Unmyelinated fibers in sural nerve biopsies of chronic inflammatory demyelinating polyneuropathy

Summary Seven patients aged 29 to 76 years with various clinical subtypes of chronic inflammatory demyelinating polyneuropathy (CIDP) were investigated. Sural nerve biopsies were performed between 7 months and 19 years after onset of disease. Quantitative electron microscopy revealed involvement of primary unmyelinated fibers (UF) in all cases. When compared with age-matched controls from the literature and two controls of our own, there was an increase of degenerating primary UF in all cases, a definite decrease of density per mm² or number per nerve after subtraction of regenerates of myelinated and unmyelinated fibers in five cases, an increase of denervated Schwann cell complexes of the unmyelinated type in three cases, and an increased incidence of a high ratio (3) of primary UF per Schwann cell complex in five cases. Presumably due to the small number and heterogeneity of cases, the results did not correlate with type and duration of CIDP, but were obviously influenced by the degree of demyelination. The possible causes of UF damage in CIDP are discussed.     

Nonmyelinating Schwann cell involvement with well-preserved unmyelinated axons in Charcot-Marie-Tooth disease type 1A

Electron microscopic examination was performed to compare morphologic changes of nonmyelinating Schwann cells and unmyelinated axons in patients with Charcot-Marie-Tooth disease type 1A (CMT1A) with peripheral myelin protein 22 duplication (n = 27) and normal control individuals (n = 14). Complete transverse sural nerve cross-sections were obtained in 16 patients and the total number of axons and Schwann cells in each cross-section was estimated. In patients with CMT1A, the number of myelinated axons was significantly decreased, whereas unmyelinated axons were well-preserved and did not show any marked changes. The numbers of nuclei, subunits, and profiles of nonmyelinating Schwann cells were all increased significantly in patients with CMT1A, whereas the numbers of axons per unmyelinated axon-containing subunit were significantly decreased. Schwann cell subunits consisted of layers of flattened cytoplasmic profiles wrapped around unmyelinated axons in the patient with CMT1A. The numbers of nonmyelinating Schwann cell profiles were increased and the numbers of axons per unmyelinated axon-containing subunit were reduced even in young patients with well-preserved myelinated fibers. In conclusion, there is marked alteration of the population and morphology of nonmyelinating Schwann cells, and axon-Schwann cell interactions seem to be regulated differently between myelinated and unmyelinated fibers in CMT1A.     

Marklose nervenfasern im Senium und im Spätstadium der Thalidomid-Polyneuropathie: quantitativ-elektronenmikroskopische Untersuchungen

Summary Sural nerve biopsies of four patients, aged 54–76 years, with a predominantly sensory type of neuropathy following high dosages of thalidomide were examined by light and electron microscopy. The present study includes a qualitative and quantitative evaluation of unmyelinated nerve fibers. Despite severe neuropathy, increased numbers of small unmyelinated axons per endoncurial area were noted in all patients. This numerical increase appeared to be independent of aging, since it was not seen in two senile controls, studied at the age of 83 and 88 years. The increase in the endoneurial density of unmyelinated axons, especially of small sized fibers, is likely to be related to regeneration following degeneration of unmyelinated axons although endoneurial shrinkage secondary to loss of large myelinated fibers could have caused an additional increase in the number of axons per endoneurial area.Axonal sprouting, despite degeneration of large numbers of myelinated and unmyelinated fibers, appears to be consistent with some of the characteristic clinical features of thalidomide neuropathy such as paresthesias, hyperesthesia for pain and temperature, and disturbances of autonomic functions. On the other hand, a variable number of empty Schwann cells (bands of Büngner) and pockets at the surface of many Schwann cells noted in the four patients with neuropathy were also seen in both senile controls with no signs of neuropathy. Thus, it is obvious that pockets and empty Schwann cells may be related to aging or other causes of slow axonal wasting with Schwann cell proliferation and are not necessarily associated with clinically manifest neuropathy.

