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.     

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.     

Relative growth and maturation of axon size and myelin thickness in the tibial nerve of the rat

Summary Morphometric observations have been made on the medial plantar division of the tibial nerve (MPD) and on the motor branches of the tibial nerve to the calf muscles (MBC) in rats ranging in age from weaning (3 weeks) to 12 months. Axon size, assessed by measurements of circumference and cross-sectional area, increased rapidly until 3 months with further slight increases between 3 and 9 months and a slight fall between 9 and 12 months. Axon size distributions were unimodal throughout in the MPD but bimodal for the MBC except at 3 weeks. Distributions of myelin thickness were bimodal throughout for both nerves. Scatter plots of g ratios (axon diameter: total fibre diameter) confirmed the presence of two fibre populations: a group of small fibres with relatively thin myelin sheaths, and a group of larger fibres within which sheath thickness was relatively less on the larger than on the smaller axons. These two fibres populations were less easily separable in the MBC than in the MPD nerves. These results document morphometrically the normal growth changes in the rat tibial nerve and also provide control data for the analysis of the effects of experimental procedures on the growth and maturation of peripheral nerve fibres.Supported by an EEC Twinning Grant and by the Nuffield Foundation     

Early and preventable changes of peripheral nerve structure and function in insulin-deficient diabetic rats.

Structure and function of peripheral nerve in early insulin deficient streptozotocin diabetic rat were examined. Quantitative studies of myelinated fibres from electron micrographs showed that the fibre calibre and slope coefficients of regression lines between axon and myelin areas were reduced and quantitation of unmyelinated fibres revealed a 40% reduction in area fraction of smooth endoplasmic reticulum. The difference in conduction velocity between diabetic rats and control rats increased with time just as does the difference in fibre calibre. Insulin treatment was able to prevent the decrease in conduction velocity as well as the reduction in fibre calibre. It is concluded that the early structural axon abnormalities results from the diabetic state and cannot be explained by poor general condition of the animals.     

Electrophysiological aspects of peripheral nervous system development]

Different aspects of the maturation of the peripheral nervous system have been studied in infants, essentially during the first year of postnatal life. Absolute refractory periods which enable indirect estimation of excitability have been measured in both nerve and muscle fibers. It appears that already at birth, the absolute refractory period is characteristic for a given group of nerve fibres. In each group of nerve fibres, the absolute refractory period is not correlated to conduction velocity (ie fibre diameter) and remains rather constant during development. However, the absolute refractory period of the most excitable fibres is smaller than the absolute refractory period of motor fibres independent of the subject's age. On the other hand, the absolute refractory period of muscle fibres is slightly higher in premature in comparison with full-term neonates and adults. The diameter growth of different categories of nerve, fibers (motor--IA--non nociceptive cutaneous) is assessed by measurement of conduction velocities. At each age the IA fibre conduction velocities are the highest. The marked difference which exists between length development and postnatal increase of the fibre diameter explains the particular evolution of conduction times.     

Peripheral nerve abnormalities related to galactose administration in rats.

Electrophysiological, biochemical, and morphometric observations were made on the peripheral nerves of rats after galactose feeding. Motor nerve conduction velocity was found to be reduced. This was associated with an accumulation of galactitol in the peripheral nerves and a diminution in their myoinositol content. An increased water content and fascicular area, taken in conjunction with a probable increase in the area of the endoneurial spaces, indicated overhydration of the peripheral nerves. Morphometric observations on the myelinated fibre population in the tibial nerve showed no loss of fibres and although both the maximal and the average diameter of the myelinated fibres was slightly less than in age-matched controls, this was insufficient to explain the reduction in conduction velocity. Segmental demyelination was not detected and the relationship between myelin thickness and axon circumference was not altered. Electron microscope observations revealed no ultrastructural changes in the myelinated fibres and, in particular, no abnormalities at the nodes of Ranvier or indication of abnormal hydration of the Schwann cells. The relevance of these findings to the peripheral nerve changes in human and experimental diabetes is discussed.     

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.     

A useful programme in BASIC for axonal morphometry with introduction of new cytoskeletal parameters.

Interest in the structure of axons and quantification of their components has been growing over the last years. However, the existing literature contains few reports of available computer programmes to facilitate such studies. This paper presents a fully comprehensive BASIC programme for the morphometric analysis of electron micrographs of cross-sectional nerve fibres. From drawings of fibre and axonal contours and dots of the microtubules and neurofilaments, the programme calculates the following parameters: area, diameter and form factor of the fibres and axons, number and density of microtubules and neurofilaments, proportion between microtubules and neurofilaments (R-proportion), myelin thickness and the diameter of the axon relative to its sheath (g-ratio). The programme also introduces three new parameters to analyse the degree of uniformity of microtubule and neurofilament distribution: distances between microtubules and between neurofilaments, equilateral index and cytoskeletal intermingling index. The programme is written in Microsoft BASIC Interpreter for Apple Macintosh (Microsoft Corporation) but can be used on other computers. Although the programme has been tested on adult rat optic nerve fibres, it can be used for different projects concerning axonal morphometry.     

