Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography.

Cross-anastomoses and autogenous grafts of unmyelinated and myelinated nerves were examined by electron microscopy and radioautography to determine if Schwann cells are multipotential with regard to their capacity to produce myelin or to assume the configuration seen in unmyelinated fibres. Two groups of adult white mice were studied. (A) In one group, the myelinated phrenic nerve and the unmyelinated cervical sympathetic trunk (CST) were cross-anastomosed in the neck. From 2 to 6 months after anastomosis, previously unmyelinated distal stumps contained many myelinated fibres while phrenic nerves joined to proximal CSTs became largely unmyelinated. Radioautography of distal stumps indicated that proliferation of Schwann cells occurred mainly in the first few days after anastomosis but was also present to a similar extent in isolated stumps. (B) In other mice, CSTs were grafted to the myelinated sural nerves in the leg. One month later, the unmyelinated CSTs became myelinated and there was no radioautographic indication of Schwann cell migration from the sural nerve stump to the CST grafts. Thus, Schwann cell proliferation in distal stumps is an early local response independent of axonal influence. At later stages, axons from the proximal stumps cause indigenous Schwann cells in distal stumps from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously unmyelinated nerves to produce myelin while Schwann cells from the previously myelinated nerves become associated with unmyelinated fibres. Consequently, the regenerated distal nerve resembled the proximal stump. It is suggested that this change is possible because Schwann cells which divide after nerve injury reacquire the developmental multipotentiality which permits them to respond to aoxonal influences.     

Guanethidine adrenergic neuropathy: an animal model of selective autonomic neuropathy

Chronic administration of guanethidine to adult rats induces a selective autoimmune adrenergic neuropathy. Physiological and biochemical features of this disorder in the peripheral nervous system were explored in young adult Sprague-Dawley rats given daily intraperitoneal guanethidine monosulfate for 5 weeks. Control rats received daily saline injections. The guanethidine-treated animals gained less weight, had ptosis, and had a lower mean arterial blood pressure in the supine and upright tilted positions. Norepinephrine was depleted in the peroneal, sural, tibial, and vagal nerves, the nutrient artery to the tibial nerve and in the superior cervical sympathetic ganglion of the drug-treated animals. On light microscopy, there was an inflammatory cell infiltrate and neuron loss in the superior cervical ganglion. Caudal and sciatic-tibial nerve conduction values were well preserved in the guanethidine-treated animals as was the ‘C’ potential derived from unmyelinated vagal fibers recorded in an in vitro chamber. The ‘C’ potential recorded from the cervical sympathetic trunk, however, was reduced in amplitude correlating with the loss of norepinephrine content in the harvested contralateral superior cervical sympathetic ganglion. The findings further support the view that guanethidine produces a selective adrenergic neuropathy in the rat — providing a useful standard with which to gauge autonomic involvement in other models of neuropathy. In addition, loss of the cervical sympathetic ‘C’ potential suggests that this presumed preganglionic structure also contains postganglionic adrenergic fibers.     

Unmyelinated axon subpopulations in the rat peripheral nervous system

Using the neurotoxin capsaicin, we examined subpopulations of unmyelinated axons in mixed (sciatic), cutaneous (sural), and muscular (nerve to soleus) nerves. Administration of capsaicin to neonatal rats caused reduction of the sciatic nerve immunoreactive (IR)-substance P (by 45%) and IR-somatostatin (by 84%) contents. This correlated with a substantial reduction in unmyelinated axons in the sciatic and sural nerves (45% and 65%, respectively), although there was no significant decrease in unmyelinated axons in the nerve to soleus. In a parallel study, we have shown that sympathetic ganglia-derived unmyelinated axons account for about 20 to 25% of the total unmyelinated axon population in both the sural nerve and the nerve to soleus. Thus, in the sural nerve, the majority of unmyelinated axons are dorsal root ganglia-derived, contain either substance P or somatostatin, and are capsaicin-sensitive; whereas in the nerve to soleus, the majority of unmyelinated axons are dorsal root ganglia-derived but are insensitive to capsaicin and do not contain substance P or somatostatin. These latter unmyelinated axons presumably contain a yet to be defined neurotransmitter and may be the axons connecting with muscular ergoreceptors, a subpopulation of unmyelinated axons that are biochemically and functionally distinct from the unmyelinated axons of cutaneous nerves.     

Unmyelinated nerve fibers in sural nerve in pure autonomic failure

We examined sural nerve biopsy specimens from 7 patients with pure autonomic failure (PAF). The mean unmyelinated nerve fiber diensity in these patients was 40% less than in age-matched controls. Increased numbers of clusters of collagen pockets not containing unmyelinated axons were the most prominent finding in PAF. This appears to reflect recent dropout of a group of sympathetic efferents and suggests grouping of unmyelinated fibers by modality at the level of the sural nerve trunk.     

