MYELINATED AND UNMYELINATED ENDONEURIAL AXON QUANTITATION AND CLINICAL CORRELATION AMIR DORI, MD, PhD,1,2 GLENN LOPATE, MD,2 RATI CHOKSI, MS,2 and ALAN PESTRONK, MD2 1 Department of Neurology, Talpiot medical leadership program, Chaim Sheba Medical Center, Tel HaShomer, Israel, 52621 and Joseph Sagol neuroscience center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Accepted 15 June 2015 ABSTRACT: Introduction: Different disease patterns result from loss of myelinated and unmyelinated axons, but quantitation to define their loss has been difficult. Methods: We measured large and small endoneurial axons in axonal neuropathies by staining them with peripherin and comparing their area to that of nonmyelinating Schwann cells stained with neural cell adhesion molecule (NCAM). Results: Loss of myelinated and unmyelinated axons was typically proportional, with predominant myelinated or unmyelinated axon loss in a few patients. Myelinated axon loss was associated with loss of distal vibration sense and sensory potentials (P < 0.0001) and was selective in patients with bariatric and bowel resection surgery (P < 0.001). Unmyelinated axon measurements correlated with skin (ankle P 5 0.01; thigh P 5 0.02) and vascular (nerve P < 0.0001; muscle P 5 0.01) innervation. Conclusions: Myelinated and unmyelinated axons can be quantitated by comparing areas of axons and nonmyelinating Schwann cells. Clinical features correlate with myelinated axon loss, and unmyelinated axon loss correlates with skin and vascular denervation. Muscle Nerve 53: 198–204, 2016
Evaluation of large versus small axon loss in nerve biopsies is important for their association with clinical syndromes such as ataxia and pain, respectively. This is usually based on qualitative visual inspection, while more quantitative measurements have required laborious morphological evaluation of fixed nerves and visualized ultrastructure.1,2 We recently found that unmyelinated perivascular axons could be quantitated by labeling the neurofilament peripherin and comparing their area to their associated nonmyelinating Schwann cells labeled with neural cell adhesion molecule (NCAM).3 Peripherin is also present in myelinated axons and is, therefore, suitable for comparison of neurofilament loss in myelinated versus unmyelinated axons. In this study, we used and validated similar methods to quantify endoneurial myelinated and unmyelinated peripherin-containing Abbreviations: DAPI, 40 ,6-diamidine-2-phenylidole-dihydrochloride; ICC, intraclass correlation coefficient; IENFD, intraepidermal nerve fiber density; LFN, large fiber neuropathy; MAS, myelinated axons to nonmyelinating Schwann cell cytoplasm areas; NCAM, neural cell adhesion molecule; PBS, phosphate-buffered saline; RGBB, red, green, blue; ROC, receiver operating characteristic; SFN, small fiber neuropathy; UAS, unmyelinated axons to nonmyelinating Schwann cell cytoplasm areas. Key words: myelinated axons; neuropathy; sural nerve biopsy; unmyelinated axons; vascular innervation Correspondence to: A. Dori; e-mail: [email protected] Additional Supporting Information may be found in the online version of this article. C 2015 Wiley Periodicals, Inc. V
Published online 16 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24740
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axons in nerves from patients with neuropathies who also had skin and/or muscle biopsies. We compared quantities of endoneurial myelinated and unmyelinated axons with clinical and electrophysiological features, axon counts in skin, and innervation of epineurial and perimysial vessels. METHODS AND MATERIALS Patients. We retrospectively reviewed records of 34 consecutive patients (19 men, 15 women; ages 15 to 81 years, mean 55) who were evaluated between July 2008 and March 2013 at Washington University in Saint Louis for axonal neuropathy and had sural nerve and muscle or skin biopsies (Supplementary Table S1, which is available online). Biopsies of muscle were performed in 33 and skin in 12 patients (ankle and thigh 8, and ankle only 4). Neuropathy diagnoses based on electrodiagnostic studies or skin biopsy were selective small fiber neuropathy (SFN, 6 patients) and large 6 small fiber syndromes (LFN, 28 patients), including distal polyneuropathy (19), multiple mononeuropathies (7), and lumbosacral plexopathy (2). The 12 patients with skin biopsies (8 men, 4 women, ages 15 to 69 years, mean 49), were diagnosed with SFN (4) or LFN (8). Biopsies. All nerve, muscle, and skin biopsies were processed and evaluated at the Washington University Neuromuscular Laboratory. Muscle and nerve biopsies were all performed on the same day. Skin biopsies were performed within 16 months (median 7 weeks) of muscle biopsy. Muscles evaluated included quadriceps (21), gastrocnemius (7), deltoid (4), and biceps brachii (1). Nerves and muscles were rapidly frozen, and cryostat sections were collected and processed for histochemistry and immunohistochemistry in our standard manner.4,5 Toluidine blue plastic sections of nerves were processed following fixation in glutaraldehyde as previously described.6,7 Muscle and nerve pathologic assessments were all performed by AP. Skin biopsies (3.0 mm punch) were performed at 2 sites: at the ankle, 10 cm above the lateral malleolus; and at the thigh, 10 cm above the patella (8 patients); or only at the ankle (4 patients). Skin was fixed and processed using standard procedures and techniques to determine the intra-epidermal nerve fiber density (IENFD).8,9 IENFD quantitation was performed by MUSCLE & NERVE
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GL. Normal values for skin biopsies were based on published data.10 An IENFD at or below the fifth percentile per age was defined as abnormal. The human studies committee of Washington University in St Louis approved all procedures. Informed consent was not required. Quantitation of Unmyelinated and Myelinated Axons.
