Internodal myelin volume and axon surface area: A relationship determining myelin thickness?

Internodes from normal, remyelinated and regenerated nerve fibres have been isolated from rat spinal roots and sciatic nerve. The internodes have been examined quantitatively by light and electron microscopy to determine their internodal length, myelin thickness, and the circumference and cross-sectional area of both the axon and fibre. Comparison of these measurements of the axon and myelin sheath has revealed a close relationship between the volume of myelin comprising the internode and the area over which the Schwann cell and axon are in close proximity, i.e. the surface area of the axolemma beneath the internodal myelin sheath. The same relationship described not only the internodes on normal nerve fibres, where internodal length is proportional to axon diameter, but also the short and thinly myelinated internodes formed in the adult animal on remyelinated and on regenerated axons. Examination of data presented by Berthold (1978) revealed that a closely similar relationship is also present in feline nerve fibres. In view of the constancy of the relationship between such different types of internode it is suggested that the regulation of myelin volume, and thereby of myelin thickness, may be mediated via the area of the axolemma or of the Schwann cell membrane beneath the myelin sheath.     

Regenerative axonal sprouting in the cat trochlear nerve

Following peripheral trochlear nerve axotomy in the cat, the normal number of myelinated axons is restored despite significant motor neuron death, suggesting regulation of the number of myelinated axons in the regenerated nerve. In this study we used light and electron microscopy to examine the production and maintenance of axonal sprouts at different locations in the nerve and at different postoperative intervals. Despite proliferative sprouting and an overproduction of nonmyelinated axons in the regenerating trochlear nerve, the number of myelinated axons was strictly regulated. Only ～1,000 regenerated axons were eventually remyelinated, but many nonmyelinated axons were still present 6–8 months postaxotomy. Regenerated axons were remyelinated in a proximal-to-distal direction between 3 and 4 weeks postaxotomy. We also examined the maturation of regenerated myelinated axons by measuring axon diameter and myelin index (an expression of myelin thickness). Mean myelinated axon diameter remained significantly below normal in long-term regenerated nerves. Mean myelin index was not different from normal at 4 weeks postaxotomy but was significantly decreased at long postoperative intervals, reflecting a slightly thicker myelin sheath relative to the axon diameter. This relative increase in mean myehn thickness could serve to restore normal conduction velocity despite the decrease in mean axon diameter. We suggest that the regulation of the number of myelinated axons at the normal number despite cell death and the increase in mean myelin thickness may both be compensatory mechanisms that function to restore preoperative conditions and maximize functional recovery. © 1995 Wiley-Liss, Inc.     

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.     

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.     

Early neuron differentiation in the mouse of olfactory bulb. I. Light microscopy

The relation between the growth of axons and the development of their myelin sheaths was determined in rats for normal, myelinating sciatic nerves, and for an experimental model allowing to retard or accelerate axon growth. Axon caliber was measured, and sheath development was determined from measurements of thickness (light microscopy), from counts of the number of turns of myelin lamellae (electron micrographs), and from the rate of the incorporation of acetate-H³ into ether-ethanol extractable lipids. An excellent correlation between changes in sheath thickness and acetate-H³ incorporation was obtained for all experiments. For the myelinating nerves the changes in the rate of acetate-H³ incorporation were related specifically to the rate with which new length of myelin leaflet was added to the existing turns of sheath. The rate of axonal growth was manipulated by applying a snug ligature around the nerve by the fourteenth day, allowing the nerve to compress itself by its own growth. Most axons passed the constriction without interruption, but they were markedly hypoplastic distal to the constriction. After removal of the ligature these axons regrew to their normal caliber ranges. Examination of myelin sheath development in this model showed that retardation of axon growth retarded sheath growth, while acceleration of axon growth accelerated sheath growth. Thus, the rate of axon growth appeared to be the factor controlling the rate of myelin formation by the sheath cells. An appendix describes a model consisting of two interrelated feedback mechanisms by which expansion of the axon may directly control the number of turns of myelin lamellae formed by the sheath cell. The model correspons on all points to established features in the fine structure of fibers.     

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     

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.     

Quantitative examination of internodal length of remyelinated nerve fibres in the central nervous system

The internodal length of remyelinated internodes was examined by observations on teased CNS nerve fibres following primary demyelination induced by intraspinal injections of lysolecithin into the white matter of cats. A remyelinated internode was identified as a thinly-myelinated internode, where a node of Ranvier was bounded by a thickly-myelinated internode on one side and a less-thicklymyelinated internode on the other; as an internode of less than 100 μm in length or below 2 standard deviations from the normal regression line of internodal length against fibre diameter; as an internode joined to one of similar myelin sheath thickness which fulfilled either or both of the previous two criteria.Using the above criteria, remyelinated internodes were found to be shorter than normal; some were very short with no relationship to axon diameter while others were longer, falling within the predicted range for a given fibre diameter.This study illustrates that examination of teased CNS fibres in pathological situations can yield valuable information. However, sampling and technical difficulties are far greater than in comparable studies on peripheral nerve fibres.     

