Collagen Pockets Research Articles

Myelin protein zero mutation-related hereditary neuropathies: Neuropathological insight from a new nerve biopsy cohort.

Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.

Clinicopathological features in two families with MARS-related Charcot-Marie-Tooth disease.

Mutations in MARS gene cause dominant Charcot-Marie-Tooth disease (CMT) 2U. The aim of this study is to investigate phenotypic heterogeneities and peripheral neuropathology of MARS-related CMT patients. We identified a heterozygous p. R199Q mutation and an already reported heterozygous p. P800T mutation of MARS gene in two unrelated families using targeted next-generation sequencing. The first pedigree comprised three patients over three generations and the second pedigree comprised two patients over two generations. In addition of an asymptomatic carrier in the second pedigree, all patients presented with childhood-onset length dependent sensorimotor neuropathy with pes cavus. Nerve conduction studies revealed slowing of motor nerve conduction velocities (MNCV) of the median nerve indicating intermediate neuropathy in the patient with the p. R199Q mutation, and normal MNCV with reduced compound muscle action potential indicating axonal neuropathy in the patient with the p. P800T mutation. Magnetic resonance imaging detected a pattern of nerve changes similar to those in demyelinating polyneuropathies in intermediate type (p. R199Q mutation) patients compared with normal in the axonal type (p. P800T mutation) patients. Additionally, sural nerve biopsy revealed loss of myelinated axons with onion bulb formation in both mutations. By electron microscopy, a marked decrease of myelinated and unmyelinated fiber, neurofilaments aggregate with degenerating mitochondria and microtubule loss in axons were frequently found. Denervated Schwann cell complexes and few collagen pockets indicated involvement of unmyelinated Schwann cells. Therefore, the investigated MARS mutations cause not only the known axonal type but also intermediate type neuropathy with involvement of both axons and Schwann cells. Those findings are useful for the differential diagnosis of CMT patients with unknown MARS variants.

The spectrum of clinicopathological features in pure autonomic neuropathy

We assessed the clinicopathological features of nine patients with pure autonomic neuropathy, that is, neuropathy without sensory or motor deficits. The duration from symptom onset to diagnosis ranged from 1 month to 13 years. Of eight patients in whom serum antiganglionic acetylcholine receptor antibody was determined, four were positive. All patients who tested positive for this antibody manifested widespread autonomic dysfunction, with the exception of one patient who only experienced orthostatic hypotension. However, patients who were negative for the antiganglionic acetylcholine receptor antibody presented with partial autonomic failure. One of these patients had diffuse parasympathetic failure and generalized hypohidrosis but no orthostatic hypotension, which is clinically compatible with postganglionic cholinergic dysautonomia. Electron microscopic examination revealed a variable degree of reduction in unmyelinated fibers. Compared with normal controls, the patients had a significantly increased density of collagen pockets (p < 0.05). Additionally, the percentage of Schwann cell subunits with axons (out of the total number of Schwann cell subunits associated with unmyelinated fibers) was significantly decreased (p < 0.01). The density of unmyelinated fibers tended to decrease with increasing time between the onset of autonomic symptoms and biopsy (p < 0.05). In conclusion, the clinical and pathological features of pure autonomic neuropathy vary in terms of progression, autonomic involvement, presence of the antiganglionic acetylcholine receptor antibody, and loss of unmyelinated fibers.

Ultrastructural Changes of Schwann Cells during Nerve Regeneration Following a Crush Injury of the Sural Nerve in Rats

Abstract Twenty-four male albino rats (200 to 250 g in weight) were used in the present study. The left sural nerve of 18 rats was subjected to crush injury while the sural nerves of 6 animals were used as control. After one week of the crush injury the Schwann cells showed multiple cytoplasmic processes. Those with long cytoplasmic processes wrapped only one axon while those wrapping multiple unmyelinated axons showed shorter processes. Some of these processes wrapped or surrounded collagen bundles “collagen pockets” and degenerated myelin, and some contained electronlucent vacuoles. Schwann cell cytoplasm showed asymmetric hypertrophy and contained dilated rough endoplasmic reticulum and ribosomes. Also, electronlucent vacuoles and whorls of degenerated myelin were seen in the cytoplasm of some Schwann cells. Schwann cells were surrounded by basal laminae which may be redundant. Two weeks post-crush, the number of regenerating Schwann cells increased and the myelin sheaths covering the myelinated axons were thicker. Schwann cells possessed long cytoplasmic processes that wrapped unmyelinated axons. After the third week of the crush injury, the Schwann cells wrapped shrunken myelinated axons with degeneration of the myelin of such axons. The number of myelinated axons increased, together with the thickness of their myelin sheath. It could be concluded that the Schwann cells play a phagocytic role during regeneration of peripheral nerves which is indicated by the presence of cytoplasmic vacuoles and degenerated myelin. Such phagocytic process might be performed by the use of their cytoplasmic processes.