An electronmicroscopic analysis of the optic nerve in the golden hamster.

The electronmicroscopic examination of sections taken from the hamster's optic nerve 5 mm behind the globe indicated that the nerve contains 110,165 +/- 4,177 (p less than 0.05) fibres of which 96.4% are myelinated. The fibre diameter distribution is unimodal with a peak at 1.2 micrometer and axon diameters ranging from 0.20 micrometer to 3.93 micrometer. Fibres of all sizes are distributed uniformly throughout the cross section of the nerve. The thickness of the myelin sheath surrounding a given axon is highly (0.80) correlated with axonal diameter and the degree of myelination for a fibre of a given size is nearly constant throughout the nerve's cross section. In nerve sections taken just posterior to the globe most (64%) of the fibres counted are unmyelinated and the percentage of unmyelinated axons is highest near the peripheral boundary of the nerve. The process of myelination is essentially complete in sections taken 3.5 mm behind the eye. These differences in the myelination of the proximal and distal nerve most probably account for the discrepancy between the results reported here and those provided by a previous study (Tiao and Blakemore, '76) concerned with the structure of the optic nerve in this species.     

Myelin sheath thickness and conduction latency groups in the cat optic nerve

In many animals, the optic nerve has multiple conduction latency groups of fibres yet displays a unimodal frequency distribution of fibre sizes. An attempt has been made to resolve this discrepancy in the cat. Examination of teased preparations of fixed cat optic nerve fibres by polarizing interference light microscopy indicated that a fiber's size may change abruptly along its length. Furthermore, the diameters of single nerve fibres followed for distances of up to 30 μm in serial transverse sections of cat optic nerve showed variations of up to 100% of a fibre's average diameter over that distance. On the other hand, the myelin sheath thickness of these fibres appeared to be relatively constant along any one fibres. A population of cat optic nerve fibres which had a unimodal axon diameter frequency distribution was found to have a myelin sheath thickness frequency distribution with five modes. These modes have been interpreted as indicating the existence of five groups of fibres in the cat optic nerve. Four of these groups may be related to four negative peaks seen in the antidromic compound action potential recorded at the margin of the cat optic disc following stimulation of the optic chiasm. The fifth myelin thickness group appears to represent the slowly conducting fibres which do not make an obvious contribution to the antidromic compound action potential. It is concluded that for the cat optic nerve, the conclusion latency groups seen in the compound action potential may be more directly correlated with the frequency distribution of fixed nerve myelin thickness than with the frequency distribution of fixed nerve fibre diameter.     

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.     

Glycosaminoglycan Supplementation Promotes Nerve Regeneration and Muscle Reinnervation

This study shows that treatment of rats with exogenous glycosaminoglycans stimulates peripheral nerve regeneration, increases the abundance of mRNAs for myelin proteins and promotes muscle reinnervation. After the sciatic nerve had been crushed the number of regenerating axons in the distal stump was markedly and highly significantly increased by glycosaminoglycan treatment throughout the experimental period. The increased number of axons was correlated with increased axon and fibre (axon + myelin) diameter. The abundance of mRNAs for P_o protein and myelin basic protein of regenerating nerves was also affected by treatment with glycosaminoglycans. The increase in mRNA was also observed in the contralateral unlesioned nerve. Such a phenomenon did not occur in saline-treated rats. Glycosaminoglycan treatment markedly increased the number of muscle fibres reinnervated and accelerated the restoration of muscle twitch tension elicited by nerve stimulation. The effect was particularly evident during the early stages (16 and 21 days after nerve crush) of muscle reinnervation.     

The optic nerve of the brush-tailed possum, Trichosurus vulpecula: fibre diameter spectrum and conduction latency groups

The principal findings of this report on the morphology and electrophysiology of the possum optic nerve are: (i) There are about 230,000 fibres in the optic nerve. This fibre count, based on electron microscopy, is slightly less than a previously reported estimate of the total number of ganglion cells in the possum retina. (ii) The majority (greater than 98%) of the fibres of the optic nerve are myelinated axons of retinal ganglion cells. The diameters of these fibres range from 0.4--4.6 micrometer (axon diameter range: 0.3--3.8 micrometer) and the frequency distribution of the fibre diameters (and axon diameters) is positively skewed and unimodal. (iii) The antidromic compound action potential of the possum optic nerve shows four negative peaks following stimulation of the optic chiasm. These peaks are associated with four conduction latency groups of fibres which have been designated t1, t2, t3 and t4 in order of increasing conduction latency. (iv) The mean peak conduction velocities of the fibres in the conduction latency groups are 13.1 ms-1 (t1), 8.1 ms-1 (t2), 5.7 ms-1 (t3) and 3.1 ms-1 (t4). (v) There is no direct correlation between the frequency distribution of fibre (or axon) diameters as measured by electron microscopy of transverse sections of fixed optic nerve and the conduction latency groups. (vi) The reconstruction of the possum optic nerve compund action potential on the basis of either axon or fibre diameter frequency distribution does not provide an acceptable, indirect correlation between the morphology and the electrophysiology of this optic nerve.     

