffin and epon sections of lesions from patients with progressive multifocal leukoencephalopathy (PML) with antisera to papovaviruses, oligodendroglial myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) according to the peroxidase-antiperoxidase method. In paraffin sections from a rapidly progressive case of PML, hyperimmune JC virus antiserum stained single oligodendroglia which were located in white matter that appeared normal histologically and stained normally with MAG and MBP antisera. In zones surrounding areas of demyelination, virus containing oligodendroglia were most numerous and MAG staining of periaxonal regions was decreased, but there was little change in MBP staining. In demyelinated regions, both MAG and MBP staining were severely altered; also there was much less JC virus staining. In tissue from three other chronic cases, viral antiserum stained fewer oligodendrocytes and the differences in MAG and MBP staining were much less striking. In epon sections from two biopsies of central nervous system tissue, we studied the electron microscopic appearance of oligodendroglia that also had been stained by JC virus antiserum. Virions were present in all nuclei and in some cytoplasmic regions. The results suggest that changes in MAG distribution are useful indicators of early oligodendroglial abnormalities which can cause myelin breakdown.

Aged↗
PubMed1982

Immunocytochemical study of myelin-associated glycoprotein (MAG) and basic protein (BP) in acute experimental allergic encephalomyelitis (EAE).

To compare distributions of oligodendroglial myelin-associated glycoprotein (MAG) and myelin basic protein (BP) during demyelination in acute experimental allergic encephalomyelitis (EAE). Lewis rats were sensitized with an emulsion containing guinea pig spinal cord and complete Freund's adjuvant. The rats were examined clinically and groups were perfused with fixative before symptoms appeared (7d), within 48 h of symptom onset (10d) and during more severe illness (14d, 21d). Paraffin sections of cerebrum, brainstem, and spinal cord were immunostained with antisera to MAG and BP. Other sections in each serially mounted series were stained with luxol fast blue, hematoxylin and eosin or with the Bodian method to correlate MAG and BP results with histological changes. No abnormalities were detected 7d after sensitization. After 10d, small perivenular inflammatory cell infiltrates were present in white matter, displaced myelinated fibers, but their MAG stained periaxonal regions and BP-stained myelin sheaths appeared normal. In pontine grey matter lesions, there were focal abnormalities in a few myelin sheaths. After 21d, demyelinating lesions were present that were largest in pontine grey matter. Decreased MAG staining was present in areas of myelin sheath loss. MAG-stained fragments were found in zones of active myelin breakdown but no decrease or other change in MAG staining extended beyond the margins of demyelinating lesions into areas with normally stained myelin sheaths. Thus, in contrast to multiple sclerosis (Itoyama et al. 1980), changes in periaxonal oligodendroglial MAG are not present in acute EAE before inflammatory cell infiltrates or myelin sheath changes appear. Our findings suggest that myelin sheaths are the primary targets in EAE-induced demyelination. Oligodendroglia appear to be relatively unaffected and are available to remyelinate axons.

Animals↗
PubMed1982

Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin.

To localize basic protein (BP) in the lamellar structure of central and peripheral myelin, we perfused newborn and 7-11-day rat pups with a phosphate-buffered fixative that contained 4% paraformaldehyde and 0.05 or 0.2% glutaraldehyde. Teased, longitudinally split or "brush" preparations of optic and trigeminal nerves were made by gently teasing apart groups of myelinated fibers with fine forceps or needles. Some of these preparations were immunostained without pretreatment in phosphate-buffered antiserum to BP according to the peroxidase-antiperoxidase method. Others were pretreated in ethanol before immunostaining. Then, all of them were dehydrated, embedded in Epon, and sectioned for electron microscopic study. In optic and trigeminal nerves that were not pretreated, myelin, glial cells, and their organelles were well preserved. BP immunostaining was present on cytoplasmic faces of oligodendroglial and Schwann cell membranes that formed mesaxons and loose myelin spirals. In compact central and peripheral myelin, reaction product was located in major dense line regions, and the myelin periodicity was the same as that observed in unstained control myelin that had been treated with preimmune serum. In ethanol-pretreated tissue, the myelin periodicity was reduced but dense line staining still was present. Our immunocytochemical demonstration of dense line localization of BP in both CNS and PNS myelin that was not disrupted or pretreated with solvents is important because of conflicting evidence in earlier immunostaining studies. Our results also support biochemical and histochemical evidence suggesting that BP exists in vivo as a membrane protein interacting with lipids on the cytoplasmic side of the bilayer in the spirally wrapped compact myelin membrane.

