Henry deF. Webster

The relationships between interphase Schwann cells and axons before myelination: A quantitative electron microscopic study

Abstract Electron micrographs of transversely sectioned sciatic nerves removed from newborn, 3-day-old, and 7-day-old rats were used to make montages of comparable areas in the marginal bundle of the posterior tibial fascicle. At each age, the number of axons, their diameter, their relationships with Schwann cell processes, and their degree of myelination were determined. Also, three-dimensional reconstructions of representative fiber groups in the newborn nerve were made from similar montages at 5 additional transverse levels. The results showed that outgrowth of axons and migration of Schwann cells continued after birth. Families of Schwann cells, each surrounded by a common basal lamina, formed the sheaths that subdivided the bundles. Axons to be myelinated appeared to progress radially from a bundle to a 1 : 1 relationship with a Schwann cell at the sheaths outer margin. Sheaths containing multiple Schwann cells became smaller and more numerous as axon bundles were subdivided. Almost all of the isolated Schwann cells, which were separated from their neighbors by collagen were myelinating single large axons.

Variation of proteins, enzyme markers and gangliosides in myelin subfractions

A discontinuous sucrose gradient was used to separate adult rat brain myelin into light, medium and heavy subfractions. Basic proteins decreased sharply, proteolipid potein changed very little, and high molecular weight proteins increased from the light to the heavy fraction. The concentration of monosialoganglioside GM1 was the highest in the middle fraction. The amount of carbohydrate in the major myelin-associated glycoprotein per mg total myelin protein increased 3.5-fold from the light to the heavy fraction. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase, which is related to myelin or the oligodendroglial membrane, and acetylcholinesterase, which is in neural membranes such as the axolemma, both increased between the light and the heavy fraction, although their relative distributions among the three fractions were different. The glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase had similar distributions suggesting that they were concentrated in similar locations, possibly in the loose myelin and oligodendroglial plasma membrane. Electron microscopic examination of the subfractions was consistent with this interpretation.

Mitotic Schwann cells in developing nerve: their changes in shape, fine structure, and axon relationships.

Abstract The shape of mitotic Schwann cells and their relationships with axons and their interphase neighbors were studied in electron micrographs of longitudinally and transversely sectioned newborn rat sciatic nerves. During prophase, the radial extent of sheetlike processes surrounding axons was progressively reduced until the Schwann cell was spindle shaped. Numerous 100 nm vesicles were found in the cytoplasm as the surface area decreased; they disappeared during subsequent reextension of the radial processes. The axis of mitosis was parallel to the long axis of the cell and transverse cleavage of the cytoplasm occurred between the daughter nuclei. The earliest cytoplasmic outgrowth was identified in serial sections through an anaphase cell which was traced distally from its mid nuclear region. A slender process, originating near the spindle pole, arched over and grew longitudinally beside and beyond the dividing nucleus. Observed also in telophase, this process was thought to represent a daughter cells new axial process and to be important in establishing the cells longitudinal symmetry. Radial processes that surrounded axons reappeared in telophase; they began forming in the nuclear region and subsequently grew radially and extended peripherally toward both ends of the cell. Our results show that the changes in shape that occur in dividing Schwann cells and their interphase neighbors increase contacts between these Schwann cells and the axons they surround. These surface interactions probably are important in the selection of axons to be myelinated.

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 Freunds 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.

Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in vivo and in vitro

Abstract Protein synthesis and ultrastructure were studied in the cultured epithelium and in the intact lens of the 6-day chick embryo. Proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lens epithelium synthesized proteins which ranged in molecular weights from more than 200,000 to less than 20,000. Major species had molecular weights of about 91,000, 65,000, 27,000, and 20,000, with a principal component of approximately 45,000 containing about 35% of the new protein. After 1 day in culture, the epithelial cells doubled in length and synthesized a smaller proportion of protein with molecular weights of about 91,000 and 65,000. Approximately 50% of newly synthesized protein had a molecular weight near 45,000. This pattern of synthesis remained for 3 days in vitro as the cells elongated. In the fibers of the intact 6-day lens, some 80% of the new protein had a molecular weight of about 45,000. Fiber cell formation in vivo and in vitro was associated with a decrease in organelle density, a clustering of ribosomes and an alignment of longitudinally oriented microtubules along the cell membrane. Thus, cells in the cultured lens epithelium of the chick embryo undergo changes that are characteristic of epithelial cells differentiating into lens fibers.

Phase and electron microscopic studies of experimental demyelination. I. Variations in myelin sheath contour in normal guinea pig sciatic nerve.

Phase and electron microscopic study of normal guinea pig sciatic nerves reveals numerous variations in the myelin sheath contour. For the most part, these variations consist of infoldings into axoplasm or protrusions into Schwann cell cytoplasm and can be seen as isolated myelin ovoids in a single plane of section. They are most numerous in the juxtanodal region of the largest fibers and are of particular importance to neuropathologists because of their resemblance to myelin forms seen with the light microscope in early demyelinative lesions. Several other features of peripheral nerve ultrastructure are briefly described.

Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: Temporally distinct expression of midkine and pleiotrophen

Heparin‐binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin‐binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF‐2) and one of its receptors (FGFR1/ flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF‐2 during EAE and determined the cellular origin of FGF‐2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF‐2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF‐2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed co‐localization of FGF‐2 to activated microglia/ macrophages with strongest expression in the macrophage‐rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/ microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/ macrophage in an autocrine fashion, to induce the expression of other factors like insulin‐like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/ or regeneration of oligodendrocytes within the lesion area. GLIA 24:390–397, 1998.

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.

Human Immunodeficiency Virus (HIV) Distribution in HIV Encephalitis: Study of 19 Cases with Combined Use of In Situ Hybridization and Immunocytochemistry

Abstract 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.

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.