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.     

Myelin-axon relationships in the rat phrenic nerve: longitudinal variation and lateral asymmetry.

It is known that the myelin sheath thickness-axon perimeter relationship varies between peripheral nerves. This study examines the possibility that that relationship may vary between levels along a given nerve or between corresponding levels of the right and left examples of the same nerve. The relationship is examined for large and small fibre classes at well separated upper and lower intrathoracic levels in the rat phrenic nerve. The study shows that the myelin-axon relationship differs between levels along the same nerve bundle in the same (intrathoracic) environment. Thus, for a given increase in the perimeter of large axons, sheath thickness increases significantly more at lower than at upper levels. In addition, myelin sheath thickness shows a statistically significant lateral asymmetry in favour of the left side for the large fibre class at the upper thoracic level. The setting of the myelin sheath thickness-axon perimeter relationship also differs between the large and small fibre classes at each level examined. Large fibres have proportionately thicker sheaths than small fibres and this difference is reflected in the significantly smaller g-ratio of the former. Systematic differences in the setting of the myelin sheath thickness-axon perimeter relationship between large and small fibre classes may be a widely occurring phenomenon. It may be concluded that the myelin-axon relationship varies significantly both within and between nerves and also between fibre classes. Accordingly, morphometric studies of normal or pathological nerves should take into account possible consistent longitudinal variation or lateral asymmetry in fibre parameters and myelin-axon relationships within a given nerve bundle or fibre class, in order to avoid introducing systematic bias and to minimize variance between samples.     

Impulse conduction in multiple sclerosis: a theoretical basis for modification by temperature and pharmacological agents

The simplest model for explaining conduction defects in multiple sclerosis (MS) and other demyelinating diseases assumes that the only abnormality present is loss of myelin. The consequences of such an assumption have been investigated by numerical solution of a well-known set of differential equations describing conduction in a model demyelinated axon. In agreement with clinical findings, we show that this model predicts that the temperature at which conduction block occurs is a steep function of the extent of demyelination, so that small temperature increases may block large numbers of conducting fibres. Decreasing calcium concentration (or increasing pH) is calculated markedly to improve the conduction velocity of conducting demyelinated fibres and will, in addition, restore conduction in blocked fibres. The effects of other pharmacological agents have also been computed. The presence of a demyelinating lesion in a nerve fibre is shown greatly to impair the ability of the fibre to conduct repetitive impulses, conduction failing at much lower frequencies than in normal fibres. These calculations provide some insight into the nature of conduction defects in demyelinated nerve, demonstrate that many clinical features of MS are the expected consequence of loss of myelin and do not require the presence of other defects for their explanation, and provide a useful approach to the search for a symptomatic therapy.     

Quantitative studies on myelinated and unmyelinated nerve fibres in the interatrial septal region of aged rat hearts

Intracardiac nerve fibres from the interatrial septum were studied quantitatively and qualitatively by electron microscopy of transversely sectioned nerve bundles in male Wistar rats of 4 and 24 months. No significant changes were found in the myelinated fibre diameters, myelinated axon diameters, myelin sheath thicknesses, g ratios, myelinated fibre areas, unmyelinated axon diameters and unmyelinated axon areas. However, there was evidence of structural changes to the nerve fibres and Schwann cells at 4 and 24 months, increasing in prevalence with age: some myelinated fibres showed infolds, disruptions and clefts of the myelin sheath and accumulation of electron dense myelin-like fragments in the axoplasm. Unmyelinated axons showed fewer changes in structure but also contained similar fragments in the axoplasm. The numbers of neurotubules and neurofilaments per microm2 in unmyelinated intracardiac axons was significantly greater than in those in samples of the cervical vagal trunk. This may be an adaptation to the continuous mechanical stress experienced by these intracardiac nerves. It is concluded that there is little structural evidence to suggest that the conductive properties of intracardiac nerve fibres are adversely affected in aged rats.     

Conduction velocity and spike configuration in myelinated fibres: computed dependence on internode distance.

It has been argued theoretically and confirmed experimentally that conduction velocity (theta) should be proportional to nerve fibre diameter for myelinated fibre tracts, such as normal peripheral nerve, exhibiting 'structural' similarity'. In some axons, however, the nodes of Ranvier are more closely spaced than in normal peripheral nerve. Analytic arguments have suggested that when internodal distance (L) alone is changed, the plot of theta versus L should have a relatively flat maximum. This was confirmed by several previous computer simulations of myelinated axons, but internode lengths of less than half the normal case were not examined. In order to gain insight into impulse propagation in myelinated and remyelinated fibres with short internodal lengths, the present study examines the conduction velocity and spike configuration for a wide range of internodal lengths. As L becomes large, theta falls and finally propagation is blocked; as L becomes small, theta decreases more and more steeply. From this, it is predicted that for fibres with very short internodal lengths, small local changes in L should affect substantially the conduction velocity.     

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.