The precise geometry of large internodes

The interrelations, in each given internode, between internodal length, axon caliber, axoplasmic volume and several parameters of sheath thickness were studied in a set of 37 large internodes from the sciatic nerve of the German shepherd dog, using multiple electron-microscopic measurements of isolated fibers. Internodal length varied in a nonlinear relation with axon caliber. Nonlinear relations were also found for the number of lamellae in the sheath as related to either axon caliber or to internodal length, particularly for large internodes: the deviations observed for axon caliber (thick fibers had fewer myelin lamellae than would correspond to axon caliber) were the opposite of for internodal length (thick fibers had more lamellae than would correspond to internodal length). A near linear relationship was found if the volume of myelin per internode was related to both the length and the circumference of the axon segment ensheated by it, i.e. axolemmal surface area.The deviations in the proportions of large internodes probably indicate the existence of upper limits of internodal growth conditioned by the enormous metabolic demands placed on the individual Schwann cell by the growth of internodes beyond 1.5–2 mm length or 100 thousand μm³ of myelin, respectively. The demands on myelin metabolism from unabated internodal growth and the resultant logistic problems of the Schwann cells are demonstrated from measurement of observed internodes and calculations of extrapolated growth.     

A morphometric study of optic axons regenerated in a sciatic nerve graft of adult rats

PURPOSE: In the present study we have morphometrically examined a regeneration model in which axons normally residing in CNS have regrown and are interacting with Schwann cells from the PNS. This study will not only provide morphometric data on regenerated optic fibers but also shed light on possible factors in determining the fiber morphometry. METHODS: The optic nerves of rats aged 6 weeks were cut intra-orbitally and replaced with a autologous sciatic nerve. After a survival period of 9 months, the graft or "regenerated" nerves containing the regenerated optic axons and Schwann cells were processed for morphometric measurements. RESULTS: The mean myelinated axon diameter of regenerated nerve (1.8 +/- 0.2 micro m) was significantly (P < 0.05) greater than that of the optic nerve (0.9 +/- 0.03 micro m). However, unmyelinated regenerated optic axons had a smaller mean axon diameter (0.49 +/- 0.04 micro m) than normal myelinated optic axons. This may suggest that myelinating glial cells exert an influence on axon caliber and Schwann cells seem to have greater effect than oligodendrocytes. The mean g-ratio showing the relative myelin sheath thickness was found to be the highest in the optic nerve (0.78 +/- 0.003), least in the sciatic nerve (0.6 +/- 0.009) and intermediate in the regenerated nerve (0.68 +/- 0.01). The results indicated that Schwann cells myelinating the regenerated optic axons have produced a thinner myelin sheath. Intra-axonally, no significant difference was detected in the number of axonal microtubules and neurofilaments between the regenerated and optic nerves. Therefore the disposition of microtubules and neurofilaments into axon may be intrinsically determined. CONCLUSIONS: In this study, we have identified some of the extrinsic and intrinsic factors in determining the fiber morphometry of the regen-erated nerve. The axon-size and myelination by glial cells were determined through the external axon-glial interactions, whereas the number of axonal microtubules and neurofilaments were intrinsically determined.     

A quantitative electron microscopic study of myelination in the pyramidal tract of rat

The ultrastructure of fibers during myelin formation in the pyramidal tract of rats is described. The distribution of fiber classes based on counts of myelin lamellae was determined for newborn, young and mature rats. In newborn rats (2–12 days), growth of the axon was extremely rapid in fibers undergoing early myelination, resulting in greater variation in the relation between axon circumference and sheath thickness and, also, in the presence of myelin sheaths that were unusually thin in relation to the size of the axons. In young rats (12 days to 8 weeks), the numbers of myelin lamellae present in the sheaths increased in proportion to the increase in axon circumference. In adult rats, the numbers of myelin lamellae present in the sheaths was in linear relation to axon circumference for all sizes of myelinated fibers. Approximately 20% of the fibers were nonmyelinated. The number of glia cells per axon at the onset of myelination was approximately 20% of the adult ratio. During growth, myelination gliosis resulted in a steady increase in the number of glia cells per axon until adult levels were ultimately achieved. Our observations suggest that formation of myelin lamellae by oligodendroglia cells may be controlled by the caliber of the axon.     

Arrest of myelination and reduced axon growth when Schwann cells lack mTOR

In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.     

