Focal myelin swellings and tomacula in anti-MAG IgM paraproteinaemic neuropathy: Novel teased nerve fiber studies

Focal myelin swellings and tomacula in teased nerve fibers from a case of IgM anti-myelin-associated glycoprotein (MAG) paraproteinaemic neuropathy were examined using a novel technique. Five different morphologic abnormalities were identified--myelin sheath outfolding, myelin sheath infolding, enlargement of the adaxonal space, myelin degeneration, multiple increased concentric loops--and a combination of these structural abnormalities often occur in association with myelin degeneration. Similar structural changes were found in externally normal segments of teased fibers without evidence of myelin swelling or tomacula from the same case. These structural abnormalities are consistent with a disturbance of the normal adhesion functions of MAG in the maintenance of axon-myelin relationships.     

Peripheral myelin protein-22 expression in charcot-marie-tooth disease type 1a sural nerve biopsies

Peripheral myelin protein-22 (PMP22) is expressed in myelinating Schwann cells and shows significant homology to murine growth arest-specific gene gas3. Charcot-Marie-Tooth disease type 1a (CMT1a) is a common hereditary demyelinating neuropathy. Recently it was demonstrated that the gene for PMP22 is duplicated in CMT1a patients. A gene dosage mechanism has been postulated to cause CMT1a. According to this hypothesis, the increase in copy number of PMP22 gene would lead to an elevated expression of PMP22 and thereby cause the demyelinating phenotype of CMT1a. In the present communication we analyzed PMP22 mRNA and protein expression in sural nerve biopsies from CMT1a patients and normal controls. We show that PMP22 mRNA expression in CMT1a is not uniform. We found both elevated as well as normal PMP22 mRNA levels in patients. Interestingly, the highest PMP22 mRNA level was found in the least affected patient. In contrast to the mRNA levels, PMP22 was clearly reduced in all CMT1a patients as shown by immunohistochemistry. Thus the CMT1a phenotype may not be strictly correlated with increased PMP22 mRNA and protein expression. Possible roles of PMP22 in the pathogenesis of CMT1a are discussed. © 1994 Wiley-Liss, Inc.     

Sequelae of sural nerve biopsies

Sequelae of sural nerve biopsy were examined in 24 patients. Fourteen subjects reported persisting pain or dysaesthesias for more than one year. In nine patients the symptoms were mild, in five severe. Hypaesthesia of the lateral aspect of the foot was found in 17 out of 18 patients with otherwise normal or only slightly impaired sensory function. In one patient sural nerve biopsy did not cause permanent sensory loss. Pain and dysaesthesia were not significantly related to post-biopsy or generalized hypaesthesia.     

Polyglucosan bodies in sural nerve biopsies

Summary The presence of polyglucosan bodies in sural nerves collected over a 16-year period was studied in relation to age, sex, presence of polyneuropathy, and concomitant presence of central nervous system disorder. Polyglucosan bodies have been seen in only one patient without a polyneuropathy. This patient was suffering from Lafora's disease. In all other sural nerves positive for polyglucosan bodies a polyneuropathy was present. Within this group the prevalence of polyglucosan bodies was positively correlated with age, and if a central nervous system disorder was associated, this prevalence was more distinct. With semiquantitative measurements of the surface of polyglucosan bodies a significant correlation was found between age and percentage of large bodies.     

Early paranodal myelin swellings (tomacula) in an avian riboflavin deficiency model of demyelinating neuropathy

Introduction

Disruption of the complex architectural and molecular organization of the paranodal region of myelinated peripheral nerve fiber may initiate the evolving time dependent process of segmental demyelination. In support of this notion was the finding of focal paranodal myelin swellings (tomacula) due to redundant folding of myelin sheaths, early in the time course of an avian riboflavin deficiency model of demyelinating neuropathy.

Methods

Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin (control group) or a riboflavin-deficient diet containing 1.8 mg/kg riboflavin. Riboflavin concentrations in the liver were measured at postnatal day 11. Peripheral nerves were morphologically examined at days 6, 11, 16 and 21 using light and electron microscopy and teased nerve fiber techniques.

Results

Riboflavin-deficient chickens showed signs of a neuropathy from days 8 and pathological examination of peripheral nerves revealed a demyelinating neuropathy with paranodal tomacula formation starting on day 11. Paranodal tomacula consisted of redundant myelin infoldings or outfoldings, increased in size and frequency after day 11. After day 16, the paranodal swellings showed prominent degenerative changes accompanied by an increased frequency of myelinated fibers showing demyelination.

