Peripheral nerve structure and function in experimental diabetes

Observations have been made on the peripheral nerves of rats in which diabetes had been induced by streptozotocin or alloxan. Motor nerve conduction velocity was found to become reduced, the reduction developing within a few days in severely diabetic animals. Conduction velocity remained diminished during survival times of up to 1 year.Histometric studies of the myelinated fibre population of the sural and tibial nerves showed no loss of fibres or reduction in their calibre. Evidence of segmental demyelination was not detected and myelin/axon ratios were normal. Cation binding at the nodes of Ranvier was unaltered. No explanation for the reduced nerve conduction velocity in morphological terms was therefore obtained.Observations on the abdominal vagus nerve revealed no loss of unmyelinated axons or reduction in their calibre. Measurements of Schwann cell and endoneurial capillary basal laminal thickness showed no differences between diabetic and control animals. Ultrastructural examination of other features of the peripheral nerves failed to define any pathological alterations in the diabetic animals. It is therefore concluded that the extrapolation of biochemical findings in experimental diabetes in an attempt to explain the origin of human diabetic neuropathy, where there are associated structural changes, should be made with caution.     

The relationship between axon diameter, myelin thickness and conduction velocity during atrophy of mammalian peripheral nerves

The atrophy of cutaneous (sural) and muscle (medial gastrocnemius) nerves proximal to a ligation were studied in cats for periods up to 9 months, using light and electron microscopy, conduction velocity measurements and computer simulations. As atrophy proceeds, nerve fibres become increasingly non-circular. Cross-sectional areas of axons and fibres (axon + myelin) were measured. The diameters of equivalent circles (having the same axon and fibre cross-sectional area) were then calculated. A linear relation was found between axon diameter and fibre diameter, but the slope decreased as atrophy continued. This indicates that the axon cross-sectional area decreases relatively more than the total fibre area. Reduction in conduction velocity correlates more closely with reduction in axon diameter than fibre (axon + myelin) diameter.The ratio of the inner (axon) perimeter to the outer (myelin) perimeter remains constant at or near the optimal value of 0.6 for conduction in all groups of fibres at all periods of atrophy. Futhermore, the thickness of the myelin remains constant for a given perimeter over the entire period of atrophy studied. This suggests that the number of turns of myelin and the length of each turn remain unchanged during peripheral nerve atrophy. A simple geometric model explains how this can occur without gaps developing between the axon and myelin or between the turns of myelin. The Frankenhaeuser-Huxley equations for conduction in myelinated nerve fibres predict changes in conduction velocity similar to those observed, if the axons atrophy without changes in myelin. The advantages of this mode of atrophy are discussed.     

Postnatal development of conduction velocity and fibre size in the rat tibial nerve

The maximum conduction velocity (CV) and fibre diameters (D) were determined in the tibial nerve of developing rats. In 1-day-old rats CV of the fastest motor and sensory fibres (assessed separately) was 1.4 m/sec on the average and increased to 35 m/sec by postnatal day 30. The maximum conduction rate in adult rats ranged from 60 to 84 m/sec. Diameters of at least 100 nerve fibres in each age group were measured in electronmicrographs. The calibre of myelinating fibres in 1-day-old rats was 0.5–1.5 μm. By day 90 after birth the range of myelinated fibre size extended to 1.5–12.5 μm. The factor relating conduction rate and total fibre diameter of the largest fibres (i.e. the value of ) was found to vary with age, increasing from 1.1 to 6.2 between postnatal days 1 and 90. These results indicate that functional and morphological properties of peripheral nerve fibres in the rat undergo considerable changes during postnatal ontogeny until they reach adult values.     

Axonal transport in early experimental diabetes

In the present study fast axonal transport was examined in streptozotocin rats with 4 weeks duration in diabetes. Tritiated leucine and 14C-labelled glucosamine were injected into the fifth lumbar ganglion and TCA-soluble as well as insoluble activity were measured in segments of the sciatic nerve at various time intervals. (1) Time from injection until start of fast axonal transport was prolonged in diabetic rats whereas anterograde transport velocity was unchanged. (2) Incorporation of labelled leucine was reduced by 40%, whereas labelled glucosamine incorporation was unchanged. (3) Alterations observed in accumulations of labelled glycoconjugates proximal and distal to a collection crush might represent a decreased amount of retrograde transported material. The changes found in protein and glycoconjugate synthesis and transport could be related to the early reduction in axon calibre and conduction velocity in peripheral nerve of streptozotocin-diabetic rat.     

Nerve biopsy and conduction studies in diabetic neuropathy.