Mit Unterstützung der Deutschen Forschungsgemeinschaft, Bonn-Bad Godesberg (Schr 195/1)     

Myelinated fiber regeneration after crush injury is retarded in sciatic nerves of aging mice

To compare nerve regeneration in young adult and aging mice, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two weeks later, both groups of mice were perfused with an aldehyde solution, and, after additional fixation, the sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. The same level was sectioned in unoperated contralateral nerves; these nerves served as controls. Electron micrographs and the Bioquant Image Analysis System IV were used to measure areas of posterior tibial fascicles and count the number of myelinated axons, the number of unmyelinated axons, and their frequency in Schwann cell units. In aging mice, the total number of regenerating myelinated axons was significantly reduced, but totals of regenerating unmyelinated axons in aging and young adults did not differ significantly. In aging mice, the frequency of Schwann cells that contained a single unmyelinated axon was greater, suggesting that before myelination began, Schwann cell ensheathment of axons also was slowed. After axotomy by a crush injury, the area of the posterior tibial fascicle was less than that in young adults and the distal disintegration of myelin sheath remnants also appeared to be retarded. The results indicate that responses of neurons, axons, and Schwann cells could be important in slowing the regeneration of myelinated fibers found in sciatic nerves from aging mice.     

Trigeminal alveolar nerve of the lower jaw in the cichlid Tilapia mariae: evidence for continual axon generation and presence of exceptionally small myelinated axons

Cross sections from the trigeminal alveolar nerve of the lower jaw in the cichlid Tilapia mariae were examined by electron microscopy. The nerve fibers are arranged in groups with a core of unmyelinated and small myelinated axons, surrounded by myelinated axons of varying sizes. The core contains large bundles of unmyelinated axons collectively ensheathed by circumferentially located Schwann cells, as well as smaller bundles of unmyelinated axons partly separated from each other by Schwann cell processes. Among the unmyelinated axons, occasional scattered profiles resembling growth cones are seen. The total number of axons in this tooth-related nerve increases from approximately 1,500 to 5,000, as the animals grow in length from 4.5 to 21.5 cm. Some 24-49% of the axons are unmyelinated. The myelinated axons have maximum diameters of 1.0-3.0 micron, depending on body size. Most myelinated axons have diameters less than 1.0 micron and the smallest ones reach down to 0.3 micron. These results show that there is a continual addition of axons to the alveolar nerve of the lower jaw in Tilapia mariae and that the critical diameter for myelination in this peripheral nerve is similar to that typically found in the mammalian CNS.     

Selective loss of small myelinated and unmyelinated fibers in Shy-Drager syndrome

Summary The number and sizes of myelinated and unmyelinated fibers in biopsied sural nerves in cases with Shy-Drager syndrome were studied in comparison with cases with olivopontocerebellar degeneration not having autonomic dysfunction. In Shy-Drager syndrome, there was a tendency for both small myelinated and unmyelinated fiber densities to be reduced in comparison with cases with olivopontocerebellar degeneration. Unmyelinated fibers more than 0.5 m in diameter were significantly reduced in Shy-Drager syndrome, a fact suggesting unmyelinated fiber degeneration. Multilamellated Schwann cell processes, isolated Schwann cell processes, and collagen pockets were more numerous and conspicuous in cases with Shy-Drager syndrome. It was concluded that unmyelinated fibers and small myelinated fibers in the peripheral nerves were involved selectively in Shy-Drager syndrome. The significance of the findings was discussed in terms of autonomic dysfunction observed clinically.     

Chlordecone intoxication in man. II. Ultrastructure of peripheral nerves and skeletal muscle.

Tremors, mental changes, opsoclonus, muscle weakness, gait ataxia, incoordination, and slurred speech developed in several employees in a Virginia chemical plant during the summer of 1974. Epidemiologic and clinical studies suggested that the chlorinated insecticide chlordecone (Kepone) was responsible. Severity of symptoms seemed directly related to dose and duration of exposure. Five sural nerve and six muscle biopsy specimens were examined by light microscopy and electronmicroscopy. The sural nerves were also evaluated by computerized morphometry, which showed considerable decrease in the number of unmyelinated fibers and lesser abnormalities of myelinated fibers. Compared with the nerves of the control subjects, those of patients may have had an increase in Reich and Elzholz bodies, and a modest increase in endoneurial collagen. There were occasional "collagen pockets," stacks of Schwann cell cytoplasmic membranes, redundant Schwann cell cytoplasmic folds, and fewer unmyelinated axons. The skeletal muscles contained increased amounts of lipofuscin and lipidlike droplets in subsarcolemmal areas and within intermyofibrillary spaces; the significance of this is unknown. Fiber size variability, type I predominance, and type grouping were present in three cases. All results strongly suggest that chlordecone is a neurotoxic agent predominantly affecting Schwann cells and unmyelinated fibers of peripheral nerves.     