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.     

Specificity in central myelination: evidence for local regulation of myelin thickness

The ventral funiculi of normal and X-irradiated 13-day-old rats were studied by electron microscopy. In both tissues, oligodendrocytes form myelin sheaths around multiple axons, with a single oligodendrocyte associated with several axons of different sizes. Despite their origin from the same glial cell, the myelin sheaths are thicker for larger axons. Polyribosomes and rough endoplasmic reticulum are observed in distal oligodendrocyte processes, in proximity to the forming myelin sheaths. These results indicate that myelin sheath thickness is matched to axon size via local mechanisms, and suggest a role of polyribosomes and/or rough endoplasmic reticulum in myelin formation.     

Membrane flow within the myelin sheath in IDPN neuropathy

This report describes some aspects of beta,beta'-iminodipropionitrile (IDPN) neuropathy in rats as observed by ultrastructural methods and X-ray diffraction. Light microscopy shows gross swelling of the axons in proximal lumbar spinal roots 8 days after intraperitoneal injection of IDPN. Mean axon cross-sectional area and mean axon perimeter increased to 280% and 160% of their control values, respectively. At the same time, myelin membrane packing was not visibly disturbed. In addition, X-ray diffraction patterns, recorded under physiological conditions, demonstrate that the myelin lipid bilayer thickness and widths of the aqueous spaces between bilayers did not change. Related observations are made on posterior tibial nerve (PNS myelin) and ventral spinal cord (CNS myelin). The various observations together are interpreted in terms of a fluid myelin membrane. It is proposed that the myelin membrane flows during axon swelling even though normal membrane-membrane contacts are maintained within the sheath. Membrane flow and slippage between membranes are explained in terms of a molecular model of the myelin multilayer.     

Myelination, demyelination and re-myelination in the central nervous system

The myelin sheaths that surround axons in the CNS are made and maintained by oligodendrocytes. These glial cells can form variable numbers of myelin segments (internodules): from 1 to 200 so that when one oligodendrocyte is destroyed with preservation of the axon, many internodules can be lost, constituting a demyelinating process. As a consequence of the destruction of myelin and sheath cells a rapid and abundant cell response takes place. The response is made up by resident (microglia) and haematogenous phagocytes which phagocytose myelin and cellular debris leaving the axons demyelinated. Demyelinated axons may either stay demyelinated and clumped together or they may be separated by astrocytic processes, yet they can be remyelinated. The occurrence of remyelination depends upon the intensity and time of exposition to the demyelinating agent. Remyelination in the CNS with complete restoration of conduction may be made by oligodendrocytes or Schwann cells which invade the CNS when astrocytes are destroyed.     

Effects of undernutrition on myelination in rat corpus callosum

Rats were undernourished by halving the mother's food intake from the sixth day of pregnancy onwards and through lactation. Subsequently, the young animals were maintained up to 48 days on half the normal diet. The effects of this regime of treatment on myelination in the corpus callosum were investigated by light and electron microscopy. In comparison with controls, the percentage of axons myelinated at 15, 21, and 48 days was reduced in undernourished rats. The number of lamellae constituting the myelin sheath was also reduced at 15 and 21 days, but at 48 days no difference was seen between control and treated rats, suggesting that a catch-up in myelination had occured. A linear relationship between myelin sheath thickness and axonal diameter was observed in both groups of animals. However, a long-term effect on axonal growth was suggested by findings in the 48-day undernourished animals; in comparison with controls, axonal diameter was reduced, relative to myelin sheath thickness.     

Schwann cells and oligodendrocytes read distinct signals in establishing myelin sheath thickness

Schwann cells and oligodendrocytes produce myelin sheaths of widely varying sizes. How these cells determine the size of myelin sheath for a particular axon is incompletely understood. Axonal diameter has long been suspected to be a signal in this process. We have analyzed myelin sheath thickness in L5 lumbar root and spinal cord white matter of a series of mouse mutants with diminished axonal calibers resulting from a deficiency of neurofilaments (NFs). In the PNS, average axonal diameters were reduced by 20-37% in the NF mutants. Remarkably, the average myelin sheath thickness remained unchanged from control values, and regression analysis showed sheaths abnormally thick for a given size of axon. These data show that a genetically induced reduction in axonal caliber does not cause a reduction in myelin sheath thickness in PNS and indicate that Schwann cells read some intrinsic signal on axons that can be uncoupled from axonal diameter. Interestingly, myelin sheaths in the spinal cord of these animals were not abnormally thick, arguing that axonal diameter may contribute directly to the regulation of myelination in the CNS and that oligodendrocytes and Schwann cells use different cues to set myelin sheath thickness.     