A new approach toward analyzing peripheral nerve fiber populations. II. Foreshortening of regenerated internodes corresponds to reduced sheath thickness

The new approach used in this study is based on the concept that axon caliber is not the only factor affecting the thickness of the myelin sheath. It is necessary to consider the entire geometric proportions of the internode, since sheath thickness corresponds to the relationship between axon caliber and the length of the internode. This type of analysis was applied to the regenerated internodes in rat sciatic nerves. Survival periods of 4, 9, 18 and 36 weeks were studied after lesions had been placed in young adult rats. The data show significantly thinner sheaths for regenerated fibers as compared with normal nerves, consistent with previous observations. This reduction in sheath thickness, however, corresponded quantitatively to the degree of foreshortening of internodes in the regenerated nerves. An average reduction of 10 in the quotient internode length/fiber caliber corresponded to a reduction of about 0.015 in the relative thickness of the sheath (quotient axon diameter/fiber diameter). This means that regenerated myelin sheaths are not truly hypoplastic; rather, they are adapted to the reduced internode length, and have the same relationship found for normal fibers. In partially damaged nerves there was a clear distinction in terms of sheath thickness between regenerated fibers and undamaged fibers. Demonstration of this phenomenon by scatter diagrams opens new possibilities for the quantitative assessment of neuropathies.     

Changes of the ratio between myelin thickness and axon diameter in human developing sural,femoral, ulnar,facial, and trochlear nerves

Summary Previous studies on sural nerves were extended to human femoral, ulnar, facial and trochlear nerves. An asynchronous development of axon diameter and myelin sheath thickness was noted in all nerves studied. Whereas axons reach their maximal diameter by or before 5 years of age, maximal myelin sheath thickness is not attained before 16–17 years of age, i.e., more than 10 years later. The slope of the regression lines for the ratio between axon diameter and myelin thickness is significantly steeper in older than in younger individuals; it also differs if small and large fibers with more or less than 50 myelin lamellae are evaluated separately. The number of Schmidt-Lanterman incisures during later stages of development is related to myelin thickness, but the length of the spiral of the myelin lamella, thought to unrolled, in relation to its width, i.e., internodal length, varies considerably during development. The changes of the relationship between axons and myelin sheath thickness during normal human development have to be taken into account if hypomyelination is considered as a significant pathological phenomenon in peripheral neuropathies, especially in children. The implications of the present findings concerning conduction velocity of peripheral nerve fibers and other electrophysiologic parameters are discussed.Supported in part by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Schr 195/5-7)Presented in part at the IXth International Congress of Neuropathology, September 5–10, 1982, in Vienna     

The effect of myelinating Schwann cells on axons

Myelinating Schwann cells control the number of neurofilaments and elevate the phosphorylation state of neurofilaments in the axon, eventually leading to the typical large axon caliber. Conversely, absence of myelin leads to lower amounts of neurofilaments, reduced phosphorylation levels, and smaller axon diameters. In addition, myelinating Schwann cells mediate the spacing of Na(+) channel clusters during development of the node of Ranvier. When axons are associated with mutant Schwann cells in inherited neuropathies, their calibers are reduced and their neurofilaments are less phosphorylated and more closely spaced. Also, axonal transport is reduced and axons degenerate at the distal ends of long nerves. Myelin-associated glycoprotein may mediate some aspects of Schwann cell-axon communication, but much remains to be learned about the molecular bases of Schwann cell-axon communication.     

Hereditary hypertrophic neuropathy in the Trembler mouse: Part 2. Histopathological studies: Electron microscopy

The Trembler mouse suffers from a dominantly inherited hypertrophic neuropathy. Electron microscopy, including a quantitative analysis of myelination was performed on the nerves of Trembler mice from birth to senility and compared with the findings in control mice. Axons in adult Trembler nerves were thinly myelinated and were surrounded by very few myelin lamellae which in turn were often uncompact circumferentially and longitudinally. Schwann cell cytoplasm was copious and had a normal content of organelles. Well-developed “onion-bulb” formations which consisted of thinly myelinated axons surrounded by empty membrane configurations were frequently seen.

The initiation of myelination was studied. The diameter distribution of promyelin fibres of control and Trembler sciatic nerve at ages day 2, 4, and 7 was calculated Myelination in Trembler nerves commenced on axons of larger diameters than controls.