The numbers of unmyelinated and myelinated axons in normal and regenerated rat saphenous nerves

Counts have been made of the numbers of unmyelinated and myelinated axons in the proximal and distal stumps of regenerated rat saphenous nerves and from equivalent sites in normal nerves. In the proximal part of normal nerves there were averages of 1 045 myelinated axons and 4 160 unmyelinated ones. Regenerated nerves contained the same number of myelinated axons in their proximal stumps but there was a 40% reduction in the unmyelinated axon count. In the distal stumps of these nerves the myelinated axon count had increased by an average of 620; this comes about because some regenerated myelinated axons support more than one process in the distal stump. In contrast, the number of unmyelinated axons was reduced further, from a mean of 2 476 in the proximal stump to one of 2 219.

The sizes of Schwann cell units in the normal and regenerated nerves were also noted. Schwann cell units in the proximal and distal stumps of the regenerated nerves were smaller than those in the normal ones.

These changes associated with unmyelinated axons in regenerated nerves are likely to contribute to the sensory, vasomotor and sudomotor abnormalities that sometimes occur after peripheral nerve injury and regeneration.     

Histochemical demonstration of neural carbonic anhydrase activity within the mandibular molar and incisor tooth pulps of the rat

Carbonic anhydrase (CA) activity was enzyme histochemically assessed in: (1) neuronal cell bodies in the rat trigeminal ganglion that were retrogradely labeled with FITC-WGA from the mandibular molar and incisor tooth pulps; (2) cell bodies retrogradely labeled with HRP-WGA from the mandibular molar tooth pulp; and (3) nerve fibers within the decalcified mandibular molar and incisor tooth pulps. The cell size spectrum of the FITC-WGA-labeled neurons was similar for both molar and incisor tooth pulps with about 90% being medium or large (greater than or equal to 300 microns2 in cross-sectional area). About 30% of the FITC-WGA-labeled cell bodies exhibited CA activity for both tooth pulps. The HRP-WGA-labeled cells also showed CA activity. In the molar root pulp, finely myelinated nerve fibers exhibited CA activity. Histochemical reaction products were distributed in the axoplasm and the cytoplasmic pocket of Schwann cell inside the compact myelin. In the incisor pulp, CA-reactive unmyelinated axons coexisted with non-reactive axons within the same Schwann units. The Schwann cell cytoplasm directly surrounding reactive axons also exhibited histochemical stainability. In the light of known distribution of CA in the peripheral nervous system, the present data suggest that the rat molar and incisor tooth pulps receive substantial innervation of large myelinated primary afferent fibers. The finely myelinated and unmyelinated axons, that have been repeatedly shown to predominate the intrapulpal nerve fibers, are considered to represent intrapulpal, terminal and preterminal specialization of otherwise large myelinated nerve fibers.     

Segregation by modality of myelinated and unmyelinated fibers in human sensory nerve fascicles

Thin diameter concentric needle electrodes with a small recording surface were used to explore the characteristics of neighboring fibers in sensory median nerve fascicles. Fibers which were close neighbors in the fascicle, were not randomly distributed intraneurally. Instead there was an intrafascicular segregation by modality of both myelinated and unmyelinated fibers. In addition, evidence was obtained which suggested that the contents of different Schwann cells vary. Some Schwann cell systems envelop afferent C fibers whereas others hold efferent sympathetic C axons. The results imply a modality segregation amongst both A and C fibers in human peripheral sensory nerve fascicles.     

