The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves.

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.

Neurotoxicity of topically applied hexachlorophene in the young rat.

Young rats 6 to 22 days of age are extremely susceptible to the neurotoxic effects of hexachlorophene given as a daily bath of undiluted antiseptic detergent containing 3% hexachlorophene (pHiso-Hex). At this age, most rats are clinically and histologically damaged by as few as two daily baths. Younger rats are relatively resistant, probably because they have less myelin to be affected; older rats cannot be poisoned by this route, probably because the more mature liver excretes the drug more effectively. Age-dose-response curves in rats are similar to those in humans. This experimental model is potentially useful in defining other characteristics of this drug.

Immunocytochemical demonstration of glial-neuronal interactions and myelinogenesis in subcultures of rat brain cells.

Subcultures have been established from primary rat brain cell cultures and have been characterised with a range of cell-specific immunocytochemical markers. The subcultures are mainly composed of fibrous astrocytes, oligodendrocytes and neurones. The cells do not divide to any great extent giving a system where it is possible to follow culture development at the cellular level for a number of weeks. During this time oligodendrocytes colonise subpopulations of neurones, differentiate further showing the presence of myelin basic protein and elaborate myelin-like membrane; the fibrous astrocytes remain scattered uniformly throughout the cultures. Radially oriented processes emerge from the oligodendrocyte-neurone aggregates which subsequently coalesce to form fascicles that link the clusters of cells together. These fascicles react with antibodies for both neurofilament protein and myelin basic protein. The subcultures provide a straightforward system that is composed of cells derived entirely from the CNS, is free from mitotic inhibitors and yet retains a sufficiently low cell density to allow immunocytochemical identification of the cell types present. The subcultures should be useful for the study of trophic interactions between oligodendrocytes and neurones as well as the early events associated with myelinogenesis.

Appearance of myelin proteins in rat sciatic nerve during development.

Rats between 5 and 45 days of age were sacrificed and their sciatic nerves dissected. Myelin was prepared from these sciatic nerves by a procedure involving purification on discontinuous sucrose gradients. The proteins of whole sciatic nerves at different ages and the proteins derived from the myelin isolated from these sciatic nerves were examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Over half of the proteins of sciatic nerve myelin migrated in a single band on the gel (P0). There were only minor changes in the protein distribution of sciatic nerve mylein during development. In contrast, the polyacrylamide gel patterns of whole sciatic nerve homogenate changed markedly during development between 5 and 15 days of age. The amount of P0 protein as a proportion of the total sciatic nerve protein increased from 3% at 5 days of age to 13% at 15 days of age after which it remained constant. Several other proteins which were also characteristic of the isolated myelin increased in relative importance during this time period. Parallel experiments dealing with a metabolic parameter of myelinogenesis, incorporation of intraperitoneally injected [35S]sulfate into sulfatide, were conducted. The maximum synthesis of sulfatide occurred between 6 and 16 days of age, coincident with the marked accumulation of myelin proteins in sciatic nerve.

Change in a myelin-associated glycoprotein in rat brain during development: metabolic aspects.

The higher apparent molecular weight of the newly synthesized glycoprotein in immature rat myelin in comparison with that in mature myelin, which was originally demonstrated with radioactive fucose, was also shown by double labeling experiments with radioactive glucosamine or N-acetylmannosamine. In addition, periodic acid-Schiff staining of gels, on which the glycoproteins of mature and immature myelin were electrophoresed separately or mixed together, revealed that the stained glycoprotein in immature myelin had a higher apparent molecular weight. Age studies with radioactive fucose showed that the greatest difference in the apparent molecular weight of the newly synthesized glycoprotein occurred at 12 days. The magnitude of the difference decreased gradually with age until 25 days when the glycoprotein electrophoresed to the same position as that in more mature myelin. Long-term experiments, in which 12- or 13-day-old rats were injected with radioactive fucose and allowed to survive for varying periods of time before myelin isolation, also revealed a gradual decrease with age in the extent of the shift of the glycoprotein toward a higher molecular weight. The possible significance of the developmental change in the glycoprotein for the process of myelin formation is discussed.

