No abstract

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. MATERIALS AND METHODS MAG was purified from myelin isolated from the brains of 200-to 250-g Osborne-Mendel rats. The first step in MAG purification was lithium diiodosalicylate extraction of the myelin fraction and subsequent partitioning of the extract with phenol as described (7). The glycoprotein preparation obtained by this procedure was dissolved in a solution containing 2% (wt/vol) sodium dodecyl sulfate, 1.5% (wt/vol) dithiothreitol, and 8% (wt/vol) sucrose for application to a preparative 5% polyacrylamide gel. The procedure for preparative electrophoresis and extraction of MAG from appropriate gel slices was as described (8). The glycoprotein extract was lyophilized, suspended in a small volume of saline, and emulsified with an equal volume of complete Freund's adjuvant for rabbit immunization.The emulsion containing about 0.5 mg of MAG was injected intradermally in multiple sites on the rabbit's back. Similar injections were repeated twice at 3-week intervals, except that the final injection utilized incomplete Freund's adjuvant. Also, a control rabbit was injected on the same time schedule with analogous extracts of preparative polyacrylamide gels to which no glycoprotein samples were applied.A double antibody radioimmunoassay was used for measuring the level of antibodies to the glycoprotein. The MAG antigen used in the assay was purified by the lithium diiodosalicylate/phenol procedure (7) after radiolabeling in vivo by injecting adult rats intracranially with [3H]fucose or 13-day-old rats with [14C]fucose (1). The incubation mixture contained 1 ltg of MAG (1500 dpm 3H or 900 dpm 14C) and 10 ,.d of normal rat serum and was made up to a total volume of 150 ,l with phosphate-buffered saline (pH 7.4). The serum to be tested for MAG antibodies was added as 20 pl of a 1:20 dilution in phosphate-buffered saline, and the assay tubes were incubated for 1 hr at 370C. Then, 100 AI of goat anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) was added, and the tubes were incubated for another 30 min at 370C followed by 1 hr at 4VC. The mixtures were centrifuged at 2000 X g for 30 min, and the precipitates were washed with phosphate-buffered saline. The precipitates were dissolved with 1% sodium dodecyl sulfate and NCS solubilizer (Amersham) for liquid scintillation counting.Newborn to adult Osborne-Mendel rats were anesthetized with chloral hydrate and fixed by intracardiac perfusion for 10 min with a solution containing 76 ml of HgCI2 (saturated at 0'C) and 20 ml of 37% (vol/vol)

In rat sciatic nerves, a small bundle of fibers was identified in which myelin sheaths were absent at birth, appeared within 3 days, and grew rapidly for 2 wk. During this interval, nerves were removed from littermates and were sectioned serially in the transverse plane. Alternating sets of thin and thick sections were used to prepare electron micrograph montages in which single myelinating axons could be identified and traced distally. During the formation of the first spiral turn, the mesaxon's length and configuration varied when it was studied at different levels in the same Schwann cell. The position of the mesaxon's termination shifted while its origin, at the Schwann cell surface, remained relatively constant. Along myelin internodes composed of two to six spiral turns, there were many variations in the number of lamellae and their contour. Near the mesaxon's origin, longitudinal strips of cytoplasm separated the myelin layers. Thicker sheaths were larger in circumference, more circular in transverse sections, and more uniform at different levels. Irregularities were confined to the paranodal region, and separation of lamellae by cytoplasm occurred at Schmidt-Lantermann clefts. Approximate dimensions of the bundle, its largest fibers, and their myelin sheaths were measured and calculated. The myelin membrane's transverse length and area increased exponentially with time; the growth rate increased rapidly during the formation of the first four to six spiral layers and remained relatively constant during the subsequent enlargement of the compact sheath.

No abstract

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 ,ig/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 ofIGF-I-treated rats.

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 se- Insulin-like growth factor I (IGF-I), a member of the insulin gene family (1,2), has been shown to have a number ofpotent effects on cultured neural and glial cells. It promotes the mitosis of sympathetic neuroblasts, the survival of fetal neurons, and the stimulation of neurite outgrowth in motor and sensory neurons, and it induces oligodendrocyte differentiation and myelin synthesis (3-10). These effects are mediated by the type I IGF receptor, which is a membranebound tyrosine kinase with significant homology to the insulin receptor (11, 12). IGF-I gene expression is abundant in the developing nervous system (13-18). IGF-I mRNA is selectively concentrated in the large principal neurons of functionally related sensory and cerebellar relay systems during a late predominantly postnatal phase of neural development (18). The specific timing and cellular sites of neural IGF-I synthesis suggest a role for IGF-I in the synaptic maturation or the myelination of these particular systems. Evidence that IGF-I has a role in myelination is the finding that transgenic mice overexpressing IGF-I (19) have increased brain myelin content (20). Also, several studies have shown that IGFs have potent and highly specific effects on oligodendrocytes in vitro (7-10). However, except for the olfactory system, very little IGF-I is normally detected in the mature rodent brain (16-18).To investigate whether IGF-I is important in the regeneration of central nervous system myelin in vivo, demyelinating lesions were produced in the corpus callosum, superior cerebellar peduncles, and anterior commissures by adding the copper-chelating agent bis(cyclohexanone) oxaldihydrazone (cuprizone) to the diet of mice (21-23). In this model of primary demyelination, there are early severe changes in oligodendroglia and myelin sheaths in specific brain areas (21-23). Axons are relatively well preserved (21-23) and the blood-brain barrier remains impermeable to plasma proteins (24). There are no perivascular lymphocytic infiltrates (21,22) and the cessation of cuprizone tr...

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 unstaiffed 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.Basic protein (BP) is a major constituent of central nervous system (CNS) myelin (3,9,23,40). It has a molecular weight of about 18,500, is antigenic, and is thought to have an important role in the formation and maintenance ofmyelin's compact lameUar structure (5,9,23, 48). Myelin isolated from the peripheral nervous system (PNS) contains smaller amounts of a basic protein cared Pl (7,8,15). Since Pl and BP have almost the same molecular weight (7,8,15) and amino acid sequence (7), they probably are identical or very closely related proteins (4,7,8,15,49).The localization of BP in the lamellar structure of myelin is an important issue for those interested in membrane assembly and maintenance. Investigators have used immunocytochemical methods and specific antisera to study this directly and both dense line (16, 39) and intraperiod region (27, 43) localizations have been described. But in all of these experiments, immunostaining was only observed in myelin pretreated with solvents like ethanol (27), in myelin lametlae broken by tissue processing procedures (16, 39), or in myelin disrupted by isolation for biochemical study (43).Since different localizations were obtained and the methods 2 used in the above studies broke up membranes or could have extra...

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.