A quantitative ultrastructural study of dorsal root regeneration

Regeneration in rat lumbo-sacral dorsal roots was studied 5–71 days following crush lesions. Wallerian degeneration occurred up to 20 days. At 11 days degenerating myelin was found in both Schwann cells and macrophages.Myelination was first observed 4 mm central to the crush at 7 days, and myelin became compact when the mesaxon exhibited 3.5 turns about the axon (about 11 days post-operatively). At 71 days, 69% of all fibres were myelinated, compared with 36% in normal roots. An example of 2 axons myelinating within the same Schwann cell occurred at 20 days.In normal roots curvilinear relationships were found between axon diameter and fibre diameter, myelin thickness and axon diameter, and between g and fibre diameter. In contrast, linear relationships between these parameters occurred in post-operative roots up to 71 days. Curvilinearity returned at 71 days. Alterations in the relationship between axon diameter and myelin thickness during regeneration indicated that myelin growth lagged behind axon growth throughout, but was more noticeable in larger calibre fibres. By 71 days, larger fibres exhibited disproportionately thin myelin, whilst small fibres possessed abnormally thick myelin compared to normal fibres of similar calibre.Regeneration was limited by axons failing to make successful central synaptic connections and by the poor metabolic response of dorsal root ganglion cells to sectioning of their central processes.     

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.     

Cation-binding sites in trigeminal ganglia and maxillary nerve: unusual reactivity of perikarya, stem axons and satellite cells

We have used the cupric/ferrocyanide reaction to study cation-binding in trigeminal ganglia and maxillary nerve of adult rats. Unmyelinated axons did not react, whereas myelinated axons were stained at nodal, paranodal or cleft sites. At 'nodal' sites, metallic deposits were found in the axoplasm, along the axolemma, and at the extracellular interfaces of the paranodal myelin. At 'paranodal' sites, particles were concentrated in the paranodal axoplasm and in the intracellular spaces of the myelin loops. Most maxillary axons examined at successive sites had all nodal or all paranodal staining, but 13 of 51 had a mixture. In trigeminal ganglia there was no staining of perineurial sheath, endoneurial cells or mast cells. Satellite cells and their basal laminae were prominently stained, with those around small neurons more reactive than those of large neurons. Patches of neuronal membrane on cell bodies were stained, more often for small than large neurons. The axon hillock and proximal stem axon were not stained in some cases, but approximately half the neurons had staining of perikaryal cytoplasm at the axon hillock or a dense asymmetric band in the proximal stem axon. Strong intraaxonal staining was found at the junction between unmyelinated proximal and myelinated distal stem axon. In distal stem axons, staining was found at the first myelin segment and at each successively thicker myelin segment; staining was mostly weak and paranodal, with intensity proportional to myelin thickness. The T-junction between stem and main myelinated axon had nodal or paranodal patterns; unmyelinated T-junctions were not stained. The varied cation-binding patterns in trigeminal ganglia show unusual properties of satellite cells and important differences between stem and main axons. The results that the cell membrane of axon hillock and proximal stem regions of many sensory large and small neurons may have numerous sodium channels and could affect signal propagation.     

How are sheath dimensions affected by axon caliber and internode length?

The significance of internode length for sheath thickness was analyzed by electron microscopic morphometry in isolated internodes from the human roots C3 and S1. These populations differ in length but have similar caliber.The amount of myelin per internode was in linear relation with product the product of axon circumference and the length of the ensheathed axon segment. Neither one of these two vectors was in a statistically significant relationship with sheath thickness. The ratio between the axolemmal area covered by the Schwann cell and the area of the myelin leaflet averaged 1:163 for human root fibers. It was 1:177 for previous data from canine sciatic nerve.The proportions of an internode were defined by an 1/d-quotient, expressingits length as multiples of axon diameter. Relative sheath thickness (g-ratio: diamter axon/diameter fiber) relates inversely with the 1/d-quotient. For a given axon caliber, the g-ratio (sheath thickness) decreases by 0.006 for every 10.0 increase in 1/d-quotient (relative internode length). Thus, internodes relatively long for axon caliber possess slightly thicker sheaths than internodes short for axon caliber. Axon caliber and relative internode length emerge as the two key factors determining the amount of myelin in a sheath.     

OBSERVATIONS ON REMYELINATION IN THE RABBIT SPINAL CORD FOLLOWING DEMYELINATION INDUCED BY LYSOLECITHIN

The extent and type of remyelination that occurred following demyelination induced by intraspinal injection of small volumes of lysolecithin was examined in 24 rabbits. Unlike the rat and cat following this procedure remyelination is not complete even after 6 months' survival and little Schwann cell invasion occurs. The cellular environment around remyelinated and persistently demyelinated axons differed. Remyelination was associated with a good astrocytic presence and removal of myelin debris, while persistently demyelinated axons were surrounded by myelin debris and a poor astrocytic presence. It was concluded that the environment around the demyelinated axon influences oligodendrocyte remyelination.     

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.