MRI of normal brain maturation.

The unprecedented gray/white differentiation obtained with magnetic resonance imaging (MRI) has created a unique opportunity to trace the normal process of myelination. Fifty-nine children referred for evaluation of a nonneurologic problem or a nonspecific neurologic complaint were studied with MRI using spin-echo technique. Children ranged in age from term (40 weeks intrauterine) to 16 years. Scans were reviewed for quantitative and qualitative changes with age. T1 and T2 relaxation times were measured for 13 regions of interest in 37 children. With increasing age a sharp decrease in both T1 and T2 values, most pronounced during the first year of life, was seen. The prolonged relaxation times in the newborn infant correspond to the known high water content of the neonatal brain; the subsequent decline corresponds to the decrease in water content and increase in myelination observed in autopsy studies of infants. Qualitative changes in the MRI appearance of the brain with age using a spin-echo sequence (2 sec repetition time) demonstrated that the process of myelination was most rapid during the first 2-3 years of life. Myelination appeared to occur earliest in the posterior fossa, with the middle cerebellar peduncle identifiable at only 3 months. By the age of 1 year, all major white matter tracts including the corpus callosum, subcortical white matter, and the internal capsule were well defined. However, due to subtle changes in appearance, the refined configuration of the adult brain was not attained until early adolescence.

Changing relation between onset of myelination and axon diameter range in developing feline white matter.

The size spectra of unmyelinated, ensheathed and initially myelinating CNS axons were examined by electron microscopy in the spinal cord ventral funiculus and the corpus callosum of the cat during development. The first myelin sheaths appeared 4 weeks before and 3 weeks after birth in the spinal and callosal areas, respectively. De novo myelination had largely ceased by 4 months in the ventral funiculus and by 7 months in the corpus callosum. The result show that the diameter ranges, within which spinal and callosal axons undergo primary ensheathment and initial myelination, are markedly different if similar levels of myelination are compared. In both areas, these diameter ranges shift towards smaller sizes with development. However, spinal and callosal axons, which undergo primary ensheathment and initial myelination simultaneously, present comparable diameter ranges. The findings support the view that other factors than the absolute physical size of the axon trigger initiation of CNS myelination. In this respect the developmental stage of the animal appears to play an important role.

Postnatal delay of myelin formation in brains from Down syndrome infants and children.

Myelination up to the age of 6 years was studied in two groups consisting of 129 Down syndrome (DS) 17/129 fetuses and 112/129 postnatal) and 73 non-DS cases (10/73 fetuses and 63/73 postnatal). In both groups studied a similar number of congenital heart disease (CHD), gastrointestinal (GI) malformations and infections were diagnosed. Paraffin or celloidin brain sections were stained with Klüver-Barrera, Heidenhain or Loyez method. The myelination was found to be delayed in 29/129 (22.5%) DS and only in 5/73 (6.8%) non-DS cases. Myelination in fetuses and newborns in the DS and non-DS groups was not delayed. In DS the myelination was delayed between ages 2 months-6 years (17/29; 58.6%) up to 12 months, and 12/29 (41.4%) aged 2-6 years, while in non-DS aged 2-6 months only. The myelination delay affected tracts with late beginning and slow cycle of myelination, mainly the associated and intercortical fibers of the fronto-temporal lobes. In 3/7 of DS cases (ages 3, 4, 6 years) less advanced myelination of U fibers was noted. In both groups the myelination delay seemed also to depend on the systemic diseases which affected the subjects during the time of myelination. In DS where CHD was present the myelination delay was found in 14/29 (48.2%) in contrast to 3/5 (60%) non-DS. Also, in 28/129 (23%) DS cases dates regarding the developmental milestones were available and some correlation was found between developmental and myelination delay.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal myelin maturation in vitro: the role of cerebrosides.