Axonal degeneration and regeneration in sensory roots in a genital herpes model

Summary In a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, roots of the lower spinal cord were examined 5 days to 6 months after inoculation. Using immunoperoxidase methods on paraffin sections, viral antigen was found in sensory ganglia, their proximal roots and distal nerves on days 5 and 6 after infection. In Epon sections, most mice had focal sensory root abnormalities in lower thoracic, lumbar or sacral levels. At days 7 and 10, lesions showed chiefly nerve fiber degeneration, particularly of large myelinated fibers. At 2 weeks, lesions contained relatively large bundles of small unmyelinated fibers with immature axon-Schwann cell relationships. From 3 to 6 weeks, lesions again contained many more small unmyelinated fibers than normal but, in increasing proportions, axons in bundles were isolated from their neighbors by Schwann cell cytoplasm, and Schwann cells having 11 relationships with axons showed mesaxon or thin myelin sheath formation. At later times, the proportion of small unmyelinated axons decreased in parallel with increased numbers of small myelinated axons. By 6 months, affected roots showed a relative reduction in large myelinated fibers, increased proportions of small myelinated fibers and Schwann cell nuclei. Numbers of unmyelinated fibers were reduced relative to 3- to 6-week lesions. Axonal degeneration and regeneration appears to be the chief pathological change in sensory roots in this model. If regenerated fibers arise from latently infected neurons, then establishment of latency is not a relatively silent event, but is associated with major long-lasting, morphologically detectable effects.     

Hypertrophic Charcot-Marie-Tooth disease. Light and electron microscope studies of the sural nerve

Sural nerve biopsies from 15 patients with hypertrophic Charcot-Marie-Tooth disease have been studied by light and electron microscopy. There is a considerable variation in size of onion bulbs in patients from different kinships but the appearances were similar in patients from the same kinship. Comparison of sural nerve biopsies from patients within the same kinship suggests that with increasing age there is a progressive reduction in myelinated fibre density, an increased number of fibres undergoing demyelination and an increased frequency of onion bulb formations. Motor conduction velocities were reduced in all patients and were inversely proportional to the number of onion bulb lamellae, and to the proportion of demyelinated fibres found on sural nerve biopsy. Abnormalities of unmyelinated fibres were present in all the nerves studied. There was a relative increase in the density of denervated Schwann cell subunits and collagen pockets. The findings suggest that unmyelinated fibres undergo degeneration in the disease and lend some support to the hypothesis that the primary abnormality may be neuronal.     

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.     

The ultrastructure of intramuscular nerves in amyotrophic lateral sclerosis

Summary Muscle biopsies of 11 patients suffering from amyotrophic lateral sclerosis (ALS) were examined and the i.m. nerves found in seven of them were examined by electron microscopy. In atrophied muscles there was a marked decrease of myelinated fibers. The ultrastructure of the remaining myelinated axons showed changes in the neurofilaments, mitochondria, and vesicles. There was a decrease in the number of unmyelinated fibers as well as the myelinated fibers. Occasionally, there was an increase of unmyelinated fibers containing small fine axons. There were corpora amylacea in unmyelinated axons and banded structures in the extraccllular area of the Schwann cells of the unmyelinated fibers. Some of these findings were considered as the ultrastructural features of degeneration and regeneration in i.m. nerves of motoneurons in ALS.Supported in part by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan     