Animals↗
PubMed1982

Myelin formation in rotation-mediated aggregating cell cultures: immunocytochemical, electron microscopic, and biochemical observations.

Myelination was studied in aggregating cell cultures derived from mechanically 15- to 16-day fetal rat brains. Myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) were localized immunocytochemically in 20-micrometers-thick Vibratome and 1-micrometer-thick Epon sections at 15, 20, 25, and 30 days in vitro. The occurrence of these proteins was correlated with the ultrastructural appearance of oligodendrocytes and myelin sheaths and with biochemical levels of MBP, MAG, and the myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). MBP appeared in ultrastructurally immature oligodendrocyte cytoplasm at 15 days in vitro. As oligodendrocytes developed a more differentiated fine structure, MBP and MAG antisera stained oligodendrocyte processes and myelin sheaths. Immunostaining in Vibratome sections demonstrated that MBP was detectable in oligodendrocytes and myelin prior to MAG. At 25 days in vitro, all Vibratome sections contained MBP- and MAG-stained oligodendrocytes and myelin sheaths. Radioimmunoassays for MBP and MAG and enzyme assays for CNP in whole homogenates of the aggregates revealed that each of these components increased with the progression of myelination. However, MBP only reached 8% of the level in adult rat brain, while MAG and CNP increased to more than half of the adult level. The protein composition of myelin purified from 30-day aggregates resembled that of myelin purified from immature rat brain.

Animals↗
PubMed1982

Lethal encephalitis and non-lethal multifocal central nervous system demyelination in herpes simplex virus type 2 infections in mice.

Six-week-old Swiss-Webster mice were infected intracerebrally with a low dose of herpes simplex virus type 2 (HSV-2) and were studied by pathological, virological and immunological methods. One third of mice developed severe neurological disease and died during the first 31/2 weeks of infection, while the remaining two thirds survived this acute stage with relatively minor neurological signs. These survivors had multifocal demyelinative white-matter lesions in the CNS, and gray-matter lesions, if present, were few, small and usually minor. By contrast, groups of mice killed during the acute stage had a much greater proportion of gray-matter lesions, and these were frequently larger and more severe. Two subgroups could be identified in the acute stage. Mice with severe gray-matter disease, high virus titres, abundant viral antigen and later virus clearance had more severe neurological signs leading to death. By contrast, those destined to survive had pupillary signs alone, and pathologically had white-matter lesions of primary demyelination with minimal or no evidence of gray-matter involvement, low levels of detectable virus and earlier virus clearance. These results show that HSV-2 can produce non-lethal CNS disease in a high proportion of mice, even if infected by the intracerebral route, and that the lesions, which may be found throughout the CNS, are mainly in the white matter, and are demyelinative in type. Survivors of this infection may be useful in a search for evidence of herpes-virus persistence in the CNS, and for a role of this virus in chronic demyelinating disease.

Animals↗
PubMed1981

Freeze-fracture characterization of isolated myelin and axolemma membrane fractions.