Myelinating neonatal rat cerebellar explants were maintained for up to 130 days in vitro. Myelin fractions were extracted from explants of different ages and purified by density gradient centrifugation. Three fractions obtained were termed "light myelin", "heavy myelin" and "membrane fraction" and were deficient in glycolipids compared to myelin fractions prepared from 15-day-old rat cerebellum. The culture myelin has an apparently normal ultrastructure but may not be as stable as myelin with a normal glycolipid composition.

A search for the "multiple sclerosis virus"--lack of effect of brain extracts on myelin development in chickens, mice and rats.

Attempts were made to transmit possible infectious agents from tissue of MS patients into three animal species, under conditions designed to enhance the development of such an agent. Myelination was monitored by measuring CNPase activity and myelin basic protein. Although no significant effects were observed, the usefulness of a new assay for CNPase was demonstrated. Nude mice were found to have a lower level of CNPase than their heterozygous littermates.

Conformation of myelin basic protein and its role in myelin formation.

High resolution 13C and 1H NMR spectra of myelin basic protein over a range of pH and concentrations indicate that intramolecular folding of the polypeptide chain occurs in aqueous solution in the region of residues 85 to 116. At pH 4 in D2O solution, the 13C resonances due to nonprotonated carbons of phenylalanine and tryptophan are broadened and chemically shifted compared to the same resonances when the protein is dissolved in 6M guanidinium hydrochloride. These residues occur in the region of the polypeptide chain in which the intramolecular folding may occur. As the pH is raised and the positive charge on the protein reduced from 28 to 18, intermolecular aggregation occurs, which appears to involve these same folded regions. Data on T1 (longitudinal relaxation times) of protons indicate also that amino-acid sidechains vary considerably in their motional freedoms. The concentration dependence of the proton NMR spectra provides further information on association of protein monomers. The region of the protein involved in folding, polymerization and substrate specificities is conservative in various species and we can surmise that it may have a specialized role in protein-lipid interactions in the myelin membrane. We suggest that the protein forms dimers across the cytoplasmic apposition during the formation of myelin. Estimates of the repulsive energies of interaction between approaching membranes suggest that some special mechanism of this kind is required to overcome the repulsive forces due to breakdown of water structure and electrostatic interaction.

Retardation of brain myelination by malnourishment and feeding low protein irradiated diet in rats.

The effect of feeding low protein diet, and the low protein irradiated diet on the deposition of myelin in the brains of rats over two generations showed that malnourishment lowered the CNP enzyme activity when compared to the normal (high protein) diet fed control rats during early postnatal period. Irradiated low protein diet, when fed within 21 days of irradiaton (15 Krads Gamma radiation, 60Co rays), produced still lower CNP enzyme activity. This also enhanced other effects of retarding the brain development, by lowering the protein and lipid contents of the brain during a period from the 4th to 25th day of postnatal development. This suggests the possibility that feeding irradiated low protein food to malnourished developing mammals could cause serious problems like mental retardation.

Biosynthesis of myelin and neurotoxic factors in the serum of multiple sclerosis patients.

The in vitro synthesis of myelin proteins has been studied by measuring the incorporation of [3H] lysine in developing rat brain slices. This incorporation system has been used to assay potentially gliotoxic and myelinolytic agents. A reduced incorporation of the labelled amino acid into myelin proteins occurs in the presence of anti-myelin anti-serum and anti-basic protein anti-serum. Diphtheria toxin has been found to inhibit the synthesis of myelin basic and proteolipid protein in the white matter slices of developing rats. Recent experiments with serum samples from multiple sclerosis patients in exacerbation suggest the presence of a factor which interferes with the synthesis of myelin in white matter slices.

Lipid and fatty acid composition of human cerebral myelin during development.