Staining of axons with peripherin and of nonmyelinating Schwann cells with NCAM was performed as previously described.3 Briefly, fresh frozen 16-mm sections were mounted on glass slides, fixed in cold acetone, and incubated with antibodies to peripherin and NCAM followed by incubation with Alexa Fluor 488 Goat anti-mouse IgG1 and Alexa Fluor 594 Goat anti-mouse IgG2b, and mounted with antifade reagent with DAPI (40 ,6-diamidine-2phenylidole-dihydrochloride). Image acquisition was based on obtaining consistent and similar signal intensities for peripherin and NCAM staining within an exposure range that avoided both oversaturation and high background. Exposure times in the photographed field for NCAM labeled Schwann processes (red) were adjusted using Olympus camera controller software (CellSens) to a color intensity of 200 on the RBG scale. Exposure times in the photographed field for peripherin-labeled axons (green) were similarly adjusted to color intensity of 200 on the RBG scale, or to the highest level that maintained the background below 10 on the RBG scale. For digital quantitative analysis, endoneurium and perivascular nerves from acquired images were selected with a “hand-drawn free select tool” of the GIMP software. An intensity threshold of 128 on the RGB (red, green, blue) scale was used to transform each color to a binary, “all or none” mode. Resulting images had 4 colors: red, green, yellow, and black. Quantitation of peripherin- and NCAMlabeled areas was performed by measuring selective color pixel areas. The pixel areas of yellow (axons embedded within nonmyelinating Schwann cells), red (nonmyelinating Schwann cell area without axons), and green (myelinated axons not embedded in nonmyelinating Schwann cells) were measured separately. Ratios of unmyelinated and myelinated axons to nonmyelinating Schwann cell cytoplasm areas (UAS3 and MAS, respectively) were calculated. A minimum of 8 image samples of endoneurium from randomly selected different fascicles or from nonadjacent sections and perivascular nerves were used to obtain mean ratios. Measurements and calculations were made without knowledge of IENFD or clinical data. Myelinated axon loss on toluidine blue plastic sections was rated on a 0–6 scale as follows: no loss, 0; minimal/mild, 1; mild–moderate, 2; Endoneurial Axon Quantitation
moderate, 3; moderate–severe, 4; severe, 5; nearly complete loss, 6. Statistical Analysis. Comparisons for correlations were performed using 2-sided Pearson product correlations. Intraclass correlation coefficient (ICC) was used to assess rater consistency and reliability; and inter-rater agreement was determined by kappa (quadratic weighted) analysis. Spearman coefficients were used to assess rank correlation (rho). The diagnostic yield of axon loss analysis was estimated by area under the curve analysis of receiver operating characteristic (ROC) curves. The Fisher exact test was used to calculate P-values for frequency. Statistical analyses were performed using MedCalc for Windows, version 13.1.2.0 (MedCalc Software, Ostend, Belgium). Values are expressed as mean 6 standard deviation. RESULTS Unmyelinated Are
and
Differentially
Myelinated Labeled
Endoneurial
by
Axons
Peripherin/NCAM
Immunofluorescence. In normal appearing sural nerve endoneurium, peripherin labeled large and small axons (Fig. 1A). Small and large axons were qualitatively differentiated by double staining of nonmyelinating Schwann cells with NCAM (Fig. 1B).11 Merged images (Fig. 1C) showed yellow, colabeled, small axons within red NCAM-labeled nonmyelinating Schwann cells (arrowheads in Fig. 1D–G). Large and intermediate size axons were green, labeled only by peripherin, and surrounded by a black halo, consistent with myelin sheaths (small arrows in Fig. 1D–G). In normal nerves, clusters of larger green axons separated dense groups of unmyelinated axons which appeared as islands throughout the endoneurium. With loss of large (NCAM-negative) axons, the area of large green peripherin-labeled axons was reduced (Fig. 1H–K). With loss of unmyelinated axons, yellow small axons within red NCAM-labeled Schwann cells were reduced (empty arrows in Fig. 1L–O). Endoneurial
Unmyelinated
and
Myelinated
Axon/
Nonmyelinating Schwann Cell Area (UAS and MAS) Ratios Are Reliable Measures. We quantitated unmyelinated axons in endoneurial Remak bundles by calculating UAS ratios. Low UAS ratios represent a loss of unmyelinated axons with preserved nonmyelinating Schwann cells. Consistency analysis of these measurements was performed by comparing 2 raters. The ICC of 20 endoneurial UAS ratio measurements (by A.D.) was 0.80 for absolute agreement among average measurements and ICC 5 0.81 for consistency among average measurements. Inter-rater agreement (kappa) showed a good correlation (k 5 0.71; standard error 5 0.17) between our 2 raters (A.D. and R.C). Myelinated MUSCLE & NERVE
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FIGURE 1. Peripherin-stained, unmyelinated axons colocalize with NCAM-stained Schwann cells, while myelinated axons do not. Fluorescent immunolabeled images of green peripherin (A,D,H,L) and red NCAM (B,E,I,M), with yellow colocalization (C,F,J,N) and multicolor digital transformation (G,K,O). D–G shows a nearly normal nerve. H–K shows a nerve with predominant myelinated axon loss. L–O shows a nerve with predominant small unmyelinated axon loss. Ratios of green to red (G/R) determine myelinated axon/nonmyelinating Schwann cell area ratios. Ratios of yellow to red (Y/R) determine unmyelinated axon/nonmyelinating Schwann cell area ratios. Scale bars 5 50 mm. NCAM, neural cell adhesion molecule.
axons were quantitated by calculating the MAS ratios, using the same denominator used for quantitating unmyelinated axon loss. The ICC for MAS measurements of the same nerves (by A.D.) was 0.77 for absolute agreement among average measurements and ICC 5 0.74 for consistency among average measurements. Inter-rater agreement (kappa) showed a good correlation (k 5 0.84; standard error 5 0.054) between our 2 raters (A.D. and 200
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R.C). MAS ratios showed a correlation (rho 5 0.655, P < 0.0001, 95% confidence interval 0.41–0.81) with the severity of myelinated axon loss on toluidine blue plastic sections. Predominant Endoneurial Unmyelinated, or Myelinated, Axon
Loss
Is
Associated
with
Different
Clinical
UAS and MAS ratios showed a general correlation between loss of Neuropathy Characteristics.
