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Biomedical subjects

H D Webster

Publications and source records attributed to H D Webster.

At least 37 records · Page 2Linked to original sources
PubMed1992

Brain vascular endothelial cells express JC virus large tumor antigen in immunocompetent and cyclophosphamide-treated hamsters.

When injected intracerebrally into newborn hamsters, the human polyomavirus JC virus (JCV) establishes a nonproductive infection resulting in brain tumor formation. Using immunostaining methods to detect the JCV regulatory protein, large tumor antigen (T antigen), we have now demonstrated JCV infection of brain vascular endothelial cells (EC) in infected hamsters. JCV T antigen was detected in lectin-labeled EC as well as in von Willebrand factor-expressing EC in both cyclophosphamide-treated and nonimmunosuppressed hamster brains 16, 21, and 31 days after birth. Cyclophosphamide-treated hamsters exhibited a greater number of JCV-infected EC, whereas T-antigen expression in nonvascular cells was not affected. The influence of cyclophosphamide was most pronounced in the cerebellum where increased numbers of JCV-infected EC were located predominantly at the internal granular layer-white matter junction, also a prominent location for T-antigen-expressing neoplastic foci. The hamster model demonstrates in vivo infection of EC by a human polyomavirus and directs interest toward the role of these cells in human JCV infection.

Animals↗
PubMed1992

Insulin-like growth factor I gene expression is induced in astrocytes during experimental demyelination.

To investigate insulin-like growth factor I (IGF-I) and IGF-I receptor gene expression during experimental demyelination and myelin regeneration, young mice were fed cuprizone (( bis(cyclohexanone) oxaldihydrazone )). This copper-chelating agent produces demyelination in the corpus callosum and superior cerebellar peduncles, and when treatment is stopped, there is rapid remyelination. At intervals during cuprizone treatment and recovery, brain sections were hybridized with specific probes and immunostained with antibodies to determine the localization and relative amounts of IGF-I and IGF-I receptor mRNAs and peptides. In untreated littermates, IGF-I and IGF-I receptor mRNAs and peptides were not detected in white matter. In cuprizone-treated mice, high levels of both IGF-I mRNA and peptide were expressed by astrocytes in areas of myelin breakdown. Astrocyte IGF-I expression decreased rapidly during recovery and oligodendroglial expression of myelin-related genes increased. In severely demyelinated areas, immature oligodendroglia exhibited a transient increase in IGF-I receptor mRNA and peptide immunoreactivity during early recovery. This highly specific pattern of IGF-I induction in astrocytes during demyelination and the expression of the IGF-I receptor in regenerating oligodendrocytes during recovery suggest that IGF-I functions in the regulation of oligodendrocyte and myelin metabolism in vivo.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Myelinated fiber regeneration after sciatic nerve crush: morphometric observations in young adult and aging mice and the effects of macrophage suppression and conditioning lesions.

To study myelinated nerve fiber regeneration during aging, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two, 4, and 8 weeks later, both groups of mice were perfused. The sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. Two weeks after axotomy, fascicles of aging mice contained significantly fewer regenerated myelinated fibers than those of young adults. After 4 weeks, the difference in the number of myelinated fibers was less. However, measurements of myelinated fibers in fascicles of aging mice showed that areas of Schwann cell cytoplasm and myelin were significantly reduced at all intervals. In contrast, although axon diameters in aging mice were somewhat less 2 weeks after crushing, the difference decreased with time, suggesting that in nerves of aging mice, regenerative responses of Schwann cells were more affected than those of axons. Other experiments in young mice showed that myelinated fiber regeneration could be retarded by suppressing macrophage responses and was not significantly changed by conditioning lesions before crush injury.

Adaptation, Physiological↗
PubMed1991

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice.

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.

Aging↗
PubMed1991

In vitro changes in the fine structure and protein composition of light myelin fractions isolated from guinea pig brain.