Conclusion

Tomacula due to redundant myelin folds are generally considered a remyelination phenomenon, yet in this avian riboflavin deficiency model of demyelination, the paranodal tomacula occurred early in the course of demyelination.     

HLA-DR-expressing cells and T-lymphocytes in sural nerve biopsies

Thirty-five sural nerve biopsies were stained immunohistochemically for HLA-DR antigen. HLA-DR was expressed on nonmyelinating Schwann cells, macrophages, vascular endothelium, and perineurium. By means of double immunofluorescence staining the identity of the HLA-DR presenting structures was confirmed. HLA-DR expression was found in all biopsies and thus was not restricted to any particular type of neuropathy. The HLA-DR expression appeared to correlate with severity and activity of the neuropathy. HLA-DR-expressing macrophages wrapping myelinated fibers were prominent in primary demyelinating neuropathies. T-cells were found in 6 out of 15 nerves examined. Their presence correlated with moderate to strong HLA-DR expression of nonmyelinating Schwann cells, and they occurred during active disease.     

Complications following sural and peroneal nerve biopsies

Nerve biopsy is used as part of the investigation of patients with peripheral neuropathy and is particularly useful in confirming the diagnosis of peripheral nerve vasculitis. Previous studies have suggested that sampling the peroneal nerve, in combination with peroneus brevis, is more sensitive than the sural nerve for this diagnosis but there are no published data on the complication rate of peroneal nerve biopsies. We have assessed the complications in 50 patients undergoing nerve biopsy, and have shown that although biopsy of the peroneal nerve may result in a larger area of sensory loss in some patients, other complications are not increased when compared with sural nerve biopsy.     

Dysmaturation neuromyopathy: correlation with minimal neuropathy in sural nerve biopsies

Dysmaturation (neuro)myopathy without specific histochemical or cytoarchitectural characteristics accounts for many cases of hypotonia in infancy. We obtained a maturation profile for type I and type II fibers from birth to 6 years of age, from which a classification of fiber dysmaturation based on fiber-type hypotrophy and coefficient of fiber variation is presented. We analyzed the morphometric, ultrastructural, and single fiber teasing findings in the sural nerve of ten infants with hypotonia and dysmaturation myopathy based on the above classification. Data on endoneurial area, myelinated fiber density, proportion of large (greater than 6 micron) myelinated fibers, unmyelinated fiber density, regression analyses of myelin area:axon area, and mean myelin thickness were developed. Abnormalities in large myelinated fiber density (3 cases), disturbances in myelination index (5), and single fiber teasing abnormalities in internode length (1) were found. Ultrastructural abnormalities were observed and classified as group 0 changes similar to the five control nerves; group 1, in which rare fibers showed degeneration or basement membrane duplication; and group 2, which was characterized by multiple abnormalities including degenerate nuclei, amyelinate fibers, degeneration/regeneration, excessive basal lamina reduplication, and hypomyelination. This study provides evidence that minimal neuropathic abnormalities are present in the sural nerve of infants carrying a diagnosis of dysmaturation neuromyopathy.     

Clinico-pathologic correlations in 78 biopsies of the sural nerve

Peripheral nerve biopsies when processed with conventional techniques for paraffin embedding usually do not provide sufficient data for the diagnostic conclusion. However, if the nerve is processed for resin embedding for semi and ultra-thin sections and teasing of fibres, several aspects can be analysed including quantitative and morphometric data. We studied the sural nerve biopsy of 78 patients examined at the Antonio Pedro University Hospital, Niterói RJ, applying those techniques and we found that in 55 cases (70.5%) the pathologic diagnosis was conclusive, in 11 (14.1%) although the nerve had abnormalities it was not possible to establish a diagnosis, and in 12 (15.4%) the nerve was normal. In 68 cases there was a clinical diagnosis which was confirmed in 49 but not in the remaining 19, since 8 had non-specific changes and 11 were normal. From the 10 cases which did not have a clinical diagnosis the biopsy was conclusive in 6, showed non-specific changes in 4, and was normal in 1 case. The pathologic conclusion in most of our cases was possible because not only we had the clinical data but all the nerves were processed for resin embedding.     