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myelinated axon loss was calculated by ROC analysis (Fig. 2B). An MAS ratio of 0.13 predicted distal vibration sense loss with sensitivity of 96% and specificity of 70% (area under curve 5 0.88; P < 0.0001). An MAS ratio of 0.10 predicted absent distal SNAPs with sensitivity of 94% and specificity of 79% (area under curve 5 0.95; P < 0.0001). Unmyelinated axon loss determined by UAS ratios showed a less strong association with distal vibration sense loss (P 5 0.02). Patients with diagnoses of small fiber neuropathy were common above a myelinated-unmyelinated axon loss regression line, at the top of, or above the 95% confidence interval (Fig. 2A). Nine patients with predominant myelinated axon loss were identified below the 95% confidence interval. Five of them had a history of gastrointestinal surgery (3 bariatric, 2 colon resection). Three patients with gastrointestinal surgery had documented nutritional deficiencies (copper in 2, and vitamin B1, B6, and B12 deficiencies in another). One additional patient with a history of small bowel resection presented with near-complete loss of both myelinated and unmyelinated axons. None of the remainder of the patients in this cohort had a history of bowel resection. A history of bariatric or bowel resection surgery was associated with markedly predominant myelinated peripherin-axon loss (P < 0.001). Correlations of Small Axon Loss among Different
FIGURE 2. Endoneurial myelinated versus unmyelinated axon loss and clinical correlation. (A) Myelinated versus unmyelinated axon/nonmyelinating Schwann cell area ratios. Vibration sense, fiber type of neuropathy, and history of bowel surgery are shown. Regression lines and 95% confidence intervals are marked. (B) Comparison of ROC curves of myelinated and unmyelinated axon/nonmyelinating Schwann cell area ratios classifying vibration sense loss. Myelinated axon/nonmyelinating Schwann cell area ratios area under curve 5 0.88; standard error 5 0.07; 95% confidence interval 5 0.72 to 0.97. Unmyelinated axon/nonmyelinating Schwann cell area ratios area under curve 5 0.72; standard error 5 0.10; 95% confidence interval 5 0.55 to 0.87. GI 5 gastrointestinal.
unmyelinated and myelinated axons (Fig. 2A; r 5 0.56, P 5 0.001). Some nerves, however, showed more prominent loss of myelinated or unmyelinated axons. Patients with severe loss of myelinated axons showed distal loss of vibration sensation even when unmyelinated axons were relatively spared. The diagnostic efficiency and optimal cutoff value of low MAS ratios for the diagnosis of Endoneurial Axon Quantitation
Tissues. To validate the UAS quantitative measurement of endoneurial axons, we compared the UAS ratio with IENFD counts in skin biopsies from 12 patients (Table S1, available online). Endoneurial UAS ratios correlated with IENFD in both ankle (r 5 0.70; P 5 0.01) and thigh (r 5 0.80; P 5 0.02) skin (Fig. 3A). Epineurial vessels in nearly normal-appearing sural nerves, were surrounded by unmyelinated axons (Fig. 4A–D). Perivascular unmyelinated axons were more abundant around arteries than veins. Peripherin antibodies also labeled smooth muscle cells in some vessel walls. Endoneurial small axon quantitation by UAS ratios correlated (r 5 0.78; P < 0.0001) with UAS ratios of epineurial vascular innervation in 34 patients with varying severities of neuropathies (Fig. 3B). Endoneurial UAS ratios also correlated (r 5 0.43; P 5 0.01) with UAS ratios of perimysial perivascular nerves in 33 patients (Fig. 3B). Correlation of myelinated axon loss with epineurial and perimysial vascular innervation was significant but less strong (p 5 0.02; P 5 0.03; respectively). Bonferroni correction for multiple comparisons of endoneurial UAS ratios remained significant for correlation with IENDF at the ankle and UAS MUSCLE & NERVE
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FIGURE 3. Endoneurial unmyelinated axons correlate with skin (A) and vascular (B) innervation.
ratios of epineurial and perimysial vascular innervation (P < 0.0125), but not for IENFD at the thigh. Bonferroni correction for endoneurial MAS ratio multiple comparisons was nonsignificant for small axon loss in the various tissues but significant for myelinated axon loss on toluidine blue analysis and for a history of bariatric or bowel resection surgery. DISCUSSION
We found that endoneurial myelinated and unmyelinated axons can be quantitated by compar-
ing their peripherin-labeled area to their neighboring or embedding nonmyelinating Schwann cells labeled by NCAM. We previously used a similar approach to measure vascular innervation by unmyelinated axons in muscles. The results of the current study show that loss of endoneurial myelinated and unmyelinated axons is proportional in most cases and is usually accompanied by similar degrees of denervation of skin and vessels. Our measures of myelinated endoneurial axon loss had a better correlation with loss of distal vibration sense and with their loss on toluidine staining.