Autonomic and electrophysiological studies in patients with signs or symptoms of diabetic neuropathy

Thirty-five patients with symptoms or signs of diabetic neuropathy were tested for autonomic neuropathy by measuring heart rate and blood pressure changes during an orthostatic tilt test, a single deep breath and the Valsalva manoeuvre and these results were related to electrophysiological measurements made on the common peroneal and sural nerves. The sural sensory action potential (SAP) was more strongly correlated with these tests of autonomic function (particularly with the brake index of the orthostatic tilt test (P less than 0.001) and the fall in systolic blood pressure 1 min after tilt (P less than 0.001], than the common peroneal nerve compound motor action potential, its minimum F wave latency or nerve conduction velocities. Patients with a detectable sural SAP had significantly higher brake indices than those with absent sural SAPs. Significant correlations were also obtained with the common peroneal motor nerve conduction velocity (MNCV) and autonomic tests and patients with MNCV less than and greater than 38 m/sec showed significant differences in many autonomic tests. The sural SAP amplitude, being less susceptible to factors that influence nerve conduction velocity, may be useful in identifying patients with an autonomic neuropathy.     

Three dimensional analysis of unmyelinated fibers in normal and pathologic autonomic nerves.

The technique of serial-section electron microscopy and diagrammatic three dimensional reconstructions has been used to assess normal and pathological unmyelinated nerve fibers from a peripheral autonomic nerve: the rat cervical sympathetic trunk. Within this predominantly unmyelinated nerve, there is a complex arrangement of axons into longitudinally oriented bundles brought together by chains of Schwann cells. Each bundle is subdivided into smaller components by the cytoplasmic processes of Schwann cells; such subdivisions, which are basal lamina-enclosed masses of Schwann cell cytoplasm, when viewed on cross-sectional electron micrographs, are termed Schwann cell units. The size and shape of each Schwann cell unit varies along the length of fibres, but the diameter of individual axons shows little variation over the segments studied. Axonal branching was not observed in normal unmyelinated nerves. Crush injury and x-irradiation produces different patterns of alteration in the axon-Schwann cell relationships of unmyelinated nerves. Following crush injury, Schwann cell processes increase in diameter and contain numerous small diameter axonal sprouts. Many of the regenerating axons remain thin while others reacquire a normal diameter. X-irradiation affects Schwann cells leading to retraction of their processes and the appearance of naked axonal segments.     

Conduction velocity in hexachlorophane neuropathy: correlation between electrophysiological and histological findings

After 2-3 weeks exposure to hexachlorophane, maximum motor nerve conduction velocity in sciatic nerves of rats was reduced by 7.5% and evoked muscle action potential amplitude by 9%. Histological examination at this stage showed intramyelin oedema affecting some fibres and axonal degeneration of other fibres. After longer periods of exposure velocity and amplitude fell further. Velocity was reduced by 27% after 6-7 months treatment. In addition to intramyelin oedema and axonal degeneration, segmental demyelination was present in animals intoxicated for more than three months. There was no correlation between the degree of oedema and reduction of conduction velocity. It is concluded that intramyelin oedema has little or no effect on conduction velocity. Nodes of Ranvier are normal in the early stages of the lesion and this may contribute to the preservation of normal conduction. The electrophysiological findings can be attributed to secondary changes of axonal degeneration and segmental demyelination.     

Motor and sensory nerve conduction velocity in the baboon: normal values and changes during acrylamide neuropathy

Nerve conduction velocity and the amplitude of nerve and muscle action potentials have been measured in the median and anterior tibial nerves of normal adult and infant baboons. The effect of altered temperature on velocity has also been investigated. Seven adult baboons were intoxicated with acrylamide. In animals given 10-15 mg/kg/day, the gradual development of a peripheral neuropathy was accompanied by a decline in the amplitude of both muscle and nerve action potentials. There was also a gradual fall in conduction velocity. In some cases maximal motor velocity in the median nerve fell by as much as 34%, and in the anterior tibial nerve by as much as 49%, the largest falls being seen in animals showing the greatest reductions in response amplitude. Histological studies, reported elsewhere, have shown that the main pathological change in our animals was a degeneration of the peripheral nerves, with little demyelination. Fibre diameter histograms indicated that large fibres were particularly severely affected, and it seems likely that the reduced maximal conduction velocities were due to this selective loss of large-diameter fibres.     