Cytokines in sural nerve biopsies from inflammatory and non-inflammatory neuropathies

Proinflammatory cytokines are supposed to play a major role in the pathophysiology of vasculitis and in the development of neuropathic pain. Here we studied the cytokine expression in sural nerve biopsy specimens from patients with vasculitic and other inflammatory and non-inflammatory neuropathies, and investigated whether an increased cytokine expression was correlated with the presence of neuropathic pain. We used immunohistochemistry including double labeling and morphometry to localize and quantify the expression of interleukin-1 beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF) in sural nerve biopsy samples of 41 patients with vasculitic neuropathy (VANP), chronic inflammatory demyelinating neuropathy (CIDP), non-inflammatory chronic axonal neuropathy (CANP), and 3 controls. Overall cytokine immunoreactivity was highest in VANP, less strong in CIDP and lowest in CANP. Cytokine immunoreactivity was directly correlated with the degree of axonal degeneration, endoneurial macrophages and epineurial T cells. In VANP and CANP, a higher cytokine content was associated with neuropathic pain.     

Application of immunohistochemical techniques to sural nerve biopsies.

The role of immunohistochemistry in the day-to-day diagnostic work of a peripheral nerve laboratory is not yet clearly established, although for conditions such as amyloid neuropathy, immunohistochemistry appears to be a useful adjunct to conventional techniques. Immunohistochemistry has provided new information about some neuropathies in which immune dysfunction is believed to play a central role. Immunohistochemical data about normal human nerve are scarce; a better appreciation of the normal cellular constituents of nerve, particularly the endoneurium, is needed. In the future, the techniques may be a means to understand better the pathogenesis of other types of neuropathy, such as inherited or toxic neuropathies, or to examine fundamental pathologic events such as axonal degeneration.     

Digital electron microscopic examination of human sural nerve biopsies

Diabetic peripheral polyneuropathy is characterized by axonal degeneration and regeneration as well as by Schwann cell and microvascular changes. These changes have been described at both the light (LM) and the electron microscopic (EM) levels; however, EM has not been applied to large clinical trials. Our goal was to adapt the rigorous techniques used for quantifying human biopsies with LM image analysis to accommodate ultrastructural analyses. We applied digital image capture and analysis to the ultrastructural examination of axons in sural nerve biopsies from diabetic patients enrolled in a multicenter clinical trial. The selection of sural nerve biopsies was based on the quality of specimen fixation, absence of physical distortion, and nerve fascicle size (> or =100,000; < or =425,000 microm2). Thin sections were collected on formvar-coated slot grids, stabilized with carbon and scanned on a Phillips CM100 transmission electron microscope. Digital images were captured with a Kodak Megaplus 1.6 camera. A montage was constructed using software derived from aerial mapping applications, and this virtual image was viewed by EM readers. Computer-assisted analyses included identification and labeling of individual axons and axons within regenerating clusters. The average density of regenerating myelinated axon clusters per mm2 was 65.8 +/- 5.1, range of 0-412 (n = 193). These techniques increase the number of samples that may be analyzed by EM and extend the use of this technique to clinical trials using tissue biopsies as a primary endpoint.     

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.     

Quantitative analysis of endoneurial T-cells in human sural nerve biopsies.

We used immunocytochemical methods on sural nerve biopsies from 42 patients with peripheral neuropathy to identify mononuclear cells, determine whether lymphocytic infiltration occurs in a variety of neuropathies, and identify the subtypes of lymphocytes. Immunostained cells were present in 76% of nerve biopsies. CD3+ cells (T lymphocytes) were greatest in density (cells/mm2). In patients whose CD4:CD8 T cell ratio was measured also in blood and cerebrospinal fluid, the CD4:CD8 T cell ratio was similar in all three compartments. These findings suggest that T lymphocytes are frequently present in nerves obtained from patients with various types of neuropathies and raise questions about factors that attract T lymphocytes into nerve that may be important in pathogenesis.     