The effectiveness of myelination was studied by relating the number of turns of myelin to the axon area of control and Trembler sciatic nerves from age 2 days to adult mice. At all ages Trembler axons were less well myelinated than controls and the difference was more pronounced with age.

Schwann cell activity was examined by relating the area of the Schwann cell cytoplasm to the area of the axon it invests. The relative amount of Schwann cell cytoplasm decreased progressively in control axons with age and as the axon became better myelinated. By contrast, that of Tremblers did not undergo a similar reduction as the animal matured and the relative amount of Schwann cell cytoplasm was markedly increased in adult Tremblers when compared with controls.

The periodicity of control and Trembler compact myelin was compared. The myelin period of Trembler mouse was significantly greater than that of controls. The defect in Trembler peripheral nerves was considered to be that of dysmyelinogenesis. The Schwann cell was active but ineffective in the synthesis, compaction and maintenance of myelin.     

Multiply myelinated axons in the optic nerve of mice deficient for the myelin-associated glycoprotein

We recently reported that some retinal ganglion cell axons in mice deficient for the myelin-associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon-to-fiber ratio. © 1995 Wiley-Liss, Inc.     

The corpus callosum during postnatal undernourishment and recovery: A morphometric analysis of myelin and axon relationships

This study was designed to compare morphometric relationships between myelin lamellae and axons in undernourished and well nourished developing rats, and in rats nutritionally rehabilitated for two weeks. Although sampling techniques employed in this study were not specifically designed to compare numbers of myelinated fibers in test and control populations, we did observe a trend indicating a reduction in the numbers of myelinated fibers. The mean numbers of myelin lamellae, from an average of all myelinated axons, were not different in control and test population. However, regression analysis of axon sizes by numbers of myelin lamellae revealed significant differences from the normal in 21-day-old undernourished rats. For callosal axons of any size, there were too few myelin lamellae in the undernourished rats. A partial recovery was observed in relatively small fibers by 35 days of age, but no recovery was observed in larger sized fibers. Comparison of the frequency distribution of axon circumferences of myelinated fibers revealed an increase in average axonal caliber. Computation shows that although mean numbers of lamellae were not altered by undernourishment, the axons themselves are increased in size by about 10%. This unexpected result indicates that the relationship normally governing the numbers of myelin lamellae is altered by postnatal nutritional deprivation, and that the relatively larger axon calibers do not produce in the ensheating oligodendroglia any compensatory increase in the layers of myelin.     

Effects of ethanol exposure on myelination and axon numbers in the L2 dorsal root of the neonatal rat.

Previous studies have demonstrated that exposure to ethanol during development delays the rate at which axons in certain central nervous system tracts acquire myelin. This delay appears to be related to an alteration in oligodendrocyte function and not to an aberrancy in axon size or number. The present study was designed to determine if alterations similar to those observed in the central nervous system also occur in peripheral nerves, specifically the L2 dorsal root. Dams were fed either an ethanol-containing or control liquid diet 2 weeks prior to pregnancy and throughout gestation. The pups born to the pregnant dams were artificially reared from postnatal day (PD) 4 to PD 10 on a similar ethanol-containing or control diet. The pups were sacrificed on PD 10, L2 dorsal roots removed and processed for electron microscopy. The numbers of axons in various states of myelination were quantified. No difference was observed in the number of unmyelinated axons in the L2 dorsal roots from ethanol-exposed and control pups. In roots from ethanol-exposed pups, there was a significant decrease in the number of axons possessing myelin arranged in compact lamellae, but a significant increase in the number of axons surrounded by myelin lamellae in which the Schwann cell cytoplasm had not yet been extruded (noncompact). However, when the number of axons possessing noncompact myelin and a compact myelin sheath were summed, no significant difference was observed. These data suggest that the delay in myelination following ethanol exposure may be a ubiquitous phenomenon throughout the nervous system.     