Morphological findings in sural nerves were related to nerve conduction in 12 patients with diabetic neuropathy, five with mainly sensory involvement, four with severe, symmetrical sensory-motor polyneuropathy, and three with multiple mononeuropathy. All had loss of large and small myelinated and of unmyelinated fibres, even early in the disease; segmental remyelination was the most prominent myelin alteration in teased fibres, segmental demyelination was found in only a few fibres. Axonal degeneration and Schwann cell damage seem to proceed independently of each other. The relation between recorded conduction velocity and that expected from the diameter of the largest fibres indicated that slowing of 20 to 30% was due to causes other than fibre loss; a grossly diminished conduction velocity was caused mainly by fibre loss. Electrophysiological findings in the sural nerve were largely representative of findings in other nerves, though abnormalities were less marked in the median nerve. In half the endoneurial vessels from diabetic neuropathy the perivascular space was thickened or contained more layers of basal laminae than normal. The same abnormalities were found in one-quarter of the endoneurial vessels from other acquired neuropathies.     

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.     

The conduction velocity of slower and the fastest fibres in infancy and childhood.

Motor nerve conduction velocity of the ulnar nerve was measured in 54 infants of various ages. Conduction velocity of slower fibres was measured using Hopf's technique, and maximal conduction velocity was measured with the usual method. Both maximal conduction velocity and conduction velocity of slower fibres increased with maturation. The former was about 65 m/s at the age of about 3-5 years, but thereafter it decreased slightly to about 60 m/s. Conduction velocity of slower fibres increased gradually until about 3 or 4 years of age, and then ranged about 40-50 m/s. The difference between the two velocities increased until 7 or 9 months of age; during the next 8 years, it tended to decrease to about 8-16 m/s. The increasing difference in size between the largest and the smallest myelinated fibres with maturation correlated with the developmental increase of the difference between maximal conduction velocity and conduction velocity of slower fibres. The anatomical evidence of the slight decrease of the axon diameter of the large fibres at the age of 7-14 years may correspond to the slight reduction of maximal conduction velocity after about 5 years of age.     

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.     

The effect of pancreatic islet transplantation on experimental diabetic neuropathy.

Quantitative light and electronmicroscopical morphometric techniques were used to determine the effect of pancreatic islet transplantation on experimental diabetic neuropathy. Groups of STZ-diabetic rats were given islet transplants at 3 weeks after diabetes onset (prevention) and at 6 months after diabetes onset (reversal). Comparisons were made with onset controls, age-matched non-diabetic controls and untreated diabetic controls 6 months later (n = 8 for all groups). Euglycaemia and normal levels of glycosylated haemoglobin were achieved in both groups of diabetics after islet transplantation. Loss of body weight in diabetic animals was prevented by early islet transplantation, but was only partially reversed following delayed islet transplantation. Normal growth of myelinated fibres and axons during development was retarded in untreated diabetics, but was normal in rats given islet transplants soon after the onset of diabetes (cross-sectional perimeter and area). Diabetics transplanted with islets after a delay had myelinated fibres and axons with diminished calibre. Teased fibre preparations of nerves from diabetics which had received islet transplants showed no excess of abnormalities. This study has shown that the development of certain structural abnormalities of peripheral nerve fibres is prevented in diabetic rats which receive transplants of islets of Langerhans soon after the onset of diabetes. However, once established abnormal fibre morphology can not be completely ameliorated merely by achieving and sustaining euglycaemia through delayed islet transplantation.     

Prechiasmatic Reordering of Fibre Diameter Classes in the Retinofugal Pathway of Ferrets

This study examines the distribution of fibre diameter classes at various sites along the retinofugal pathway of adult ferrets. Light microscopic observations were made on semi-thin sections, and regional fibre diameter spectra were constructed from diameter measurements taken from electron micrographs of thin sections of the intraorbital optic nerve (2.5 mm from the optic disc), the intracranial optic nerve (1 mm rostral to the fusion of the nerves), and the optic tract (just caudal to the optic chiasm).
Whereas diameter types are relatively evenly distributed behind the eye in the postoptic nerve, they begin to segregate along its prechiasmatic course. Within this prechiasmatic region, coarse and fine calibre fibres are confined increasingly to more ventral locations in the nerve, leaving a dorsal band populated predominantly by intermediate calibre fibres. In conjunction with this redistribution of axon size classes, the fascicular arrangement of axons which is present distally, changes to a non-fascicular organization. The prechiasmatic organization of fibre types approximates that found in the optic tract where the coarse and fine calibre fibres lie further ventrally towards the pial surface.
The prechiasmatic region can be viewed as a region of transition where the order of fibres in the nerve (retinotopic) starts to change to that present in the optic tract (chronotopic), resulting in the first-born beta cell axons becoming segregated dorsally, and rostral to the coarse and fine calibre classes which segregate at further caudal locations. Further, since the sorting of fibres according to diameter appears before the fibres reach the optic chiasm, the segregation of diameter classes is not dependent on the chiasmatic sorting of fibres according to their crossed or uncrossed course.     