Quantitative and qualitative study of sural nerve biopsies in Krabbe's disease

Summary Sural nerve biopsies from two infants with Krabbe's disease were examined morphologically, and quantitatively analyzed to obtain the density and size distribution of myelinated and unmyelinated nerve fibers as well as the variation in internodal lengths along the course of teased nerve fibers. When compared with age-matched control material, these sural nerves revealed a 50% reduction in density of myelinated fibers with a relative preponderance of small myelinated fibers. It was estimated that each nerve contained about 15% of the large (7–10) nerve fiber population found in control nerve. No reduction in the density of unmyelinated fibers was detected.Studies of teased preparations revealed an intermittent shortening of myelin internodes with resulting increased disparity of internodal lengths along individual fibers. These changes were indicative of widespread and severe demyelination and remyelination involving peripheral myelinated nerve fibers of all sizes in Krabbe's disease. No evidence of axonal or Wallerian degeneration was detected.Lipid accumulations were seen within endoneurial macrophage and within Schwann cells of myelinated fibers in both sural nerves and in the sympathetic chain removed at autopsy in case 2. The Schwann cell deposits were osmiophilic and appeared as focal collections of paranodal granules along myclinated fibers of teased preparations.Supported by U. S. P. H. S. Grants No. NB 08620 and NS 08054, and grants from the Allen P. and Josephine B. Green Foundation, Mexico, Missouri.     

Is quantitation necessary for assessment of sural nerve biopsies?

In this study we measured the reliability and accuracy of visual assessment of certain features of sural nerve pathology. Three raters visually assessed 20 sural nerves over two sessions. Four features were categorized: (1) myelinated fiber (MF) density; (2) size loss of MFs; (3) thinly myelinated axons; and (4) axonal clusters. Intra- and interrater reliabilities for the categories were determined. Quantitative data were compared with the visual assessments. Percentage agreements for single raters between the two sessions ranged from 35% to 100% and 9 times out of 12 were > or =70%. Interrater reliability, however, showed a kappa-value range of 0.03 (poor) to 0.49 (medium). In 85% of cases, visual ratings were within one category of the quantitated MF loss. However, visual categorization was poor compared with quantitation for determining size loss of MFs and myelin thickness. Quantitation needs to be considered to aid peripheral nerve pathologists in the assessment of some of the features of sural nerve biopsy specimens.     

Lymphocytes in sural nerve biopsies from patients with plasma cell dyscrasia and polyneuropathy.

Sural nerve biopsies were examined in 36 patients with plasmacell dyscrasia and polyneuropathy. The M-component isotype was IgM in 19, IgG in 16 (one patient had both IgM and IgG) and IgA in 2 patients. Five of the IgM patients had Waldenstr?m's disease, one of the IgG cases a myeloma and two lymphoma. The remaining 28 patients had monoclonal gammopathy of uncertain significance (MGUS). Nerve conduction studies showed signs of mixed axonal/demyelinating polyneuropathy in most cases. The biopsies were evaluated with regard to nerve fibre loss, segmental demyelination and inflammatory cell infiltration. Inflammatory cells infiltrating the nerves were found in 7 of 19 IgM cases and in 6 of 16 IgG cases. Immunohistochemical staining with monoclonal antibodies to B-lymphocytes and to subsets of T-lymphocytes was performed in 33 cases. An equal distribution of CD4 and CD8 positive T cells, but no B cells, were found among the inflammatory cells. The findings indicate a possible role for cell mediated immunological mechanisms in some patients with plasma cell dyscrasia associated with polyneuropathy.     

Epidemic neuropathy in Cuba: Morphological characterization of peripheral nerve lesions in sural nerve biopsies

More than 50 000 patients were affected in Cuba during an epidemic outbreak of peripheral neuropathy from January 1992 until September 1993. The disease presented as either a retrobulbar optic neuropathy, a predominantly sensory peripheral neuropathy, a dorsolateral myeloneuropathy, or as mixed forms. The morphological findings in sural nerve biopsies from 34 patients with various forms of the disease are presented here. Frozen, paraffin and semi-thin sections were prepared for light and electron microscopy, immunohistochemistry and morphometric analysis. Every case presented morphological alterations ranging from mild axonal dystrophy (9 cases, or 27%) to moderate and severe axonal damage (25 cases, or 73%). In 6 cases (18%), axonal damage was accompanied by perineural fibrosis and vascular abnormalities. Axonal regeneration was noted in 8 cases (23%) and remyelination in 9 (26%). Morphometric analysis showed a predominant loss of myelinated fibers in 92% of the patients. Quantification of myelinated fiber loss in 11 patients revealed a remarkable decrease in large caliber fibers. Scarce mononuclear cells were observed in 17 cases. No virus-like elements were seen. The morphological features found in this study indicate that, regardless of the clinical presentation, peripheral nerve lesions of the epidemic neuropathy in Cuba correspond to an axonal neuropathy. These lesions are compatible with nutritional, toxic, or metabolic etiologies. An inflammatory etiology would be unusual with these lesions.