Relation between myelin sheath thickness, internode geometry, and sheath resistance

The thickness of the myelin sheath of nerve fibers was traditionally assessed solely as a function of axon caliber. Studies concerning the additional effect of variation in internode length are of relatively recent date. Carefully calibrated measurements of sheath thickness and internode geometry were used in this study to define an equation to predict the approximate number of lamellae from axon caliber and internode length, for normal and regenerated peripheral nerve fibers, and for fibers from hypomyelinated murine mutants. The definition of sheath thickness thus obtained was compared with different assumptions on the biophysical nature of myelin sheath resistance. The observed relations between sheath thickness and internode geometry were not compatible with an effective adjustment of sheath thickness to a radial flow of current across the sheath. Conversely, sheath thickness was found to vary in such a way that the resistance of the spiral path between the lamellae was matched precisely to axonal current density. The calculated resistance of the spiral leakage path, furthermore, was equal to measured sheath resistance. This new concept reconciles low sheath resistance with a high resistance of the myelin leaflet, yielding, at the same time, a fine tuning of sheath resistance to variations of internode geometry.     

SCHWANN CELL RESPONSES DURING REGENERATION AFTER ONE OR MORE CRUSH INJURIES TO MYELINATED NERVE FIBRES

The responses of Schwann cells during regeneration of myelinated nerve fibres were studied ultrastructurally in the distal segment of mouse phrenic nerve after a single or repeated localized crush injury. Chronological observations on nerves after a single crush confirmed the occurrence of myelination of only single regenerating axons among many that appeared in individual Büngner bands. The redundant axon sprouts often showed the structural features of degeneration and decreased in number with time. During the process, supernumerary Schwann cells not related to myelin formation were produced. They commonly failed not only to make a one-to-one relationship with an axon, but they also failed to acquire a new basal lamina of their own. With time, they showed shrinkage of their cytoplasm and became arranged circumferentially around the myelinating axon with unipolar or bipolar cytoplasmic processes. Electron microscopic, quantitative assessment of the nuclear population of Schwann cells following repeated crushes up to four times, clearly indicated a progressive and predominant increase in the number of the supernumerary Schwann cells with the number of crushes. Also, they were found to form separate concentric cytoplasmic lamellae around the myelinating axons, developing structures resembling onion-bulbs. It was concluded that essentially the same regenerating process as that observed after a single crush was repeated following re-crush, thereby resulting in the successive accumulation of supernumerary Schwann cells around a myelinating axon.     

Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: evidence for axonal sprouting

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 microm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system.     

Relation between the number of myelin lamellae and axon circumference in fibers of vagus and sciatic nerves of mice

Nerve fiber populations of the vagus and sciatic nerves of mice were classified according to the number of myelin lamellae present in the sheaths. This method for classifying fiber populations was superior to others used previously since it provided a more sensitive procedure for the analysis of individual fibers and better control over the technical factors involved in tissue processing. The relationship of the number of myelin lamellae in the sheath to axon circumference was found to be linear. In fresh tissue there was one myelin lamella for every 0.24 μ increase in axon circumference above da value of 2.32 μ (the mean circumference of an average-sized nonmyelinated fiber). A formula was proposed which may be useful for understanding how axons control myelin development and interpreting developmental stages, as well as for evaluating pathologic conditions affecting the peripheral nervous system. The critical diameter above which fibers were found to be myelinated was about 0.8 μ for fixed nerve and 1.1 μ for fresh nerve. The ratio of axon diameter to fiber diameter ranged between 0.5 and 0.9 and was not related to fiber size.     

The Schmidt-Lantermann incisures of the myelin sheath of Mauthner axons: site of longitudinal myelin growth]

The myelin sheath of the Mauthner axons in the spinal cord of young and adult fish belonging to the family Cyprinidae was examined by phase and electron microscopy. The sheath thickened considerably with age, the number of lamellae increasing from 200-230 in young fish (length 2--4 cm) to 250-300 in adult animals (length 20-25 cm). During this growth, the myelin sheath remained fairly compact and of optimal thickness for impulse propagation, the axon/fiber thickness ratio being 0.67-0.72 in young and 0.76-0.78 in adult fish. As already observed by previous investigators, the Mauthner axons lacked nodes of Ranvier. However, Schmidt-Lantermann's clefts (SLC) were present at irregular intervals. Within the SLC, parallel arranged osmiophilic bands of variable length and with a thickness of approx. 42-44 nm were observed to form a transitional zone between the cytoplasmic areas of the oligodendrocyte and the typical myelin structure. Between such adjacent 'D-bands', which thinned out to build up the major dense lines, an electron translucent area measuring approx. 25--26 nm in width contained a thin 'I-band' (8--10 nm) which was continuous with the intraperiod line. It is speculated that, in the Mauthner axon, the SLCs may be sites where freshly synthesized myelin is added to the lamellae already present, thus permitting their longitudinal growth.     