To find out if in vitro maintenance produces changes in the electron microscopic appearance, protein composition and phosphorylation properties of guinea pig CNS myelin fractions, we incubated them for 10 min, 4 hr, 24 hr, and 48 hr in phosphate-buffered saline (pH 7.4) or in 20 mM Hepes, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothreitol, and 20 mM NaCl at 4 and 30 degree C. Aliquots were processed for electron microscopic study, were analyzed for protein content by gel electrophoresis, and were assayed for endogenous protein phosphorylation. Before incubation, electron micrographs of fractions contained two types of multilamellar whorls with the periodicity of CNS myelin sheaths. The first type of whorl was separated from nearby whorls; the other type had surface lamellae that were connected to other multilayered membrane fragments. After incubation at 4 degree C for 24 hr, the number of both types of multilamellar whorls in micrographs had increased approximately 3- to 4- fold. Counts per unit area showed that the observed increase was both time- and temperature-dependent. In aliquots studied by gel electrophoresis, only minor degradation of myelin proteins was observed. The endogenous protein phosphorylation properties of the myelin fragments also remained functional, suggesting that the activities of protein phosphotransferases were not altered. We conclude that the incubation conditions described here favor interactions of proteins and lipids that lead to the formation of multilayered aggregates of CNS myelin membranes.

Animals↗
PubMed1991

Distribution of protein kinase C isozymes in rat optic nerves.

Light (LM) and electron (EM) microscopic immunocytochemical methods were used to study the distribution of protein kinase C (PKC) isozymes in adult rat optic nerves. In cryostat and vibratome sections examined by LM, type II (beta) isozyme was localized almost exclusively in the axons. In the EM, immunoreaction products were found to associate with microtubules and neurofilaments. The inner surface of axonal membranes were occasionally stained. Analysis of PKC isozyme composition of the optic nerves by using immunoblot techniques revealed that type II (beta) isozyme accounted for approximately 80% of the total immunoreactivity. By contrast, type III (alpha) isozyme, which accounted for the remaining 20% of PKC, was found mainly in the astrocytes. Astrocytic processes next to blood vessels and between myelinated axons were stained. In the EM, immunoreaction products were found in the cytoplasm and along astroglial filaments. Segments of plasma membranes also were stained; but nuclei were unstained. Adult glial cells were not stained by an antibody to type II (beta) isozyme except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunostaining was observed in sections treated with a monoclonal antibody to type I (gamma) isozyme. Immunoblot analyses also did not reveal this subspecies. The absence of type I (gamma) isozyme in optic nerves is not due to a down-regulation of the enzyme during development. In developing (5 and 11 day) rats, immunoreactivity of protein kinase C was very faint or absent. After 15 days, reaction products of both type III (alpha) and type II (beta) isozymes were found throughout the nerve. These findings suggest that type II (beta) isozyme may be involved in axonal transport whereas type III (alpha) isozyme may play a role in some astrocyte functions in mature optic nerves.

Aging↗
PubMed1991

The early development of the neopallial wall and area choroidea in fetal rats. A light and electron microscopic study.