Early sarcolemmal dysfunction in skeletal muscle amyloidosis

Summary Weakness and stiffness appeared in a 65-year-old man affected by multiple myeloma. Muscle fibre conduction velocity was recorded in situ in the biceps brachii and found to be significantly decreased. Muscle biopsy, performed in the same muscle, showed amyloid deposition and moderate atrophy of muscle fibres, which was not sufficient to explain the reduction in muscle fibre conduction velocity. The results of the study suggest that amyloid interferes with conduction along the sarcolemma and that this plays a pathogenetic role mainly in the early stages of the disease.     

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.     

Polyneuropathy. Facts and fancies.

Some conclusions are drawn from findings in 167 consecutive patients with the ordinary "garden variety" of polyneuropathy; the aetiology was unknown in 15%. Histological findings in sural nerves were related to clinical and electrophysiological abnormalities. In some patients with discrete clinical abnormalities, sensory and motor conduction and amplitudes of evoked sensory and muscle action potentials were normal, whereas the nerve biopsy showed slight but definite abnormalities. The reverse, abnormal nerve conduction and normal histological findings, did not occur. Histological findings were rarely, and electrophysiological findings were not, specific for the aetiology or type of a neuropathy. Thus, neither conduction studies nor conventional or single fibre electromyography can identify the underlying pathology: loss of large myelinated fibres (greater than 7 micrometers) was equally prominent in nerves with de- and re-myelination as in those without them. Paranodal and segmental demyelination in less than 20% of the teased fibres occurred as often in nerves with as in those without disproportionate slowing in conduction. When the recorded conduction velocity was equal to that to be expected from the fibres with the largest diameter, slowing in conduction could be explained by axonal degeneration ("proportionate" slowing, 79% of the nerves). When the recorded velocity was disproportionately slower than that expected from fibre diameter (21% of the nerves), causes other than loss of the largest fibres must be assumed to explain the slowing in conduction. Myelin abnormalities in more than 50% of the teased fibres were found only in nerves from patients with the hypertrophic type of peroneal muscular atrophy and in postgastrectomy neuropathy and can probably explain the marked disproportionate slowing in conduction. The material contained, however, only one patient with acute idiopathic polyradiculoneuropahy. In diabetic neuropathy, segmental demyelination was present in only 8 of 502 teased fibres (9 nerves), remyelination was present in 135 fibres, and could not explain the disproportionate slowing in conduction. The mechanism of disproportionate slowing, when it is not due to demyelination, is still obscure.     

Immunofluorescence and immunoelectron microscopic evidence for differences in myelination of GABAergic and cholinergic septohippocampal fibres

It is known that the rat septohippocampal projection is realised at least by GABAergic, parvalbumin containing and cholinergic fibres. The GABAergic component originates from fast-firing and fast-conducting neurons, whereas the cholinergic component represents the slow-firing, slow-conducting type. The present immunofluorescence and immunoelectron microscopic study shows that the vast majority of parvalbumin-immunoreactive, GABAergic axons are surrounded by enormously thick myelin sheaths, but choline acetyltransferase immunoreactive axons were rarely found to be myelinated. In addition, cholinergic fibres show considerably smaller diameters. Accordingly, our results are correlated with the well-known differences in conduction velocities between the GABAergic and cholinergic fibres of the septohippocampal pathway, which depend on myelination and axon calibre.     

Combined scatter diagrams of sheath thickness and fibre calibre in human sural nerves: changes with age and neuropathy.

A computer-assisted method permits collection of large numbers of measurements of fibre profiles in electron micrographs of human sural nerve biopsies. The method is based on simultaneous demonstration of fibre calibre and of sheath thickness in terms of the g-ratio (quotient axon diameter/fibre diameter), and on the recalculation of all parameters for circular fibre profiles. The fibres of small and large diameters of human sural nerves were found to form separate populations with distinctly different trends for sheath thickness and also different patterns of maturation. Preliminary data in neuropathy show that this method gives a better distinction of the extent of damage to either of the two populations; it also permits assessment of the number of regenerating or remyelinating fibres.     