Age changes in conduction velocity of unmyelinated fibers

Increase of conduction velocity of unmyelinated fibers has been measured in the cervical sympathetic trunk of growing rats. Fibers in this nerve are almost all unmyelinated.

• 1 Conduction velocity is about half adult values at ten days after birth. The velocity in some nerves reaches the adult range by 50–80 days. There is evidence that the velocity of many fibers other than the fastest also reaches the adult range by this age.
• 2 During the period of growth from 10–112 days of age, velocity increases in direct proportion to the increase in length of the nerve.

     

Behavior of Schwann cells from Trembler mouse unmyelinated fibers transplanted into myelinated nerves

In the peripheral nerves of Trembler mice, Schwann cells produce little or no myelin and continue to multiply beyond the normal neonatal period. However, in the unmyelinated (Remak) fibers of these mutants, Schwann cell morphology and multiplication are normal. To determine if such phenotypically normal Schwann cells would show the Trembler abnormalities when challenged to form myelin in nerve grafts, an unmyelinated nerve, the cervical sympathetic trunk, was transplanted into the richly myelinated sural nerve in a series of normal host animals. Two months after transplantation, regenerated grafts, composed of normal host axons and Trembler cervical sympathetic trunk Schwann cells, showed the characteristic Trembler abnormalities of myelination and multiplication whereas Schwann cells from normal cervical sympathetic trunks myelinated the host axons normally. Thus, the Trembler mutation appears to affect Schwann cell differentiation at a specific phase, the formation of myelin. This primary expression of the Schwann cell abnormality initiates a cycle of secondary effects that include demyelination, Schwann cell multiplication, and attempted remyelination.     

Sensory action potentials and biopsy of the sural nerve in neuropathy.

In 167 consecutive patients with various types of neuropathy, the amplitude of the sensory potential and the maximum conduction velocity along the sural nerve were compared with conduction in other sensory nerves, and were related to structural changes revealed by nerve biopsy. Electrophysiological findings in the sural nerve were similar to those in the superficial peroneal and the median nerve, though the distal segment of the median nerve was normal in 20 per cent of the patients when it was abnormal in the sural nerve. Quantitation of histological findings was a more sensitive method than the electrophysiological study in that two-thirds of 33 patients with normal electrophysiology in the sural nerve showed mild loss of fibres or signs of remyelination in teased fibres. The amplitude of the sensory potential was grossly related to the number of large myelinated fibres (more than 7 micrometer in diameter). Considering the 95 nerves from which teased fibres were obtained, maximum conduction velocity was abnormal in half. In 18 of these nerves, slowing in conduction was due to axonal degeneration: the velocity was as to be expected from the diameter of the largest fibres in the biopsy ("proportionate slowing"). In 9 nerves slowing was severe and more marked than to be expected from loss of the largest fibres ("disproportionate slowing"); these nerves showed paranodal or segmental demyelination in more than 30 per cent of the fibres. In 16 nerves from patients with neuropathy of different aetiology neither loss of fibres nor demyelination could explain the moderate slowing. The cause of slowing in these nerves is unknown; other conditions are referred to in which slowing in conduction cannot be attributed to morphological changes. Finally, electrophysiological and histological findings are reported in some patients with neuropathy associated with malignant neoplasm, with rheumatoid arthritis, with polyarteritis nodosa, with acute intermittent porphyria and with cirrhosis of the liver.     

Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves

The innervation of the guinea pig trachea was studied in wholemount preparations stained for acetylcholinesterase, catecholamines, and substance P immunoreactivity and by electron microscopy. The majority of parasympathetic and afferent nerve fibres arrive from the vagus via branches of the recurrent laryngeal nerves. The recurrent laryngeal nerves are composed of several fascicles comprising 600-700 small myelinated fibres (2-5 microns diameter) and about 1,000-2,000 unmyelinated fibres; both components exit from the nerve and project in fine branches to the trachea. A separate component of 200-250 large myelinated fibres (more than 5 microns diameter) runs the full length of the nerve and innervates the striated muscles of the larynx. The recurrent laryngeal nerves are slightly asymmetric in their origin, length, number, and composition of fibres, with the right nerve being shorter but with more numerous and thinner myelinated fibres. At the distal end of the recurrent nerve, a fine branch called the ramus anastomoticus connects it to the superior laryngeal nerve. In the tracheal plexus, there are on average 222 ganglion cells (range 166-327), distributed mostly in small ganglia of 12 or fewer neurons. The ganglionated plexus is situated entirely outside the tracheal wall, overlying the smooth muscle. Ligation experiments show that sympathetic nerve fibres reach the trachea with the recurrent nerves via anastomoses between the sympathetic chain and vagus nerves, or occasionally with recurrent nerves directly, the largest being at the level of the ansa subclavia. There are also perivascular sympathetic nerve plexuses. Substance P immunoreactive fibres enter the trachea from the vagus nerves and by pathways similar to those of sympathetic nerves. There are also paraganglion cells within the recurrent laryngeal nerve that contain catecholamines and are surrounded by substance P immunoreactive fibres. After cervical vagotomy, all the large myelinated fibres of the ipsilateral recurrent laryngeal nerve degenerate and so do all but 10 or 20 small myelinated fibres and all but a few unmyelinated fibres. Degenerating fibres are found within the entire tracheal plexus, indicating bilateral innervation. The small myelinated fibres that survive cervical vagotomy probably represent sympathetic or afferent nerves with their cell bodies located in sympathetic or dorsal root ganglia.     