The telencephalic wall was studied by light and electron microscopy in 11-13 day old fetal rats (E11-13). A few specimens from E14-16 were also included for comparisons. Two areas were selected: the dorso-lateral convexity of the hemispheric vesicles, called the neopallial wall, and the area choroidea, the posterior part of the telencephalic roof which unites the two hemispheres. Our observations and a review of the literature have shown that on E11-12 the neopallial wall, the telencephalic roof, and the hippocampal anlage between them form a continuous, nonstratified, cohesive monolayer of columnar and mitotic cells, which essentially is similar to epithelial monolayers elsewhere in the body. This simple structure is modified late on E12 or early in E13 in the neopallial wall when postmitotic neurons appear and migrate in order to form the cortical plate. However, bipolar radially oriented cells, which span the entire width of the wall, still predominate. These cells, now called radial glial cells, increase greatly in number and length during the period of neuronal migration. The cuboidal cells in the neural tube, the columnar cells in the early neopallial wall, and the radial glial cells in the period of neuronal migration have the same basic structure. They are axially polarized epithelial cells which are characterized by the following basic features. They have an elongated bipolar shape which is maintained by a cytoskeleton of longitudinally oriented microtubules. Opposite ends are different structurally and functionally. Thus, the apical ends, connected by tight junctions, face the fluid-filled cavity while the outer ends, covered by a basal lamina, face mesenchymal tissue including blood vessels. A polarization of cytoplasmic organelles is also evident, e.g. the Golgi apparatus has always a supranuclear position. During the early development of the telencephalon this basic epithelial structure is maintained but is modified locally in order to serve various functions. The columnar/radial glial cells in the neopallial wall are elongated and slender, have a narrow Golgi apparatus, profiles of RER and vesicles, relatively few ribosomes, and show a few examples of micropinocytosis. These cells grow continuously in length during development. On the other hand, the cells in the area choroidea have a low columnar or cuboidal shape, which does not change during development. The inner portion (between the nucleus and the ventricle) contains a voluminous Golgi apparatus, many mitochondria, RER cisternae which contain electron-dense material, SER, and many vesicles. The inner ends of the cells project into the ventricular cavity as bulbous or apical protrusions which contain many organelles, especially MVBs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Preparation of fetal rat brains for light and electron microscopy.

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: 1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. 2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11-20 days (E11-E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11-E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11-E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s-collidine buffer, followed by osmium tetroxide in s-collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.

Animals↗
PubMed1990

Prominent white matter lesions develop in Mongolian gerbils treated with 100% normobaric oxygen after global brain ischemia.

Carotid arteries were occluded bilaterally for 15 min in two groups of Mongolian gerbils. The first group received 100% oxygen during the first 3 h of reperfusion. During that period, room air was given to the second group. After 3 h, both groups received room air. Brains of gerbils that died within 14 days after occlusion were removed, fixed in formalin and embedded in paraffin. Gerbils that survived 15-28 days were perfused with formalin before their brains were removed and embedded in paraffin. Adjacent, serially cut sections were stained with luxol fast blue (LFB)-H&E, cresyl violet, according to the Bodian method, or immunocytochemically with antisera raised against myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). In brain sections of gerbils receiving 3 h of 100% oxygen, there were circumscribed white matter lesions in the corpus striatum, lateral thalamus, mesencephalon and posterior limb of the internal capsule. Myelin sheaths were swollen, fragmented and were less intensely stained by MBP antiserum. MBP and LFB-stained myelin fragments were present extracellularly and in macrophages. Many axons in these areas appeared undamaged. Previously described ischemic changes were found in gray matter and some areas of white matter in both groups. However, neurons in the deeper laminae of the cerebral cortex appeared to be better preserved in gerbils given oxygen. The results suggest that hyperoxia, if present immediately after transient brain ischemia, may damage myelin more severely than other cellular elements.

Animals↗
PubMed1990

P0 glycoprotein mRNA distribution in myelin-forming Schwann cells of the developing rat trigeminal ganglion.

A biotinylated P0 cDNA was hybridized in situ to aldehyde-fixed vibratome sections of trigeminal ganglia from day 2, day 7, day 15, day 30 and adult rats. Nickel-enhanced horseradish peroxidase (HRP) was used in an antibody sandwich method to detect hybridization. After postfixation in osmium tetroxide, the sections were dehydrated in ethanol and embedded in epon. At each age, some vibratome sections were used to count the HRP-positive and HRP-negative myelin-forming Schwann cells. The percentage of HRP-positive myelin-forming Schwann cells in ganglia from day 2, 7, 15, 30, and adult rats were 31%, 56%, 47%, 12% and 3%. In sections of ganglia from 2-day-old rats, studied by light and electron microscopy, peroxidase reaction product localizing hybridized P0 mRNA was found on profiles of granular (rough) endoplasmic reticulum (RER) in perinuclear regions of Schwann cells which had formed two to three compact myelin lamellae. Peroxidase deposits were larger and more numerous in the cytoplasm cells with thicker myelin sheaths. At day 7, some Schwann cells had long external mesaxons; the cytoplasm between these mesaxons and the cell surface often contained abundant HRP-stained profiles of RER. In sections from day 7 and day 15 ganglia, substantially more reaction product was found. In each myelin-forming Schwann cell, the amount was generally proportional to the size of the newly formed myelin sheath. HRP deposits were observed all along the outer surfaces of myelin segments at these ages, and their distribution corresponded to that of the RER.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Immunolabeling of JC virus large T antigen in neonatal hamster brain before tumor formation.