Monoamine oxidase-containing nerve fibers in the major cerebral arteries of rats

The localization of monoamine oxidase (MAO) in nerve fibers associated with the major cerebral arteries in rats was studied using a new coupled peroxidation method modified by adding nickel ammonium sulfate at the electron microscopic level. MAO was, localized in some unmyelinated axons, both in the adventitia and in the periadventitial nerve bundles. Schwann cell cytoplasm encircling myelinated axons in the periadventitial nerve bundles also contained a MAO-reactive substance. The incidences of MAO-containing axons in the adventitial layer of the anterior cerebral, middle cerebral, internal carotid and basilar arteries were 32.3%, 29.5%, 29.6% and 21.1%, respectively. In the periadventitial nerve bundles, MAO activity was also demonstrated in 10.8% among unmyelinated axons. Preincubation with clorgyline, a specific inhibitor of MAOA, suppressed staining in the axons, both in adventitia and in periadventitial nerve bundles, but not in the Schwann cell cytoplasm. Conversely, preincubation with deprenyl, a specific inhibitor of MAOB, suppressed staining in the Schwann cell cytoplasm, but not in the axons. Therefore, MAO in the axons is regarded as MAOA and MAO in the Schwann cell cytoplasm as MAOB. In immunosympathectomized rats (anti-NGF-treated rats), MAO reactivity was suppressed in axons associated with cerebral arteries, but was retained in some Schwann cell cytoplasm. The results indicate that the MAO-containing unmyelinated axons coincide with the postganglionic noradrenergic ones. Thus, histochemical MAO staining may be utilized to study postganglionic sympathetic nerve fibers presumably innervating the major cerebral arteries at the electron microscopic level.     

IgM deposits on skin nerves in anti-myelin-associated glycoprotein neuropathy

Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is a chronic demyelinating neuropathy with predominant involvement of large sensory fibers and deposits of IgM and complement on sural nerve myelinated fibers. We assessed the presence of IgM deposits on skin myelinated nerve fibers and the involvement of unmyelinated axons in anti-MAG neuropathy. Skin biopsies were performed in 14 patients with anti-MAG neuropathy, in 8 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and in 2 patients with IgM paraproteinemic neuropathy. Biopsies were taken at the proximal thigh in 20 patients, at the distal leg in 21 patients, at the proximal arm in 13 patients, and at the hand or fingertip in 10 patients. We found IgM deposits on dermal myelinated fibers in all anti-MAG neuropathy patients, with a greater prevalence at the distal site of the extremities. Deposits were located throughout the length of the fibers and at the paranodal loops. CIDP and IgM paraproteinemic neuropathies did not show any deposit of IgM. Anti-MAG neuropathy and CIPD patients showed a decrease in epidermal nerve fiber density reflecting an associated axonal loss. In anti-MAG neuropathy, both large- and small-diameter nerve fibers are affected, and specific deposits of IgM are found on skin myelinated nerve fibers.     

Sensory innervation of periodontal ligament of rat molars consists of unencapsulated Ruffini-like mechanoreceptors and free nerve endings

The trigeminal ganglion (TG) of adult rats was injected with 3H-amino acids to label periodontal receptors by axonal transport; 20-24 hours after injection, samples of molar ligament were prepared for autoradiography and electron microscopy. Four types of neurites labeled from TG were found in the avascular ligament fiber regions: large, complex, Ruffini-like endings, lacking a capsule, but with finger extensions touching ligament collagen; smaller Ruffini-like endings, lacking a capsule and neural fingers; free bundles of unmyelinated axons; and free, small, myelinated axons. The vascular channels plus associated loose connective tissue that perforate the ligament contained labeled preterminal ensheathed axons, small Ruffini endings, and free unmyelinated or small myelinated axons. The incidence of labeled endings was about 5 X greater next to the lower third of the root than in the upper two-thirds or beneath the root. The TG myelinated axons (diameter range 2-15 microns) entered the ligament in sheathed nerve bundles; these branched to form numerous small preterminal axons that were surrounded by a periaxonal fluid space and a perineurial sheath. Terminal axons branched from nodes of Ranvier, left the preterminal chamber, and followed an extended branching course through the collagen fibers. Large, complex Ruffini-like endings had numerous mitochondria and were partially covered by special lamellar Schwann cells and complex basal lamina; vesicles and multivesicular bodies were found near exposed regions of the receptor. Smaller Ruffini-like endings lacked neural fingers and had a simpler structure and less elaborate Schwann cells. The structure of Ruffini-like endings was highly varied; thus a structural continuum may exist from the largest, most complex to the smallest, simplest Ruffini-like receptor. The TG unmyelinated axons entered the ligament in ensheathed bundles; they then branched into free bundles that were found in the avascular ligament or near blood vessels. No encapsulated receptors were found.     