Source and origin of nerve fibres immunoreactive for substance P and calcitonin gene-related peptide in the normal and chronically denervated superior cervical sympathetic ganglion of the rat

Immunohistochemical studies of sympathetic ganglia have indicated that the normal rat superior cervical ganglion contains both SP-IR and CGRP-IR fibres, and CGRP- and SP-immunoreactivity coexist in some fibres. In rat sympathetic ganglia decentralization by preganglionic denervation leads to intraganglionic increase of peptidergic fibres immunoreactive (IR) for substance P (SP) and calcitonin gene-related peptide. We explored the sources of SP- and CGRP-IR fibres in normal and in chronically decentralized rat SCGs.The distribution of immunoreactivities for CGRP and SP was determined in SCGs of normal rats and of rats following preganglionic denervation followed by sensory denervation. Ganglia were studied after short-term (2–5 days) sensory denervation, and long-term (7–16 months) sympathetic denervation followed by short-term (2 days) sensory denervation. To explore for the production of SP and CGRP by intrinsic neurones within the ganglion, normal and chronically decentralized SCGs were examined following pretreatment by local in vivo application of colchicine. Normal and chronically decentralized ganglia were also injected with fluorescent tracer Fluorogold for retrograde tracing of extrinsic fibres back to their neurones of origin.The observations suggest that in normal SCG in the rat the SP-IR and CGRP-IR nerve fibres are derived via direct links from vagus and glossopharyngeal nerves and the cervical plexus, or from nerve fibres running along the cervical sympathetic trunk, and the external carotid and the internal carotid nerves.Sensory nerve inputs to the rat SCG following decentralization may contribute to the low levels of ganglionic activation observable in the autonomic failure of multiple system atrophy in man.     

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 tonic activity of single nerve fibers of the rabbit cervical sympathetic nerve

Spontaneous sympathetic activity was recorded in single fibres dissected from the cervical sympathetic nerve trunk (CSNT) of rabbit. Conduction velocities and action potential amplitudes for B-fibres were measured in the whole nerve trunk. Results of the spectral analysis suggested that activity of unmyelinated fibres depended on baroreceptor activity to a greater extent than that of myelinated fibres and that the 2-3 Hz rhythm was present in animals with intact baroreceptors simultaneously with the cardiac rhythm. That rhythm was a usual feature of activity of both B- and C-fibres. Cross-correlation analysis of the single fibre activity was estimated in 24 fibre pairs. It was found that in five fibre pairs there was a correlation associated with the presence of a common physiological rhythm, in one fibre pair there was a correlation derived from the existence of a strong common input to both distinct preganglionic neurons.     

Effects of chronic lithium treatment on the peripheral nervous system.

BACKGROUND: Although lithium carbonate is widely used in the treatment of mood disorders, symptoms suggesting toxic effects on the peripheral nervous system may emerge even in subjects whose serum lithium levels remain within the recommended therapeutic range. METHOD: Electroneuronographic (ENG) parameters (motor nerve conduction velocity of peroneal and median nerves, sensory nerve conduction velocity of sural and median nerves, amplitude of motor potential of peroneal and median nerves, and amplitude of sensory action potential of the median nerve at the wrist and the sural nerve) were investigated in 2 groups (N = 34) of patients suffering from bipolar affective disorder (DSM-III-R, DSM-IV) undergoing maintenance treatment with lithium carbonate for at least 1 year (mean = 2.06 years) in monotherapy. For 12 patients, ENG results were compared with pretreatment values, whereas in the other 22 cases, only data relevant to posttreatment were available. Fifty-four healthy subjects and 20 patients with recurrent major affective disorder (unipolar and bipolar) never treated with lithium made up the comparison groups. RESULTS: Compared with the 2 comparison groups, patients on chronic lithium treatment showed significant reduction of motor nerve conduction velocity of peroneal and median nerves, sensory nerve conduction velocity of sural and median nerves, amplitude of motor potential of peroneal and median nerves, and amplitude of sensory action potential of the median nerve at the wrist and the sural nerve. The comparison with the assessment made prior to lithium treatment also showed significant changes; after a period of treatment with lithium varying from 2 to 8 years (mean = 5.2 years), significant reductions were found on motor and sensory nerve conduction velocity and on amplitude motor potentials and sensory action potentials. CONCLUSION: Chronic maintenance treatment with lithium affects the peripheral nerves, even if the impairment rarely is such as to warrant discontinuation of treatment. Monitoring of ENG results could be useful for the early detection of neurotoxicity of lithium.     