Unmyelinated fiber sensory neuropathy differs in type 1 and type 2 diabetes

Neuropathic pain is common in diabetic patients. Degeneration of sensory C-fibers in peripheral nerve plays a prominent role in the generation of neuropathic pain. We examined degenerative changes of C-fibers in two rat models with type 1 and type 2 diabetes. Type 1 insulinopenic BB/Wor and type 2 hyperinsulinemic diabetic BBZDR/Wor-rats of 8 months duration with equal exposure to hyperglycemia were examined. Thermal hyperalgesia was monitored using an infrared thermal probe. C-fiber size, number, frequencies of denervated Schwann cells, regenerating C-fibers, type 2 axon/Schwann cell relationship and collagen pockets in the sural nerve were examined morphometrically. Neurotrophic receptor expression was examined by Western blotting. Neurotrophins and neuropeptides were examined by ELISA. Type 1 rats showed increased thermal hyperalgesia followed by a decrease. Hyperalgesia in type 2 rats showed a slower progression. These findings were associated with a 50% (p < 0.001) loss of C-fibers, increased frequencies of denervated Schwann cells (p < 0.001), regenerating fibers (p < 0.001), collagen pockets (p < 0.001) and type 2 axon/Schwann cell relationship (p < 0.001) in type 1, but not in type 2 rats. Expression of insulin receptor, IGF-1R, TrkA and C was decreased in BB/Wor rats, whereas BBZDR/Wor rats showed milder or no deficits. NGF and NT-3 in sciatic nerve and substance P and calcitonin gene-related peptide in dorsal root ganglia were decreased in type 1, but not in type 2 rats. The more severe molecular, functional and morphometric abnormalities of nociceptive C-fibers in type 1 insulinopenic rats compared to type 2 hyperinsulinemic rats suggest that impaired insulin action may play a more important pathogenetic role than hyperglycemia per se.

UNMYELINATED NERVE FIBER DEGENERATION IN CHRONIC INFLAMMATORY DEMYELINATING POLYNEUROPATHY

To determine whether unmyelinated nerve fibers escape degeneration as one might expect in an immune response exclusively directed at myelin, we performed a morphometric examination of unmyelinated axons and myelinated nerve fibers in sural nerve biopsy specimens of 14 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and of 12 age‐matched normal controls. The numbers of unmyelinated axons, myelinated nerve fibers, denervated Schwann cell units and collagen pockets were quantified and related to the clinical and electrophysiological data of the patients with CIDP. In 4 patients with a rapid onset of the neuropathy and a highly elevated CSF protein, the numbers of both unmyelinated axons and myelinated nerve fibers were decreased equally. In 8 patients we found that the unmyelinated axons were relatively spared compared with the loss of myelinated nerve fibers. In these patients, however, the presence of denervated Schwann cell units and of collagen pockets was increased. We conclude that unmyelinated nerve fibers are affected in patients with CIDP.

Unmyelinated nerve fiber degeneration in chronic inflammatory demyelinating polyneuropathy.

To determine whether unmyelinated nerve fibers escape degeneration as one might expect in an immune response exclusively directed at myelin, we performed a morphometric examination of unmyelinated axons and myelinated nerve fibers in sural nerve biopsy specimens of 14 patients with a chronic inflammatory demyelinating polyneuropathy (CIDP) and of 12 age-matched normal controls. The numbers of unmyelinated axons, myelinated nerve fibers, denervated Schwann cell units and collagen pockets were quantified and related to the clinical and electrophysiological data of the patients with CIDP. In 4 patients with a rapid onset of the neuropathy and a highly elevated CSF protein, the numbers of both unmyelinated axons and myelinated nerve fibers were decreased equally. In 8 patients we found that the unmyelinated axons were relatively spared compared with the loss of myelinated nerve fibers. In these patients, however, the presence of denervated Schwann cell units and of collagen pockets was increased. We conclude that unmyelinated nerve fibers are affected in patients with CIDP.

Morphometric and ultrastructural changes with ageing in mouse peripheral nerve.