FIGURE 4. Epineurial vascular innervation. Fluorescent immunolabeled images of green peripherin (A), red NCAM (B), with yellow colocalization (C), and DAPI (D). Peripherin stained small axons surrounding both arteries (full arrowhead) and less prominently veins (empty arrowhead). NCAM stained nonmyelinating Schwann cells that embed perivascular small axons and co-stain with peripherin. Smooth muscle cells of vessels were often stained with peripherin. Arteries showed thicker walls compared with veins with DAPI staining. Scale bar 5 50 mm. NCAM, neural cell adhesion molecule. 202
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Patients with a history of bariatric surgery had marked predominant loss of myelinated axons. Our measures of unmyelinated endoneurial axon loss had a better correlation with vascular denervation. Patients with predominant loss of small axons generally had small fiber neuropathy involving several tissues. Morphometry may play an important role in interpretation of sural nerve biopsies, particularly for early detection of predominant large or small axon loss. However, quantitation of axon loss is commonly based on general categorization during visual inspection of sural nerve cross-sections.1 This commonly focuses on myelinated axon parameters (number, density, diameter, myelin sheath thickness, and presence of regenerated myelinated axonal clusters) and not on unmyelinated axons or a comparison of their relative loss. Computer-based quantitation methods were developed to increase sensitivity and accuracy of axon counts, but these also focus on myelinated axon parameters,1,2 not on unmyelinated axons. Endoneurial unmyelinated axon quantitation is commonly based on axon counts in electron micrographs.12–17 Accordingly, this technique covers a limited area of endoneurium, rendering this technique susceptible to sampling errors. Counting of immune-stained PGP 9.5 unmyelinated axons shows a significant correlation with that obtained by electron microscopy analysis,18 but both methods are used uncommonly. We routinely stain the sural nerve with a combination of antibodies against multiple neurofilaments19,20 for qualitative assessment of myelinated and unmyelinated axon loss with light microscopy. This includes anti-neurofilament 200 kDa (NF200), anti-SMI32R, and anti-peripherin. While this combination maximizes visualization of axons, our initial observation identified NF200 and SMI32R to be more abundant in large axons, while peripherin stained both large (myelinated) and small (unmyelinated) axons. In this study, we used only anti-peripherin antibodies to stain similar neurofilaments in both large and small axons. Axon quantitation was based on the abundance of this neurofilament, extrapolating its spatial distribution to persistence of axons. Peripherin is thought to play an important role in maintenance of the neuronal cytoskeleton through heteropolymerization with other neurofilaments21–25 and regulating their diameter.26 Use of a single neurofilament stain like peripherin, which stains both myelinated and unmyelinated axons, provides reproducible and comparable quantitation of axon loss. Peripherin staining could underestimate the density of large axons if peripherin is selectively reduced, perhaps early in the disease course, while other neurofilaEndoneurial Axon Quantitation
ments or axonal structures remain intact. Whether such pathologies occur requires further study. Early ultrastructural changes in unmyelinated axons in chronic neuropathies may include an increased number of nonmyelinating Schwann cell cytoplasmic processes followed by increased numbers of “empty” Schwann processes.14,27,28 This suggests that detection of axon loss from nonmyelinating Schwann cells may be more sensitive than axon counting.28 Our quantitation of UAS ratios takes advantage of this change and makes it a sensitive tool for detecting loss of unmyelinated axons from their embedding Schwann cells. The UAS ratio measuring loss of unmyelinated axons from their embedding Schwann cells also allows quantification of epineurial vasa nervorum innervation. Denervation of the vasa nervorum in the epineurium was long thought to cause endoneurial ischemia that results with focal axonal loss as occurs in some neuropathies29–32 including diabetes.33–36 Small axon loss occurs in the early stages of diabetes,37,38 and microangiopathy may be detected before axon loss.39,40 In our patients, vasa nervorum denervation correlated with loss of small endoneurial axons. This is similar to our previous demonstration that peripherin-labeled perivascular nerves in muscles are proportional to the IENFD in small fiber neuropathies and controls. In patients with neuropathy it appears that there is usually multi-organ denervation with a general loss of unmyelinated axons in skin, muscle, endoneurium, and epineurium. This study is limited by the lack of absolutely normal sural nerve biopsies from patients with absence of clinical complaints. Incorporation of possibly normal nerves from autopsies was abandoned due to low quality of axon stains. Sural nerves from patients with selective loss of large myelinated or small unmyelinated axons due to well defined genetic disorders such as spinocerebellar degeneration or Fabry disease may have strengthened our conclusions but were unavailable. Age-dependent changes in unmyelinated axons were also not evaluated here, although sural nerves with high and low endoneurial UAS ratios were detected in similar age groups. Additionally, while regional axon loss in the endoneurium is easy to see with our staining method, we have currently not determined its quantitation. In conclusion, we show that endoneurial myelinated and unmyelinated axon loss is quantifiable by peripherin/NCAM staining and digital analysis. Identification of selective or predominant loss of myelinated or unmyelinated axons can provide important information about the etiology of neuropathies. Selective loss of large axons may be common with nutritional deficiencies. Our MUSCLE & NERVE