Using immunolabeling methods, the JC virus (JCV) early or regulatory protein, large T antigen, was demonstrated in frozen sections of neonatal hamster brains before tumor formation. Three days after intracerebral inoculation of 2500 hemagglutinating units of JCV, T antigen was expressed predominantly in nuclei of cells in the external granular layer and newly forming internal granular layer of the cerebellum and also in cell nuclei located in the hippocampus, periventricular areas, and the olfactory bulb. At 7 days postinoculation (p.i.), most cerebellar T antigen-containing cells had migrated to the internal granular layer, but by 15 days p.i., cells that expressed T antigen was greatly reduced in number or absent. However, by 30 days p.i., the internal granular layer of the cerebellum again contained T antigen-positive cells. In contrast to the scattered cells seen at 3 or 7 days p.i., these cells appeared in dense clusters thought to represent pretumor foci. Since JCV capsid proteins were not detected at any time, JCV may establish a latent or abortive infection in cells during their mitotic phase and these cells initially express T-antigen during migration or become immunoreactive later before tumor formation.

Animals↗
PubMed1989

Inhibition of protein synthesis during CNS myelination produces focal accumulations of membrane vesicles in oligodendrocytes.

Optic nerves of Xenopus tadpoles were exposed to cycloheximide to identify changes that occur during CNS myelin membrane formation when protein synthesis is inhibited. Groups of stage 51-56 tadpoles were immersed in either 10 or 20 micrograms ml-1 cycloheximide, and at specified times between 12 and 18 h after initial immersion tadpoles were killed and their optic nerves prepared for ultrastructural analysis. As early as 12 h there were alterations in oligodendrocytes from treated animals compared with control animals. The number of polyribosomes in the perikarya and cell processes was greatly reduced and the rough endoplasmic reticulum was disorganized. Mitochondria and microtubules were normal in appearance. Many oligodendroglial tongue processes at the inner margin of the myelin sheath were enlarged, occasionally indented the axon and were filled with vesicular profiles. Vesicles were noted in other cytoplasmic regions of oligodendrocytes and focal changes in the lamellar structure of myelin were found in paranodal regions. The internodal portions of the myelin sheath, axons and astrocytes appeared normal. Polyacrylamide gels of optic nerves showed that the incorporation of 35S-methionine into polypeptides had been almost completely inhibited by treatment with cycloheximide. These observations suggest that cycloheximide, by inhibiting synthesis of myelin proteins, alters the ability of oligodendrocytes to incorporate membrane components into CNS myelin sheaths.

Animals↗
PubMed1989

Non myelin-forming perineuronal Schwann cells in rat trigeminal ganglia express P0 myelin glycoprotein mRNA during postnatal development.

To determine whether P0 myelin glycoprotein mRNA is expressed in Schwann cells that ensheath neurons and do not form myelin, we probed aldehyde-fixed vibratome sections of developing and adult trigeminal ganglia with a biotinylated P0 cDNA. For probe detection, vibratome sections were treated with nickel-enhanced horseradish peroxidase (HRP). At each age, some vibratome sections were used to count numbers of HRP-positive and -negative satellite cells. The percentages of HRP-positive satellite cells at 2, 7, and 15 days were 22%, 30% and 14%. None was positive at 30 days or in the adult. Other vibratome sections were embedded for light and electron microscopic study. In semithin sections from ganglia removed from 2-day-old rats, small dot-like densities of HRP were located in perinuclear regions of a few perineuronal Schwann cells. In 7-day-old ganglia, more of these Schwann cells contained HRP. In thin sections studied with the electron microscope, peroxidase was found in cytoplasmic regions enriched in granular endoplasmic reticulum and ribosomes. At 2 and 7 days, HRP densities in perinuclear regions were larger and more numerous than at 15 days. No signal was detected in 30 day or adult perineuronal Schwann cells. The results show that early in postnatal development, P0 mRNA is expressed in some Schwann cells that ensheath neurons, that do not contain immunocytochemically detectable levels of P0 and that do not ever form myelin.

