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H D Webster

Publications and source records attributed to H D Webster.

At least 19 recordsLinked to original sources
PubMed1995

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1995

Cryogenic spinal cord injury induces astrocytic gene expression of insulin-like growth factor I and insulin-like growth factor binding protein 2 during myelin regeneration.

To study injury-induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45: 742-757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin-like growth factor I (IGF-I), IGF-I receptor (IGFR-I), IGF binding protein 2 (IGFBP-2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3-56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin-positive, GFAP-negative astrocyte-like cells in the lesion expressed IGF-I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP-positive, hypertrophic astrocytes, many of which also were vimentin-positive. Levels of IGF-I, IGFBP-2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21-28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7-14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin-positive, oligodendroglia-like cells also were immunostained by anti-IGFR-I. Our findings suggest that early astrocytic production of IGF-I and IGFBP-2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury.

Animals↗
PubMed1994

Effects of nerve segment supernatants on cultured Schwann cell proliferation and laminin production.

Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzyme-linked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti-laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant-treated cultures by using a bromodeoxy-uridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production.

Animals↗
PubMed1994

Concentric sclerosis (Baló): morphometric and in situ hybridization study of lesions in six patients.

Brain tissues from 6 patients with concentric sclerosis (Baló) were examined by in situ hybridization, immunocytochemistry, morphometry, and histological methods. The patients were 24 to 48 years old and had progressive cerebral symptoms and signs that lasted 15 to 100 days. Large demyelinative lesions, most frequent in the frontal white matter, contained alternating bands of demyelinated and partly myelinated white matter that were arranged in concentric or mosaic patterns. In the areas of demyelination, axons were relatively well preserved and there were perivascular inflammatory infiltrates. In 2 specimens, lesions contained regions with the characteristic appearance of actively demyelinating multiple sclerosis plaques. Oligodendroglial densities were highest in normal-appearing white matter, lower in partially myelinated areas, and lowest in demyelinated areas, which also contained many hypertrophic astrocytes closely associated with oligodendroglia. Messenger RNA levels for myelin-related proteins followed the same pattern; they were lowest in demyelinated areas, higher in partially myelinated areas, and highest in normal-appearing white matter beyond lesion margins. Our findings suggest that concentric sclerosis is a variant of multiple sclerosis, that oligodendroglial loss is important in the pathogenesis of demyelination, and that partially myelinated areas probably represent stages of ongoing myelin breakdown rather than remyelination of previously demyelinated areas.

Adult↗
PubMed1994

Expression of insulin-like growth factor-I and related peptides during motoneuron regeneration.

The regulation of insulin-like growth factor-I (IGF-I) and related peptides during motoneuron regeneration was examined in the facial nerve following facial nerve transection. One to 39 days after axotomy, the mRNAs and peptides of IGF-I, type-I insulin-like growth factor receptor (IGFR), insulin-like growth factor binding proteins 1-5 (IGFBP-1-5), and glial fibrillary acidic protein (GFAP) were assayed in brain stem sections by in situ hybridization and immunohistochemistry. Relative mRNA levels of IGF-I, IGFR, IGFBP-2, and GFAP in the ipsilateral facial nucleus were highest 4-7 days after transection and declined thereafter. Double immunostaining experiments showed that both IGF-I and IGFBP-2 were localized in GFAP-positive astrocytic processes, many of which were perineuronal. Peak staining intensity was found 4-7 days after transection and immunoreactivity still was present after 21-35 days. IGFR mRNA was found in some regenerating neurons; however, IGFR peptide was not detected in these neurons or in any other cells in the facial nucleus. Our findings suggest that astrocytic production of IGF-I and IGFBP-2 may accompany regeneration of neurons undergoing retrograde changes induced by axotomy.

Animals↗
PubMed1994

Astrocytes express insulin-like growth factor-I (IGF-I) and its binding protein, IGFBP-2, during demyelination induced by experimental autoimmune encephalomyelitis.

To assess the distribution of insulin-like growth-factor-related proteins during autoimmune CNS demyelination and remyelination, experimental autoimmune encephalomyelitis was produced by injecting Lewis rats with an emulsion containing guinea pig spinal cord and complete Freund's adjuvant. Tail weakness appeared at 10-12 days and was followed by hind and forelimb weakness. Paraplegia and incontinence were observed in some animals. From 8-40 days postinoculation (dpi), spinal cord sections were used to correlate lesion location and severity with mRNA distributions of insulin-like growth factor I (IGF-I), IGF-binding protein 2 (IGFBP-2), IGF-I-receptor (IGFR-I), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP). These were determined semiquantitatively by in situ hybridization. Fourteen dpi, there were inflammatory infiltrates and demyelination in both white matter (WM) and grey matter (GM). IGF-I and GFAP mRNAs were increased in these lesions and transcripts encoding myelin basic protein (MBP) were greatly reduced. Large lesions with extensive demyelination were evident in both WM and GM when mRNA levels of GFAP and IGF-I peaked 26 dpi. MBP mRNA levels began increasing 21 dpi and peaked 26 dpi, when a few thin regenerating myelin sheaths were found morphologically. Astrocytes, identified by their morphology and GFAP immunoreactivity, expressed very low levels of IGFBP-2 mRNA and peptide in normal controls; their levels were significantly higher 14 dpi, peaked 26 dpi, and then gradually decreased. Some neurons, as well as oligodendroglia in areas undergoing remyelination, expressed IGFR-I. Although levels of IGF-I, IGFBP-2, and GFAP mRNAs were highest in lesion areas, levels were also elevated around lesions and in some normal-appearing areas of WM and GM 14-40 dpi. The gene expression of both IGF-I and IGFBP-2 by hypertrophic GFAP-positive astrocytes was demonstrated 14-40 dpi by combined in situ hybridization and immunocytochemistry as well as by double immunostaining. Coexpression of IGF-I and IGFBP-2 in the same astrocyte was a frequent finding. Relative increases in both IGF-I, GFAP, IGFBP-2, IGFR-I, and MBP mRNAs peaked at about the same time. This suggests that during lesion progression and recovery, astrocytic expression of IGF-I-related peptides may reduce immune-mediated myelin injury. We also suggest that astrocytic IGFBP-2 in lesions may help target IGF-I to IGFR-I-expressing oligodendrocytes and promote remyelination of demyelinated axons.