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peripherin staining techniques enables uniform quantitation of small axons in epineurial and perimysial vascular innervation as well as in the endoneurium. Use of additional neurofilament or axon markers should allow quantitation of subpopulations of sensory or autonomic axons in different neuropathies. The study was supported by the Washington University Neuromuscular Research Fund. REFERENCES 1. Pamphlett R, Sjarif A. Is quantitation necessary for assessment of sural nerve biopsies? Muscle Nerve 2003;27:562–569. 2. Vonturkovich MA, Wadsworth MP, Pendlebury WW, Taatjes DJ. A novel combined imaging/morphometrical method for the analysis of human sural nerve biopsies for clinical diagnosis. Methods Mol Biol 2013;931:391–411. 3. Dori A, Lopate G, Keeling R, Pestronk A. Myovascular innervation: axon loss in small-fiber neuropathies. Muscle Nerve 2015;51:514–521. 4. Mozaffar T, Pestronk A. Myopathy with anti-Jo-1 antibodies: pathology in perimysium and neighbouring muscle fibres. J Neurol Neurosurg Psychiatry 2000;68:472–478. 5. Pestronk A. Testing at Washington University Neuromuscular Laboratory. http://neuromuscular.wustl.edu/over/labdis.html#labwu2015. 6. Pestronk A, Schmidt RE, Choksi R. Vascular pathology in dermatomyositis and anatomic relations to myopathology. Muscle Nerve 2010;42:53–61. 7. Manousakis G, Koch J, Sommerville RB, El-Dokla A, Harms MB, AlLozi MT, et al. Multifocal radiculoneuropathy during ipilimumab treatment of melanoma. Muscle Nerve 2013;48:440–444. 8. Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, Mellgren SI, et al. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. Eur J Neurol 2010;17:903–912; e944–e909. 9. Lopate G, Streif E, Harms M, Weihl C, Pestronk A. Cramps and small-fiber neuropathy. Muscle Nerve 2013;48:252–255. 10. Lauria G, Bakkers M, Schmitz C, Lombardi R, Penza P, Devigili G, et al. Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study. J Peripher Nerv Syst 2010;15:202–207. 11. Le Forestier N, Lescs MC, Gherardi RK. Anti-NKH-1 antibody specifically stains unmyelinated fibres and non-myelinating Schwann cell columns in humans. Neuropathol Appl Neurobiol 1993;19:500–506. 12. Ochoa J, Mair WG. The normal sural nerve in man. I. Ultrastructure and numbers of fibres and cells. Acta Neuropathol 1969;13:197–216. 13. Ochoa J, Mair WG. The normal sural nerve in man. II. Changes in the axons and Schwann cells due to ageing. Acta Neuropathol 1969; 13:217–239. 14. Behse F, Buchthal F, Carlsen F, Knappeis GG. Unmyelinated fibres and Schwann cells of sural nerve in neuropathy. Brain 1975;98:493– 510. 15. Pollock M, Nukada H, Allpress S, Calder C, Mackinnon M. Peripheral nerve morphometry in stroke patients. J Neurol Sci 1984;65:341– 352. 16. Jacobs JM, Love S. Qualitative and quantitative morphology of human sural nerve at different ages. Brain 1985;108(Pt 4):897–924. 17. Kanda T, Tsukagoshi H, Oda M, Miyamoto K, Tanabe H. Morphological changes in unmyelinated nerve fibres in the sural nerve with age. Brain 1991;114(Pt 1B):585–599. 18. Johnson PC, Beggs JL, Olafsen AG, Watkins CJ. Unmyelinated nerve fiber estimation by immunocytochemistry. Correlation with electron microscopy. J Neuropathol Exp Neurol 1994;53:176–183.
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19. Pestronk A, Watson DF, Yuan CM. Neurofilament phosphorylation in peripheral nerve: changes with axonal length and growth state. J Neurochem 1990;54:977–982. 20. Pestronk A. Nerve biopsy at Washington University Neuromuscular Laboratory. http://neuromuscular.wustl.edu/over/ labdis.html#nerve2015. 21. Athlan ES, Sacher MG, Mushynski WE. Associations between intermediate filament proteins expressed in cultured dorsal root ganglion neurons. J Neurosci Res 1997;47:300–310. 22. Athlan ES, Mushynski WE. Heterodimeric associations between neuronal intermediate filament proteins. J Biol Chem 1997;272:31073– 31078. 23. McLean J, Xiao S, Miyazaki K, Robertson J. A novel peripherin isoform generated by alternative translation is required for normal filament network formation. J Neurochem 2008;104:1663–1673. 24. Gentil BJ, McLean JR, Xiao S, Zhao B, Durham HD, Robertson J. A two-hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25-interacting protein, SIP30. J Neurochem 2014;131:588– 601. 25. Yuan A, Sasaki T, Kumar A, Peterhoff CM, Rao MV, Liem RK, Julien JP, Nixon RA. Peripherin is a subunit of peripheral nerve neurofilaments: implications for differential vulnerability of CNS and peripheral nervous system axons. J Neurosci 2012;32:8501–8508. 26. Kreplak L, Aebi U, Herrmann H. Molecular mechanisms underlying the assembly of intermediate filaments. Exp Cell Res 2004;301:77–83. 27. Ochoa J. Recognition of unmyelinated fiber disease: morphologic criteria. Muscle Nerve 1978;1:375–387. 28. Behse F. Morphometric studies on the human sural nerve. Acta Neurol Scand Suppl 1990;132:1–38. 29. Beggs J, Johnson PC, Olafsen A, Watkins CJ, Cleary C. Transperineurial arterioles in human sural nerve. J Neuropathol Exp Neurol 1991; 50:704–718. 30. Amenta F, Mione MC, Napoleone P. The autonomic innervation of the vasa nervorum. J Neural Transm 1983;58:291–297. 31. Appenzeller O, Dhital KK, Cowen T, Burnstock G. The nerves to blood vessels supplying blood to nerves: the innervation of vasa nervorum. Brain Res 1984;304:383–386. 32. Dhital K, Lincoln J, Appenzeller O, Burnstock G. Adrenergic innervation of vasa and nervi nervorum of optic, sciatic, vagus and sympathetic nerve trunks in normal and streptozotocin-diabetic rats. Brain Res 1986;367:39–44. 