Aging↗
PubMed1988

Herpes-simplex-related antigen in human demyelinative disease and encephalitis.

Using immunohistochemical methods optimized to detect herpes simplex virus type 2 (HSV-2) antigen, paraffin sections from human central nervous system tissues from 31 cases pathologically diagnosed as multiple sclerosis (MS), 34 cases of other neurological diseases, 4 adult cases of HSV encephalitis, and mouse brains infected with various HSV strains were examined. Two distinct patterns of immunoreactivity with HSV antisera were seen. In typical acute human and experimental encephalitis, antigen was readily detected using high dilutions of antisera to both HSV types -1 and -2, and was found nonselectively in both neurons and glia. Lesions were destructive, with necrosis of all neural cell types, and inflammation was a mixture of polymorphonuclear and mononuclear cells. By contrast, immunoreactivity in lesions in each of three MS cases and in one case of brain stem encephalitis was found only with HSV-2 antisera, and relatively high antiserum concentrations were required to detect it. Reactivity appeared to be largely restricted to glial cell nuclei within and near lesions that were selectively demyelinated. Only mononuclear inflammation was present. These experiments suggest that HSV-related antigen may be found in a broader spectrum of human CNS lesions than has previously been recognized, and that HSV or a related agent may be associated with a selective infection of glial cells and with CNS demyelination.

Adult↗
PubMed1988

A double-label method detects both early (T-antigen) and late (capsid) proteins of JC virus in progressive multifocal leukoencephalopathy brain tissue from AIDS and non-AIDS patients.

A new double-label immunocytochemical method detects JC virus (JCV) early (T-antigen) and late (capsid) proteins simultaneously in cryostat sections of progressive multifocal leukoencephalopathy (PML) brain tissue from both acquired immunodeficiency syndrome (AIDS) and non-AIDS patients. T-antigen is detected with a monoclonal antibody (PAb 416) followed by goat anti-mouse IgG and mouse Clono-PAP, while capsid proteins are detected by a rabbit polyclonal antiserum to capsid proteins followed by biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate. The substrates are 3,3'-diaminobenzidine and Vector Red I, respectively. With this method some infected glial cells stain for late (capsid) antigens in the nucleus, while others show early protein (large T-antigen) immunoreactivity. The latter are likely to be astrocytes infected abortively or oligodendrocytes in the early stages of a productive JCV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1988

A monoclonal antibody to SV40 large T-antigen labels a nuclear antigen in JC virus-transformed cells and in progressive multifocal leukoencephalopathy (PML) brain infected with JC virus.

Thirty monoclonal antibodies to SV40 large T-antigen were tested for reactivity on the JC virus-transformed hamster glial cell line known as HJC-15. Two of them (PAb 416 and PAb 108) detected a nuclear antigen in both SV40-transformed CCL 75.1 cells and in HJC-15 cells, but not in control cells lacking T-antigen. These same antibodies also labeled a nuclear antigen in hamster tumor tissue derived from HJC-15 cells. In addition, the monoclonal antibody PAb 416 detected a nuclear antigen in progressive multifocal leukoencephalopathy (PML) tissue infected with JC virus, but not in normal brain tissue or tissue from other neurological diseases. Staining by PAb 416 was reduced by prior incubation with hamster anti-JCV tumor serum, suggesting that the polyclonal antiserum to JCV T-antigen may compete for an epitope at or near the PAb 416 binding site.

Animals↗