VARICOSITIES IN HUMAN FETAL SCIATIC NERVE FIBRES

A morphological study performed on sciatic nerves from 10 fetuses aged 19 to 32 weeks revealed variations in axonal diameter along the length of the fibres but a uniform myelin sheath thickness. This gave the fibres a beaded appearance. The diameter of the axon in the varicosities was up to seven-times greater than that of the intervening axon. The varicosities were separated by distances up to 50 microns. Both myelinated and single unmyelinated fibres had varicosities. Neurofilaments and neurotubules were more densely packed in the axons between the varicosities. The absolute number of filaments and tubules per axon was similar in axons with equal numbers of myelin lamellae but with different diameters, as calculated from transverse sections. The varicosities were observed in all fetuses aged 19 to 24 weeks, but in only one of the two fetuses aged 28 weeks. They were not present in the 32 week fetus. They appear to be a characteristic morphological feature of nerve fibres during early fetal life and can be identified only in teased fibre preparations or in longitudinal sections of the nerve. Their presence explains the bimodal or markedly skewed distribution of myelinated fibre axon diameters that was seen in nerves from young fetuses. It also helps in understanding the discrepancies reported in size patterns between axon diameter and myelin thickness. It is possible that the varicosities may be partially artefactual but their occurrence may imply a particular vulnerability of fetal nerve fibres. Their production may be related to movements of the axoplasmic fluid which is abundant in young fetuses.     

Axons regenerated through silicone tube splices. II. Functional morphology

The recovery of axons regenerated through silicone tube splices was studied with electron microscopic and morphometric methods. Regenerated nerves contained both myelinated and unmyelinated axons of near normal morphology. The number and diameter of axons increased with postoperative time, and size-frequency histograms demonstrated that regeneration occurred in all major fiber groups. Remyelination occurred between about 4 and 6 weeks. Some of the smallest regenerated axons had unusually thick myelin sheaths, but overall regenerated axons had a slightly thinner sheath compared with similar-size normal fibers, although the ratio of sheath thickness to axon size was within the normal limits of g = 0.65 to 0.8 by 6 weeks. Axons did not, however, regain their normal size within 10 months of surgery. This deficit was apparently the primary factor limiting conduction velocity in these regenerated axons.     

The growth and myelination of central and peripheral segments of ventral motoneurone axons. A quantitative ultrastructural study.

This study compares the growth and myelination of those parts of cervical ventral motoneurone axons in the spinal cord (the intramedullary segments) and in the ventral roots of fetal and young rats (up to 21 days postnatal). The same fibre bundles are examined centrally and peripherally. Myelination begins centrally and peripherally at about birth. However, the peripheral segments of some fibres may begin to become myelinated before the central. Over the first 3 weeks after birth the minimum circumference of peripheral segments of myelinated axons remains relatively constant at 3 mum but that of central segments falls from 2.5 mum to just over 1 mum. Axons within the same fibre bundles tend to be thinner and less heavily myelinated centrally than peripherally. With ageing, axon circumference becomes more strongly correlated with sheath thickness. The thickness of the sheath surrounding an axon of a given circumference does not differ statistically from one age to another or between central and peripheral segments. Studies of myelin sheath growth rate show that in the early stages glial and Schwann cells vary independently of one another in the rates at which they add new turns to sheaths around central and peripheral segments of axons in the same bundles.     

Morphometric effects of vincristine on nerve regeneration in the rat

Administration of vincristine (200, 100 or 50 micrograms/kg/week) for 6 months during regeneration of the sciatic nerve after crush injury caused a dose-dependent reduction in nerve fibre size and failure of removal of myelin debris. Successfully regenerating neurites showed an unusual amount of shape distortion. The ratio of myelin sheath thickness to axon circumference was reduced, but the ratio of myelin sheath thickness to axon area was normal. Microtubule concentration was diminished in axons, but neurofilament density was unaffected. Unmyelinated axons were reduced in number but their axon diameter distribution was not affected. Fibres on the non-crushed side appeared normal. The toxicity of vincristine to regenerating nerves is probably related to increased blood-nerve permeability occurring both at the site of crush and along the degenerating nerve.