The macromolecular organization of membranes isolated from the rabbit optic nerve and tract was analyzed using the freeze-fracture technique. A myelin fraction and two axolemma-enriched fractions were prepared from a preparation of myelinated axons isolated by flotation in a buffered salt-sucrose medium. In the myelinated axon preparation, axolemma and myelin membranes were easily identified. Larger areas of the axon membrane and myelin membrane totally lacked intramembranous particles. The particles remaining on the myelin membrane formed patches of evenly distributed elongated and globular particles. In contrast, the particles remaining on the axolemma were globular in shape and tightly clustered. Particle clustering and particle-free areas were not characteristic of either the axolemma or myelin membrane of whole nerves fixed in situ and processed for freeze-fracture. The isolated myelin membrane fraction contained a large number of vesicles completely lacking intramembranous particles. Of the remaining membrane vesicles, profiles with dispersed elongated and globular particles predominated. A small percentage of vesicles displayed intramembranous particles of the same size, shape and clustering pattern as that seen on the axolemma of the myelinated axon preparation. The two axolemma fractions were enriched in membrane containing tightly clustered globular particles. Particle-free vesicles as well as some myelin membrane vesicles were also seen in the axolemma fractions.

Animals↗
PubMed1981

Immunocytochemical localization and developmental profile of neuron specific enolase (NSE) and non-neuronal enolase (NNE) in aggregating cell cultures of fetal rat brain.

In aggregating cultures, neuron specific enolase (NSE) was first detected biochemically at 3 days. NSE levels increased with time in aggregate cultures and at 48 days reached a level which was 33% of that found in adult rat brain in vivo. The level of non-neuronal enolase (NNE) was essentially identical in aggregate cultures and normal rat brain. Immunocytochemically, NSE(+) cells first appeared at 10 days in vitro. Their number increased until 20 days in culture and then remained constant. When the immunocytochemical localization of NSE and NNE was compared in vibratome sections of 25 day aggregates, all identifiable neurons were NSE(+), NNE(-) and glial cells were NSE(-), NNE(+). In 1 micron thick epon sections of 30 day aggregates NSE antiserum stained neuronal cytoplasm intensely. Comparison of NSE staining in 1 micron thick epon sections with the same cell in an adjacently cut thin section provided conclusive evidence that NSE(+) cells were neurons and NSE(-) cells were glia. These results demonstrate that the three-dimensional organization of aggregate cells provides an excellent environment for neuronal differentiation and also emphasize the advantages of this culture system for multidisciplinary studies of brain development.

Animals↗
PubMed1981

Effects of colchicine on myelination of rabbit optic nerve: a biochemical study.

The effects of intravitreous injections of colchicine on the myelination of the optic nerve were studied in rabbits at different times during development using biochemical and morphological techniques. Myelin basic protein concentrations and 2',3-cyclic nucleotide 3'-phosphodiesterase activities were affected in a similar way by colchicine treatment and reflected the degree of myelination of the tissue. During early development, colchicine produced axonal degeneration and secondary demyelination (Wallerian degeneration). Later, axonal and myelin abnormalities were more variable. Some demyelination was observed amd myelin formation also may have been inhibited. Thus, the effect of colchicine was proportional to the degree of optic nerve and retinal maturation (the youngest being the most sensitive) and, to a lesser extent, to the dosage, Under conditions used for this study, no remyelination was observed. In adult animals, no lesions could be detected histologically one to four months after injection of colchicine, Thus, intravitreous administration of colchicine is a useful chemical technique for producing Wallerian degeneration in optic nerves of young developing rabbits.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1980

Immunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions.

To study the distribution of myelin-associated glycoprotein (MAG) in human nervous tissue and in multiple sclerosis (MS) lesions, we used paraffin sections and our modification of the peroxidase-antiperoxidase technique. Sections of MS lesions also were treated with antiserum to basic protein (BP) and with histological stains for axons and myelin sheaths. In tissue from normal developing central nervous system, oligodendroglia, their processes, and wwly formed myelin sheaths were intensely stained by MAG antiserum. In adults, MAG was found in periaxonal regions of myelinated fibers of the central and peripheral nervous system. The most striking finding in MS lesions was the extension of decreased MAG immunostaining into white matter that appeared normal when treated with BP antiserum or luxol fast blue. In acute early MS lesions the decrease in MAG immunostaining extended far beyond the margin of acute demyelination, where the BP staining of degenerating sheaths often was increased. In chronic inactive plaques, this decrease in periaxonal MAG immunostaining was limited to relatively few fibers in a thin rim around each lesion. These observations suggest that in MS, immunoreactivity of periaxonal MAG is altered before myelin breakdown begins. Early in degeneration, myelin sheaths and their fragments often were more intensely stained by BP antiserum than normal sheaths; later the staining intensity decreased. In shadow plaques, BP antiserum stained some oligodendroglia. Their appearance and location among thinly myelinated axons suggested that these oligondendroglia were forming new sheaths around previously demyelinated axons.