Animals↗
PubMed1994

Human immunodeficiency virus (HIV) distribution in HIV encephalitis: study of 19 cases with combined use of in situ hybridization and immunocytochemistry.

Brains of 19 AIDS patients with HIV encephalitis were examined by immunohistochemistry and in situ hybridization using antisense HIV DNA and RNA probes. Double immunohistochemical labeling, using antibodies against viral and cell-type specific antigens, was utilized to study lesions in some brains. Other combined studies included use of in situ hybridization and immunohistochemical labeling of the same section, using antibodies against either viral or cell-type specific antigens. Hybridization signals were abundant and were concentrated mainly in the white matter. Heavy labeling was found in the subcortical white matter, the corpus callosum, the internal capsule, and white matter regions of the brainstem and cerebellum. Deeper cortical layers often contained cells with hybridized probe when the subcortical white matter was intensely labeled. HIV nucleic acid sequences were found almost exclusively in macrophages. Counts showed that 16-25% of macrophages contained viral antigens and exhibited hybridized HIV probe. Almost all of these macrophages contained proviral DNA, viral RNA and viral proteins; i.e. they were actively replicating HIV. We also examined brains from three AIDS cases without clinical or pathological evidence of HIV encephalitis; no HIV sequences or immunoreactive proteins were detected.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Expression of viral T-antigen in pathological tissues from transgenic mice carrying JC-SV40 chimeric DNAs.

Immunostaining methods were used to detect viral T-antigen and the cellular protein p53 in pathological tissues obtained from transgenic mice carrying JC-SV40 hybrid viral DNAs. A transgenic mouse carrying the SV40 regulatory region and JC virus (JCV) T-antigen-coding sequences exhibited an SV40-characteristic choroid plexus papilloma that expressed JCV T-antigen and p53. JCV-associated pathology was observed in two other mice in which the JCV regulatory signals directed SV40 T-antigen-induced adrenal neuroblastomas and brain neoplastic cells. However, these mice also exhibited an SV40-characteristic osteosarcoma and abdominal lymphoma that contained SV40 T-antigen and p53-positive cells. Contrasting thymic pathology was observed in the two types of mice where the SV40 regulatory region directed a JCV T-antigen-induced thymoma in one mouse, and the JCV regulatory region directed SV40 T-antigen-induced thymic hypoplasia in two other mice.

Adrenal Gland Neoplasms↗
PubMed1993

Effects of psychosine (galactosylsphingosine) on the survival and the fine structure of cultured Schwann cells.

The cytotoxicity of psychosine (galactosylsphingosine) for cultured rat Schwann cells was studied by maintaining them in medium containing 1, 10, 50, 75 and 100 microM psychosine for 24, 48 and 72 hours (h). When incubated in 50-100 microM concentrations of psychosine for 24 h, 52-99% of cultured Schwann cells did not survive. Lower concentrations (1-10 microM) did not significantly reduce Schwann cell numbers for the first 24 h. However, only 43-69% of cultured Schwann cells survived in these low concentrations for 48 h, and substantially fewer remained after 72 h of incubation. During incubations in psychosine, bipolar processes of Schwann cells retracted; the resulting oval and rounded Schwann cells still were S-100 positive. When these Schwann cells were transferred into normal medium, their processes elongated quickly. When examined with the electron microscope, the cytoplasm of Schwann cells incubated in psychosine contained numerous membranous inclusions and fewer mitochondria, some of which were swollen. There also were fewer profiles of granular endoplasmic reticulum and some had widely dilated cisternae. These results suggest that 1) exogenous psychosine in concentrations of 1 microM and greater is cytotoxic for cultured rat Schwann cells; 2) psychosine has reversible toxic effects and its turnover is rapid; and 3) psychosine produces membranous inclusions and abnormalities in the mitochondria and granular endoplasmic reticulum of cultured Schwann cells. Our findings support the hypothesis that the accumulation of psychosine in human and murine globoid cell leukodystrophy is toxic for Schwann cells, produces changes in their capacity to maintain myelin, and leads to Schwann cell dysfunction.

Animals↗
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↗