33. Grover-Johnson NM, Baumann FG, Imparato AM, Kim GE, Thomas PK. Abnormal innervation of lower limb epineurial arterioles in human diabetes. Diabetologia 1981;20:31–38. 34. Simpson LO. Altered blood rheology in the pathogenesis of diabetic and other neuropathies. Muscle Nerve 1988;11:725–744. 35. Dyck PJ, Karnes JL, O’Brien P, Okazaki H, Lais A, Engelstad J. The spatial distribution of fiber loss in diabetic polyneuropathy suggests ischemia. Ann Neurol 1986;19:440–449. 36. Dyck PJ, Lais A, Karnes JL, O’Brien P, Rizza R. Fiber loss is primary and multifocal in sural nerves in diabetic polyneuropathy. Ann Neurol 1986;19:425–439. 37. Dyck PJ, Dyck PJ, Larson TS, O’Brien PC, Velosa JA. Patterns of quantitative sensation testing of hypoesthesia and hyperalgesia are predictive of diabetic polyneuropathy: a study of three cohorts. Nerve growth factor study group. Diabetes Care 2000;23:510–517. 38. Sumner CJ, Sheth S, Griffin JW, Cornblath DR, Polydefkis M. The spectrum of neuropathy in diabetes and impaired glucose tolerance. Neurology 2003;60:108–111. 39. Malik RA, Tesfaye S, Newrick PG, Walker D, Rajbhandari SM, Siddique I, et al. Sural nerve pathology in diabetic patients with minimal but progressive neuropathy. Diabetologia 2005;48:578–585. 40. Thrainsdottir S, Malik RA, Dahlin LB, Wiksell P, Eriksson KF, Rosen I, et al. Endoneurial capillary abnormalities presage deterioration of glucose tolerance and accompany peripheral neuropathy in man. Diabetes 2003;52:2615–2622.
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Evaluation of large versus small axon loss in nerve biopsies is important for their association with clinical syndromes such as ataxia and pain, respectively. This is usually based on qualitative visual inspection, while more quantitative measurements have required laborious morphological evaluation of fixed nerves and visualized ultrastructure.1,2 We recently found that unmyelinated perivascular axons could be quantitated by labeling the neurofilament peripherin and comparing their area to their associated nonmyelinating Schwann cells labeled with neural cell adhesion molecule (NCAM).3 Peripherin is also present in myelinated axons and is, therefore, suitable for comparison of neurofilament loss in myelinated versus unmyelinated axons. In this study, we used and validated similar methods to quantify endoneurial myelinated and unmyelinated peripherin-containing Abbreviations: DAPI, 40 ,6-diamidine-2-phenylidole-dihydrochloride; ICC, intraclass correlation coefficient; IENFD, intraepidermal nerve fiber density; LFN, large fiber neuropathy; MAS, myelinated axons to nonmyelinating Schwann cell cytoplasm areas; NCAM, neural cell adhesion molecule; PBS, phosphate-buffered saline; RGBB, red, green, blue; ROC, receiver operating characteristic; SFN, small fiber neuropathy; UAS, unmyelinated axons to nonmyelinating Schwann cell cytoplasm areas. Key words: myelinated axons; neuropathy; sural nerve biopsy; unmyelinated axons; vascular innervation Correspondence to: A. Dori; e-mail: [email protected] Additional Supporting Information may be found in the online version of this article. C 2015 Wiley Periodicals, Inc. V
Published online 16 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.24740
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axons in nerves from patients with neuropathies who also had skin and/or muscle biopsies. We compared quantities of endoneurial myelinated and unmyelinated axons with clinical and electrophysiological features, axon counts in skin, and innervation of epineurial and perimysial vessels. METHODS AND MATERIALS Patients. We retrospectively reviewed records of 34 consecutive patients (19 men, 15 women; ages 15 to 81 years, mean 55) who were evaluated between July 2008 and March 2013 at Washington University in Saint Louis for axonal neuropathy and had sural nerve and muscle or skin biopsies (Supplementary Table S1, which is available online). Biopsies of muscle were performed in 33 and skin in 12 patients (ankle and thigh 8, and ankle only 4). Neuropathy diagnoses based on electrodiagnostic studies or skin biopsy were selective small fiber neuropathy (SFN, 6 patients) and large 6 small fiber syndromes (LFN, 28 patients), including distal polyneuropathy (19), multiple mononeuropathies (7), and lumbosacral plexopathy (2). The 12 patients with skin biopsies (8 men, 4 women, ages 15 to 69 years, mean 49), were diagnosed with SFN (4) or LFN (8). Biopsies. All nerve, muscle, and skin biopsies were processed and evaluated at the Washington University Neuromuscular Laboratory. Muscle and nerve biopsies were all performed on the same day. Skin biopsies were performed within 16 months (median 7 weeks) of muscle biopsy. Muscles evaluated included quadriceps (21), gastrocnemius (7), deltoid (4), and biceps brachii (1). Nerves and muscles were rapidly frozen, and cryostat sections were collected and processed for histochemistry and immunohistochemistry in our standard manner.4,5 Toluidine blue plastic sections of nerves were processed following fixation in glutaraldehyde as previously described.6,7 Muscle and nerve pathologic assessments were all performed by AP. Skin biopsies (3.0 mm punch) were performed at 2 sites: at the ankle, 10 cm above the lateral malleolus; and at the thigh, 10 cm above the patella (8 patients); or only at the ankle (4 patients). Skin was fixed and processed using standard procedures and techniques to determine the intra-epidermal nerve fiber density (IENFD).8,9 IENFD quantitation was performed by MUSCLE & NERVE
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GL. Normal values for skin biopsies were based on published data.10 An IENFD at or below the fifth percentile per age was defined as abnormal. The human studies committee of Washington University in St Louis approved all procedures. Informed consent was not required. Quantitation of Unmyelinated and Myelinated Axons.