The effects of axotomy on the conduction of action potentials in peripheral sensory and motor nerve fibres.

Medial gastrocnemius and sural nerves in one hindlimb of the cat were transected and prevented from regenerating. After periods ranging from 29-273 days, compound action potentials were recorded from axotomised and contralateral control nerves. The amplitude and integrated area of action potentials decreased and conduction velocity slowed following axotomy. The area under compound action potentials generated by stimulating sensory fibres declined significantly faster than that generated by stimulating motor fibres. Analysis of changes in whole nerve conduction velocity distributions showed that the velocities of fast conducting sensory fibres decreased at the most rapid rate. The conduction velocities of motor fibres and slow sensory fibres declined at significantly slower rates. The loss of electrical activity in the largest sensory nerve fibres following axotomy, may play a role in determining the faster rate at which their action potentials deteriorate.     

Endothelin immunoreactivity and mRNA expression in sensory and sympathetic neurones following selective denervation

The localization of endothelin (ET) in perivascular nerve varicosities supports pharmacological evidence that ET is a neurotransmitter in the autonomic nervous system. To examine the potential source of ET previously localized in cerebrovascular nerves, ganglia which send projections to these vessels were immunolabelled for ET and examined at the ultrastructural level. The trigeminal (TG) and superior cervical ganglia (SCG) were examined in control rats and following either sensory denervation or sympathectomy. In control TG, ET immunolabelling was detected throughout the cytoplasm of a subpopulation of neurones whereas in the SCG only the occasional ET-positive neurone was seen. Following sensory denervation with capsaicin, very few ET-immunoreactive nerve cell bodies or nerve fibres were detected in the TG compared with control ganglia, suggesting that ET is predominantly localized in primary afferent neurones, although some remaining myelinated nerve fibres stained positively. ET labelling of neurones in the SCG was unaffected by sensory denervation. Following selective damage to sympathetic nerves with 6-hydroxydopamine, there was a marked increase in intensity of ET-labelling of nerve fibres in the TG, probably due to increased availability of nerve growth factor for sensory nerves. There was no effect on ET immunoreactivity in the nerve cell bodies and nerve fibres within the SCG. However, in situ hybridization techniques demonstrated that 6-hydroxydopamine sympathectomy resulted in a marked increase in ET-1 mRNA expression in the SCG neurones. In conclusion, sensory nerves projecting from the TG are a more likely source of ET-positive perivascular nerves in cerebral arteries than sympathetic nerves from the SCG. Damaged sympathetic neurones markedly increase ET mRNA expression. In view of the neuroprotective properties of ET, this may represent a compensatory mechanism to promote repair.     

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.     

Spinal segmental preganglionic outflow to cervical sympathetic trunk and postganglionic cardiac sympathetic nerves

The aim of this study was to verify in which spinal cord segments the preganglionic neurones projecting to the cervical sympathetic trunk or converging onto the somata of the postganglionic cardiac sympathetic neurones are located in cats. The thoracic white rami T1 to T5 were electrically stimulated and the evoked responses were recorded in the cervical sympathetic trunks and postganglionic cardiac nerves. The responses were mostly evoked by electrical stimulation of group B preganglionic fibres. The maximum amplitude of evoked responses in the cervical sympathetic trunk was obtained when the T2 white ramus was stimulated and decreased gradually when followed by the stimulation of T1, T3, T4 and T5 white rami. In most cases the maximum amplitude of evoked responses in the left inferior cardiac nerve, right inferior cardiac nerve and left middle cardiac nerve was obtained when the T3 white ramus was stimulated. The size of the responses decreased when more cranial and caudal white rami were stimulated. It was found that the somata of the postganglionic neurones of the right and left inferior cardiac nerves were placed in the right and left stellate ganglion, respectively. Somata of the postganglionic neurones with axons in the left middle cardiac nerve were mainly located in the left middle cervical ganglion and some in the left stellate ganglion.