Qualitative and quantitative information is reported on the morphological changes that occur in nerve fibres and nonneuronal cells of peripheral nerve during the lifetime of the mouse. Tibial nerves of mice aged 6-33 mo were studied. With ageing, collagen accumulates in the perineurium and lipid droplets in the perineurial cells. Macrophages and mast cells increase in number, and onion bulbs and collagen pockets are frequently present. Schwann cells associated with myelinated fibres (MF) slightly decrease in number in parallel with an increase of the internodal length from 6 to 12 mo, but increase in older nerves when demyelination and remyelination are common. The unmyelinated axon to myelinated fibre (UA/MF) ratio was about 2 until 12 mo, decreasing to 1.6 by 27 mo. In older mice, the loss of nerve fibres involves UA (50% loss of 27-33 mo cf. 6 mo) more markedly than MF (35%). In aged nerves wide incisures and infolded or outfolded myelin loops are frequent, resulting in an increased irregularity in the morphology of fibres along the internodes. In the mouse there is an adult time period, 12-20 mo, during which several features of degeneration progressively appear, and an ageing period from 20 mo upwards when the nerve suffers a general disorganisation and marked fibre loss.

Morphometric and ultrastructural changes with ageing in mouse peripheral nerve

Qualitative and quantitative information is reported on the morphological changes that occur in nerve fibres and nonneuronal cells of peripheral nerve during the lifetime of the mouse. Tibial nerves of mice aged 6–33 mo were studied. With ageing, collagen accumulates in the perineurium and lipid droplets in the perineurial cells. Macrophages and mast cells increase in number, and onion bulbs and collagen pockets are frequently present. Schwann cells associated with myelinated fibres (MF) slightly decrease in number in parallel with an increase of the internodal length from 6 to 12 mo, but increase in older nerves when demyelination and remyelination are common. The unmyelinated axon to myelinated fibre (UA/MF) ratio was about 2 until 12 mo, decreasing to 1.6 by 27 mo. In older mice, the loss of nerve fibres involves UA (50% loss of 27–33 mo cf. 6 mo) more markedly than MF (35%). In aged nerves wide incisures and infolded or outfolded myelin loops are frequent, resulting in an increased irregularity in the morphology of fibres along the internodes. In the mouse there is an adult time period, 12–20 mo, during which several features of degeneration progressively appear, and an ageing period from 20 mo upwards when the nerve suffers a general disorganisation and marked fibre loss.

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 density 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.

MORPHOLOGICAL CHANGES IN UNMYELINATED NERVE FIBRES IN THE SURAL NERVE WITH AGE

Quantitative changes in unmyelinated nerve fibres in sural nerves obtained at autopsy were evaluated in 28 normal adults. The following conclusions were reached. (1) The density of unmyelinated axons showed no significant correlation with age, but the densities of (2) Schwann cell subunits with axons, (3) Schwann cell subunits without axons, (4) single protrusions of Schwann cells and (5) collagen pockets, and (6) the mean number of Schwann cell profiles per axon, all showed positive correlations with age. Additionally, (7) the percentage of subunits containing unmyelinated axons and (8) the mean number of axons in single axon-containing Schwann cell subunits demonstrated negative correlations with age. The density of Schwann cell nuclei related to unmyelinated fibres did not show a significant change with age. The age-dependent changes in unmyelinated nerve fibres thus mainly consist of an increased production of processes by Schwann cells in the absence of cell multiplication. A decrease in unmyelinated nerve fibre density or a compensatory increase of small unmyelinated axons did not occur in these normal adults. In terms of relative sensitivity for the detection of the earliest changes in unmyelinated fibres, the indices (6) and (7) are considered to be useful and superior to the conventional assessment of unmyelinated axon density and diameter distribution. These two indices are not influenced by postmortem swelling of the axons and Schwann cells. Measurements of unmyelinated axon density and size distribution will continue to be useful in the assessment of more advanced pathological conditions.

Endothelial Collagen Pockets in Angiosarcomas

In the most malignant areas of angiosarcomas, collagen bundles impinge on the endothelial cells, sink into ‘collagen pockets’ that migrate throughout the cytoplasm and build up cord-like bridges within the lumen.

Quantitative histologic study of sural nerves in xeroderma pigmentosum

The sural nerves of 2 siblings, 7 and 6 years of age with Group A xeroderma pigmentosum, were biopsied. The densities of myelinated fibers, 5,808/mm 2 and 5,163/mm 2, respectively, were strikingly decreased in comparison to control data. Both large and small myelinated fibers were reduced. Electron microscopy demonstrated many collagen fibers in the endoneurium and some collagen pockets. The loss of myelinated fibers was less severe than in previously reported patients. This discrepancy may be due to age differences at biopsy; our patients were biopsied at the ages of 7 and 6 years, while those patients reported previously were 10 years of age or older. The incidence of neurologic manifestations in xeroderma pigmentosum may increase after 6 years of age.