Staining of axons with peripherin and of nonmyelinating Schwann cells with NCAM was performed as previously described.3 Briefly, fresh frozen 16-mm sections were mounted on glass slides, fixed in cold acetone, and incubated with antibodies to peripherin and NCAM followed by incubation with Alexa Fluor 488 Goat anti-mouse IgG1 and Alexa Fluor 594 Goat anti-mouse IgG2b, and mounted with antifade reagent with DAPI (40 ,6-diamidine-2phenylidole-dihydrochloride). Image acquisition was based on obtaining consistent and similar signal intensities for peripherin and NCAM staining within an exposure range that avoided both oversaturation and high background. Exposure times in the photographed field for NCAM labeled Schwann processes (red) were adjusted using Olympus camera controller software (CellSens) to a color intensity of 200 on the RBG scale. Exposure times in the photographed field for peripherin-labeled axons (green) were similarly adjusted to color intensity of 200 on the RBG scale, or to the highest level that maintained the background below 10 on the RBG scale. For digital quantitative analysis, endoneurium and perivascular nerves from acquired images were selected with a “hand-drawn free select tool” of the GIMP software. An intensity threshold of 128 on the RGB (red, green, blue) scale was used to transform each color to a binary, “all or none” mode. Resulting images had 4 colors: red, green, yellow, and black. Quantitation of peripherin- and NCAMlabeled areas was performed by measuring selective color pixel areas. The pixel areas of yellow (axons embedded within nonmyelinating Schwann cells), red (nonmyelinating Schwann cell area without axons), and green (myelinated axons not embedded in nonmyelinating Schwann cells) were measured separately. Ratios of unmyelinated and myelinated axons to nonmyelinating Schwann cell cytoplasm areas (UAS3 and MAS, respectively) were calculated. A minimum of 8 image samples of endoneurium from randomly selected different fascicles or from nonadjacent sections and perivascular nerves were used to obtain mean ratios. Measurements and calculations were made without knowledge of IENFD or clinical data. Myelinated axon loss on toluidine blue plastic sections was rated on a 0–6 scale as follows: no loss, 0; minimal/mild, 1; mild–moderate, 2; Endoneurial Axon Quantitation
moderate, 3; moderate–severe, 4; severe, 5; nearly complete loss, 6. Statistical Analysis. Comparisons for correlations were performed using 2-sided Pearson product correlations. Intraclass correlation coefficient (ICC) was used to assess rater consistency and reliability; and inter-rater agreement was determined by kappa (quadratic weighted) analysis. Spearman coefficients were used to assess rank correlation (rho). The diagnostic yield of axon loss analysis was estimated by area under the curve analysis of receiver operating characteristic (ROC) curves. The Fisher exact test was used to calculate P-values for frequency. Statistical analyses were performed using MedCalc for Windows, version 13.1.2.0 (MedCalc Software, Ostend, Belgium). Values are expressed as mean 6 standard deviation. RESULTS Unmyelinated Are
and
Differentially
Myelinated Labeled
Endoneurial
by
Axons
Peripherin/NCAM
Immunofluorescence. In normal appearing sural nerve endoneurium, peripherin labeled large and small axons (Fig. 1A). Small and large axons were qualitatively differentiated by double staining of nonmyelinating Schwann cells with NCAM (Fig. 1B).11 Merged images (Fig. 1C) showed yellow, colabeled, small axons within red NCAM-labeled nonmyelinating Schwann cells (arrowheads in Fig. 1D–G). Large and intermediate size axons were green, labeled only by peripherin, and surrounded by a black halo, consistent with myelin sheaths (small arrows in Fig. 1D–G). In normal nerves, clusters of larger green axons separated dense groups of unmyelinated axons which appeared as islands throughout the endoneurium. With loss of large (NCAM-negative) axons, the area of large green peripherin-labeled axons was reduced (Fig. 1H–K). With loss of unmyelinated axons, yellow small axons within red NCAM-labeled Schwann cells were reduced (empty arrows in Fig. 1L–O). Endoneurial
Unmyelinated
and
Myelinated
Axon/
Nonmyelinating Schwann Cell Area (UAS and MAS) Ratios Are Reliable Measures. We quantitated unmyelinated axons in endoneurial Remak bundles by calculating UAS ratios. Low UAS ratios represent a loss of unmyelinated axons with preserved nonmyelinating Schwann cells. Consistency analysis of these measurements was performed by comparing 2 raters. The ICC of 20 endoneurial UAS ratio measurements (by A.D.) was 0.80 for absolute agreement among average measurements and ICC 5 0.81 for consistency among average measurements. Inter-rater agreement (kappa) showed a good correlation (k 5 0.71; standard error 5 0.17) between our 2 raters (A.D. and R.C). Myelinated MUSCLE & NERVE
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FIGURE 1. Peripherin-stained, unmyelinated axons colocalize with NCAM-stained Schwann cells, while myelinated axons do not. Fluorescent immunolabeled images of green peripherin (A,D,H,L) and red NCAM (B,E,I,M), with yellow colocalization (C,F,J,N) and multicolor digital transformation (G,K,O). D–G shows a nearly normal nerve. H–K shows a nerve with predominant myelinated axon loss. L–O shows a nerve with predominant small unmyelinated axon loss. Ratios of green to red (G/R) determine myelinated axon/nonmyelinating Schwann cell area ratios. Ratios of yellow to red (Y/R) determine unmyelinated axon/nonmyelinating Schwann cell area ratios. Scale bars 5 50 mm. NCAM, neural cell adhesion molecule.
axons were quantitated by calculating the MAS ratios, using the same denominator used for quantitating unmyelinated axon loss. The ICC for MAS measurements of the same nerves (by A.D.) was 0.77 for absolute agreement among average measurements and ICC 5 0.74 for consistency among average measurements. Inter-rater agreement (kappa) showed a good correlation (k 5 0.84; standard error 5 0.054) between our 2 raters (A.D. and 200
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R.C). MAS ratios showed a correlation (rho 5 0.655, P < 0.0001, 95% confidence interval 0.41–0.81) with the severity of myelinated axon loss on toluidine blue plastic sections. Predominant Endoneurial Unmyelinated, or Myelinated, Axon
Loss
Is
Associated
with
Different
Clinical
UAS and MAS ratios showed a general correlation between loss of Neuropathy Characteristics.