Nerve conduction changes and fine structural alterations of extra- and intrafusal muscle and nerve fibers in streptozotocin diabetic rats

Streptozotocin-induced diabetes mellitus in known to cause a reduction of both conduction velocity and axon caliber in sciatic nerves and also a decrease in muscle fiber size. The present study investigates whether the distal parts of the peripheral nervous system, including extra- and intrafusal muscle fibers, are more severely affected than the proximal segments in the diabetic state. Proximal and distal sensory nerve conduction velocities were monitored during a period of 3 months in rats rendered diabetic by injection of streptozotocin. Segments of the sciatic and ventral coccygeal nerves, and of the biceps femoris and lumbrical muscles, were studied by light and electron microscopy, including morphometric analysis. In contrast to previous studies, daily suboptimal insulin injections were given to prevent acute metabolic complications. Sensory conduction velocity in the ventral coccygeal nerve was significantly (P > 0.05) decreased in the diabetic rats compared to controls. Proximal and distal nerve segments were equally affected. Mean cross-sectional axon area of the sciatic nerve was moderately, but significantly (P < 0.05), smaller in insulin-treated diabetic rats than in controls. In both the sciatic nerve and the terminal, intrafusal nerve segments, occasional axons showed moderate dystrophic changes. Fibers of the intrafusal nerve segments appeared to be equally affected compared to the fibers in the sciatic nerve, although no quantitative comparison was made. The increase of small caliber skeletal muscle fibers in experimental streptozotocin-induced diabetes was confirmed. These findings indicate that proximal and distal segments of peripheral nerves are affected equally in the early stages of experimental diabetic neuropathy.© 1995 John Wiley &Sons, Inc.     

Motor nerve conduction velocity and nerve fibre diameter in experimental protein deprivation

Summary Motor nerve conduction velocity (MCV) and nerve fibre calibre spectra of peripheral nerves were determined at different ages, during development in normal and protein-deprived rats. The protein deprivation was achieved by feeding female rats a protein deficient diet, given ad lib from 2 weeks before conception and onwards. After weaning, the offspring were fed the same protein-deficient diet. Compared to control rats which were given a balanced diet, the protein-deprived rats showed considerably reduced body weights but moderately reduced values of albumin and total protein in serum at 6 and 15 weeks of age. Although essentially the same, the mean nerve fibre diameter of the studied dorsal tail nerve was slightly larger in the protein-deprived rats at 6 weeks of age than in controls, while the inverse relationship was found at 15 weeks of age. The increase of MCV in normal and protein-deprived rats followed essentially the same curve during the period from 5–30 weeks of age. The results indicate that the MCV and nerve fibre diameter during development are correlated to chronological age and not to body size, although protein deprivation may, to a minor extent, influence nerve fibre calibre growth.This study was supported by a grant from the Swedish Medical Research Council (to P. Sourander) (No. B 79-12X-03 488-08B), and by grants from the University of Göteborg     

Nerve fibre velocity and refractory period distributions in nerve trunks.

With the use of a double stimulus technique, nerve fibre velocity range measurements were performed over a single conduction distance in 13 normal subjects and over two conduction distances in another 12 normal subjects. The velocity ranges were found to be dependent upon the conduction distance, owing to unknown refractory period delays. Refractory period values were calculated for the 12 subjects and also direct refractory period distribution measurements were made on 15 normal subjects using a twin stimulus and automatic subtraction technique. Corrections to the velocity range measurements were made upon differing assumptions as to the correlation between refractory period and fibre conduction velocity. It was concluded that a single median value refractory period obtained from the distribution was the best correction to use, based upon the hypothesis that for group A fibres the random scatter of refractory period values is far greater than any variation due to a correlation between refractory period and fibre conduction velocity. It was found important to recognize that calculated values of velocity range are a function not only of the spread of fibre conduction velocities but also of refractory periods.     

The effect of pre- and postnatal undernutrition on the calibre growth of central motor fibres

Summary The effect of early in life instituted undernutrition on the calibre growth of cortico-spinal motor fibres was studied electron microscopically at three different developmental stages in the rat. The total diameters of these motor fibres were measured from a defined level of the medulla oblongata and plotted in histograms.In undernourished rats the calibre growth was retarded as compared to agematched control rats. Early initiated nutritional rehabilitation through controlled cross-fostering revealed that the impaired calibre growth was reversible, and thus completely restituted at the age of 180 days.It is concluded that early initiated protein-calorie undernutrition affects this central motor pathway in a similar way, as it affects a peripheral motor nerve (cf. Sima, 1974).