慢性炎性脱髓鞘性多发性神经根神经病周围神经细胞免疫与临床研究

目的研究慢性炎性脱髓鞘性多发性神经根神经病（chronic inflammatory demyelinating polyradic—uloneuropathy，CIDP）细胞免疫染色结果与临床、电生理和病理的关系。方法经周围神经活检确诊的12例CIDP神经活检标本和10例其它神经系统疾病患者的周围神经标本，用免疫组织化学染色的方法标记神经内膜的淋巴细胞、巨噬细胞和表达鼠抗人白细胞DR抗原（HLA-DR）的细胞，并分别计数，比较2组患者阳性细胞数量；分析CIDP患者3种阳性细胞数与临床、电生理和病理的关系。结果CIDP组与对照组比较，鼠抗人白细胞共同抗原（LCA）单克隆抗体、鼠抗人巨细胞（CD68）单克隆抗体、HDL-DR单克隆抗体的计数均有明显差异，P值分别为0．001、0．006和0．002；CIDP组HLA-DR阳性计数与CD68阳性计数之间有明显差异，P值为0．04，神经内膜水肿的LCA计数和无水肿的LCA计数比较有明显差异，P值为0．03，CD68阳性细胞在感觉神经传导速度减慢、神经纤维中重度减少的患者较相应的亚组有明显增高，且有显著差异，P值均为0．01，HLA-DR阳性计数在神经纤维中重度减少的患者也较相应的亚组有明显增高，有统计学差异，P值为0．01。结论CIDP患者神经内膜的炎性细胞浸润是较多见的病理特点，并与神经内膜水肿有关，巨噬细胞的浸润与感觉神经传导速度减慢以及神经纤维数量减少有关，病程较长时巨噬细胞和雪旺氏细胞都可能为HLA-II类抗原的抗原提呈细胞，雪旺氏细胞可能不仅为抗原提呈细胞，还可能同时参与对髓鞘的吞噬与破坏。     

Peripheral neuropathy in four cases of group A xeroderma pigmentosum

We describe the clinical features and findings of biopsied sural nerves of 4 cases of xeroderma pigmentosum. Nine genetic forms of xeroderma pigmentosum have been reported by complementation studies. These four cases were diagnosed as Group A xeroderma pigmentosum by complementation studies using cultured skin fibroblasts. All cases had delayed mental and motor development in areas such as head control over 4 months of age and walking without support over 18 months of age. Three cases had the gradual onset of a gait disturbance between 6 and 9 years of age. Motor conduction velocity and sensory conduction velocity of the ulnar nerve were slightly delayed. The sural nerve of the slightly impaired patient showed a normal density of myelinated fibers, but a selective reduction of the large myelinated fibers with zebra-body-like structures in Schwann cell cytoplasm. The population density of all nerve fibers was severely diminished in the severely impaired cases. Ultrastructural observation disclosed many denervated Schwann cells and pockets of collagen isolated by loops of denervated Schwann cell cytoplasm. These findings suggest that the degenerative process in peripheral nerves of xeroderma pigmentosum is axonal. Peripheral neuropathy in Group A xeroderma pigmentosum resembles that of patients with ataxia telangiectasia who are known to have a defect in the repair mechanisms of their DNA in cultured skin fibroblasts.     

Axonal branching following crush lesions of peripheral nerves of rat

Branching of myelinated and unmyelinated nerve fibers in normal and regenerating personal and soleus nerves was studied by light and electron microscopy. There were at most 2% more myelinated and 13% more unmyelinated axons in the distal as compared with the proximal nerve segments. Two to four weeks after a crush lesion the distal axons became 2-3 times more numerous; thereafter their number decreased. The number of axons in the proximal nerve segment did not change. The number of myelinated sprouts in most regenerated nerves equalled the number of myelinated fibers in the proximal nerve, while the number of unmyelinated axons after 12-19 weeks was 18-60% higher than normal. Branching was not restricted to the crush region. The results indicate that following a crush lesion all axons branch but only branches of unmyelinated fibers persist for a prolonged period of time. It is tentatively suggested that regenerating axons branch when searching for a target and that when contact is made with the target this prevents additional branching and eliminates redundant branches. Myelinated axons are guided by existing Schwann cells, whereas unmyelinated axons do not follow predetermined pathways; this may explain their greater tendency to form permanent branches.