Acute Disease↗
PubMed1979

Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1979

Remodelling of optic nerve myelin sheaths and axons during metamorphosis in Xenopus laevis.

Whole mounts and transverse sections of Xenopus optic nerves were examined with the light and electron microscopes before, during, and after metamorphosis. In stage 52--58 tadpoles, almost all myelin sheaths were circular in transverse sections. Early in metamorphosis (stages 60--61) large redundant myelin loops surrounded many large axons in central regions of the nerve. The loops subsequently were broken down into ovoids and lamellar segments that remained mostly within oligodendrocytes. These myelin changes were not observed in the chiasm or next to the eye. They were not associated with significant axonal degeneration and were no longer apparent in optic nerves of young frogs. Xenopus optic nerves also became shorter during metamorphosis. We therefore suggest that myelin sheaths with redundant loops which degenerate and disappear are being remodelled as the nerve decreases in length.

Animals↗
PubMed1979

Morphological differentiation of mechanically dissociated fetal rat brain in aggregating cell cultures.

Rotation-mediated aggregating cell cultures of mechanically dissociated fetal rat brains (15-16 days) were morphologically characterized at 4, 19, 26 and 40 days in vitro. The dissociated cells coalesced into spherical aggregates which increased in diameter from 340 micrometer at 4 days to 430 micrometer at 40 days. Cells within the aggregates developed from an undifferentiated state at 4 days to a population of morphologically mature neurons, astrocytes and oligodendrocytes (26 days in vitro) before degenerating. Synaptic contacts and myelinated axons appeared as the cells differentiated. Neurons tended to occur in clusters that were located in central regions of the aggregates, whereas astrocytes were more concentrated in the periphery. Synapses and myelinated axons were more abundant in central portions of the aggregates. The amount of myelin formed within the aggregates was less than in organotypic cultures or in vivo. These results show that the morphological differentiation of mechanically dissociated aggregates resembles the development of rat CNS in vitro. The ease with which large amounts of aggregates can be prepared provides an in vitro system which can be analyzed biochemically without the use of micro-methods. This advantage is particularly useful for multidisciplinary investigations in developmental neurobiology.

Animals↗
PubMed1979

Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat.

The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.

Animals↗
PubMed1979

Immunocytochemical localization of rat peripheral nervous system myelin proteins: P2 protein is not a component of all peripheral nervous system myelin sheaths.

Specific antibodies have been developed against P1, P2, and P0 myelin proteins and were used to study the localization of these proteins in the rat peripheral nervous system. Both peripheral and central nervous system myelin sheaths contain P1 protein. P0 and P2 proteins are found exclusively in peripheral nervous system myelin sheaths. Antisera to P1 and P0 proteins stain all peripheral nervous system myelin sheaths uniformly. P2 protein is not a component of all peripheral nervous system myelin sheaths. In sheaths that do contain P2 protein, it is concentrated in the area of the Schmidt-Lanterman incisures.

Animals↗
PubMed1978

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1978

Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation.

A specific antibody to myelin basic protein has been used to localize the protein in developing rat oligodendroglia and myelin. Basic protein is found in the oligodendroglial cytoplasm of anterior commissures of 5- and 7-day old rats before the beginning of myelination. Staining of basic protein in oligodendroglia increases, becoming most intense during early myelination; it decreases during rapid myelination. Staining intensity of oligodendroglia is dependent upon age, brain region, and nervous tract studied. In myelin, reaction of basic protein with antibody decreases when large compact sheaths are present, unless tissue sections are first treated with alcohol.

Animals↗