A development study of major lipid fatty acids in human brain myelin was undertaken and compared to those in cerebral white matter of the same region. The myelin was isolated from 23 subjects at ages from newborn to old age. The proportions of cholesterol and galactolipids increased in myelin during the first 6 months of age and up to 2 years of age in cerebral white matter. During the same periods the individual phospholipids also showed marked variations. Serine phosphoglycerides and especially sphingomyelins increased, and choline phosphoglycerides decreased. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and sphingomyelins underwent the largest maturation changes. The proportions of saturated fatty acids in EPG diminished rapidly with a corresponding increase of monoenoic acids. Fatty acids of the linoleic acid series showed a peak between 4 months and 12 months of age, and then their proportion slowly diminished to old age. The fatty acid changes in serine phosphoglycerides were much less pronounced than in EPG but of similar type. In sphingomyelin the proportion of saturated long-chain fatty acids diminished while the proportion of monoenoic acids increased--this increase continued at least to the age of 15 years. The same fatty acid changes occurred in cerebrosides and sulfatides as in the sphingomyelins, but they were less pronounced. The fatty acid changes during development were much more pronounced in white matter than in myelin but already from 1-2 years of age the lipids of myelin and white matter had the same patterns--in the galactolipids from 2 months of age. The individual variations of the lipid fatty acid patterns were small except for at the youngest ages and the variations found for this period might depend on the difficulties in determining the gestational age.

Studies of isolated, maintained oligodendroglia: biochemistry, metabolism, and in vitro myelin synthesis.

Oligodendroglia can be isolated in bulk from dissected white matter of lamb or bovine brain. Studies of the composition of the whole cell and of glial subfractions were performed to detect similarities to mature myelin. With care isolated oligodendroglia can now be maintained in culture for three to four days. While in culture the cells elaborate a form of glial myelin which has characteristics of both the intact cell and of mature myelin. Glial myelin reacts with both the antiserum to oligodendroglial surface components and with antiserum to galacto-cerebroside; it increases in amount with time in culture; if various radiolabeled substrates are added to the cells in culture, the glial myelin has both lipids and proteins which are extensively radiolabeled; the glial myelin has both basic protein and 2',3'-cyclic AMPase associated with it. Thus this model system may be an excellent system for studying myelin assembly in vitro.

Myelin deficiency in experimental phenylketonuria: contribution of the aromatic acid metabolites of phenylalanine.

Retarded body and brain growth and a deficit of myelin in the cerebral hemispheres and the cerebellum were observed in an animal model of phenylketonuria, the p-chlorophenylalanine and L-phenylalanine treated preweanling rat. These manifestations of phenylketonuria were reproduced in rats treated with phenylacetate in amounts approximating those likely to be produced in phenylketonuria. Young rats treated with equivalent amounts of other metabolites of phenylalanine, namely, phenylpyruvate, phenyllactate, and mandelate, which also accumulate in the brain during hyperphenylalaninemia, did not exhibit any toxic effects. Phenylpyruvate did not give rise to phenylacetate in the brain, but a small percentage was converted to phenyllactate. The gross composition of myelin isolated from the brains of saline and phenylacetate treated animals was similar. At various time intervals after subcutaneous injection, phenylacetate in the brain reached levels thirty times those of phenylpyruvate and phenyllactate, although animals received equivalent amounts of the three metabolites. The retarded growth of the body and brain of the young animal treated with phenylacetate may be attributed to the formation of phenylacetylcoenzyme A in the tissues. The site of action is very likely linked to acylcoenzyme A metabolism, i.e., the synthesis and utilization of acetylCoA and acetoacetylCoA, which are involved in reactions generating ATP and energy and in the synthesis of cholesterol and fatty acids. Results of this investigation indicate that growth retardation induced by phenylacetate during the period of very rapid development of the brain is responsible for the mental retardation in phenylketonuria.

Impaired Myelination in rats given excess vitamin A postnatally.

The effect of postnatal administration of 1000 I.U. of Vitamin A on 4th, 6th, 8th, and 10th day of age to rat pups has been studied on brain myelin lipid and sulphatide synthesis from Na235SO4. Administration of vitamin A reduced brain weight, free cholesterol, phosphatidal ethanolamine and the synthesis of myelin sulphatides from Na235SO4.