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

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 micrometers in diameter were significantly reduced in Shy-Drager syndrome, a fact suggesting myelinated 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.

Peripheral nerve abnormalities in aging rats.

The sciatic/tibial/plantar nerve complex of normal aged rats displays striking morphological changes that are most pronounced distally. Nerve fiber abnormalities include: (a) large numbers of axonal glycogenosomes, mitochondria, dense membranous bodies, and decorated particles; (b) adaxonal Schwann cell processes sequestering portions of axoplasm; (c) swollen demyelinated and remyelinated axons, some encircled by supernumerary cellular processes; and (d) collagen pockets, denervated Schwann cell columns, and empty basal laminae. Abnormalities a and b were encountered with increasing frequency on descent through the tibial and plantar nerves. Abnormalities b, c, and d were found in lateral and medial plantar nerves, where they were associated with an enlarged endoneurial space. Found in animals kept in cages with smooth or wire-mesh floors, the incidence of these changes increased with advancing age. They are attributed to trauma and ischemia from chronic pressure on the plantar nerve.

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.

Study on the ultrastructure and acetylcholinesterase activity in von Recklinghausen's neurofibromatosis.

The cutaneous nodules obtained from seven patients with von Recklinghausen's neurofibromatosis were investigated by electron microscopy, and ultrastructural localization of acetylcholinesterase activity was demonstrated in the nerve fibers of this tumor for the first time using Karnovsky's thiocholine method. The enzymatic activity was mainly found in unmyelinated fibers, exactly associated with their axonal membranes, the interspace between the apposing axonal and Schwann cell membrane, and some different mesaxons, which indicated their cholinergic nature. Almost all myelinated fibers and some unmyelinated fibers did not possess the activity. The relationship between axon and Schwann cell was quite similar to that of normal peripheral nervous system, but two striking alterations of the nerves existed: One is the dissociation of unmyelinated fibers, and the other is the degenerative changes of the axon and the myelin sheath. As the evidence of schwannian proliferation, onion bulb formations and collagen pockets were observed. Some signs of fibroblastic proliferation were also found. From the present study and the review of the literature, the probable histogenesis of this disease was discussed.

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.

ELECTRON MICROSCOPY OF CUTANEOUS NERVES AND RECEPTORS

Recent ultrastructural observations on the connective tissue sheaths of nerves, Schwann cell-axonal relations, and nerve terminals and receptors are reviewed. It seems likely that endoneurial collagen is formed by perineurial cells during development and postnatally. New observations on "collagen pockets" are presented. Attention is drawn to freeze-fracture studies of peripheral nerve, particularly in relation to junctional complexes associated with compact myelin, and further application of the technique is considered. Current views on Merkel cells, encapsulated endings, and free nerve terminals are discussed.

UNMYELINATED FIBRES AND SCHWANN CELLS OF SURAL NERVE IN NEUROPATHY

Electron micrographs of 45 sural nerves from patients with acquired (22) or heredodegenerative neuropathy (23) were analysed with respect to the number of unmyelinated nerve fibres, 37 nerves with respect to the number of Schwann cell sub-units and of structures connected with Schwann cells. Findings were compared with those in 6 nerves from control subjects and referred to the total number rather than to the number per mm2 to eliminate error due to increase in the transverse endoneurial area, present in more than half the diseased nerves. Ninety-one per cent of the diseased nerves showed one or several abnormalities in unmyelinated fibres of their Schwann cells. The best indicator of fibre loss was an increase in the number of Schwann cell sub-units devoid of axons, found in more than half the nerves. This was the only abnormality related with decrease in number of myelinated fibres. The increase in number of empty Schwann cell sub-units was due both to loss of unmyelinated nerve fibres and to proliferation of Schwann cells. Proliferation was indicated by the higher incidence of Schwann cell nuclei in cross-sections of diseased nerves than in controls. The earliest sign of involvement was an increase in number of profiles and of small isolated Schwann cell projections, observed in 33 of 37 diseased nerves, as the only abnormality in 7 nerves. The number of unmyelinated nerve fibres by itself was of little value to indicate loss of fibres, since regeneration often replaced or more than replaced degenerated fibres. Regeneration was indicated by an increase in number or incidence of small unmyelinated fibres, present in nearly half of 45 diseased nerves; and by an increased in the total number, present in a third of the nerves. An increase in the number of collagen pockets and of fibres undergoing degeneration (loss of organelles) and a decrease in the number of unmyelinated fibres per Schwann cell sub-units was present in only a quarter to a third of diseased nerves and was not related to other criteria of loss of fibres or of regneration.