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myelinated axon loss was calculated by ROC analysis (Fig. 2B). An MAS ratio of 0.13 predicted distal vibration sense loss with sensitivity of 96% and specificity of 70% (area under curve 5 0.88; P < 0.0001). An MAS ratio of 0.10 predicted absent distal SNAPs with sensitivity of 94% and specificity of 79% (area under curve 5 0.95; P < 0.0001). Unmyelinated axon loss determined by UAS ratios showed a less strong association with distal vibration sense loss (P 5 0.02). Patients with diagnoses of small fiber neuropathy were common above a myelinated-unmyelinated axon loss regression line, at the top of, or above the 95% confidence interval (Fig. 2A). Nine patients with predominant myelinated axon loss were identified below the 95% confidence interval. Five of them had a history of gastrointestinal surgery (3 bariatric, 2 colon resection). Three patients with gastrointestinal surgery had documented nutritional deficiencies (copper in 2, and vitamin B1, B6, and B12 deficiencies in another). One additional patient with a history of small bowel resection presented with near-complete loss of both myelinated and unmyelinated axons. None of the remainder of the patients in this cohort had a history of bowel resection. A history of bariatric or bowel resection surgery was associated with markedly predominant myelinated peripherin-axon loss (P < 0.001). Correlations of Small Axon Loss among Different
FIGURE 2. Endoneurial myelinated versus unmyelinated axon loss and clinical correlation. (A) Myelinated versus unmyelinated axon/nonmyelinating Schwann cell area ratios. Vibration sense, fiber type of neuropathy, and history of bowel surgery are shown. Regression lines and 95% confidence intervals are marked. (B) Comparison of ROC curves of myelinated and unmyelinated axon/nonmyelinating Schwann cell area ratios classifying vibration sense loss. Myelinated axon/nonmyelinating Schwann cell area ratios area under curve 5 0.88; standard error 5 0.07; 95% confidence interval 5 0.72 to 0.97. Unmyelinated axon/nonmyelinating Schwann cell area ratios area under curve 5 0.72; standard error 5 0.10; 95% confidence interval 5 0.55 to 0.87. GI 5 gastrointestinal.
unmyelinated and myelinated axons (Fig. 2A; r 5 0.56, P 5 0.001). Some nerves, however, showed more prominent loss of myelinated or unmyelinated axons. Patients with severe loss of myelinated axons showed distal loss of vibration sensation even when unmyelinated axons were relatively spared. The diagnostic efficiency and optimal cutoff value of low MAS ratios for the diagnosis of Endoneurial Axon Quantitation
Tissues. To validate the UAS quantitative measurement of endoneurial axons, we compared the UAS ratio with IENFD counts in skin biopsies from 12 patients (Table S1, available online). Endoneurial UAS ratios correlated with IENFD in both ankle (r 5 0.70; P 5 0.01) and thigh (r 5 0.80; P 5 0.02) skin (Fig. 3A). Epineurial vessels in nearly normal-appearing sural nerves, were surrounded by unmyelinated axons (Fig. 4A–D). Perivascular unmyelinated axons were more abundant around arteries than veins. Peripherin antibodies also labeled smooth muscle cells in some vessel walls. Endoneurial small axon quantitation by UAS ratios correlated (r 5 0.78; P < 0.0001) with UAS ratios of epineurial vascular innervation in 34 patients with varying severities of neuropathies (Fig. 3B). Endoneurial UAS ratios also correlated (r 5 0.43; P 5 0.01) with UAS ratios of perimysial perivascular nerves in 33 patients (Fig. 3B). Correlation of myelinated axon loss with epineurial and perimysial vascular innervation was significant but less strong (p 5 0.02; P 5 0.03; respectively). Bonferroni correction for multiple comparisons of endoneurial UAS ratios remained significant for correlation with IENDF at the ankle and UAS MUSCLE & NERVE
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FIGURE 3. Endoneurial unmyelinated axons correlate with skin (A) and vascular (B) innervation.
ratios of epineurial and perimysial vascular innervation (P < 0.0125), but not for IENFD at the thigh. Bonferroni correction for endoneurial MAS ratio multiple comparisons was nonsignificant for small axon loss in the various tissues but significant for myelinated axon loss on toluidine blue analysis and for a history of bariatric or bowel resection surgery. DISCUSSION
We found that endoneurial myelinated and unmyelinated axons can be quantitated by compar-
ing their peripherin-labeled area to their neighboring or embedding nonmyelinating Schwann cells labeled by NCAM. We previously used a similar approach to measure vascular innervation by unmyelinated axons in muscles. The results of the current study show that loss of endoneurial myelinated and unmyelinated axons is proportional in most cases and is usually accompanied by similar degrees of denervation of skin and vessels. Our measures of myelinated endoneurial axon loss had a better correlation with loss of distal vibration sense and with their loss on toluidine staining.
FIGURE 4. Epineurial vascular innervation. Fluorescent immunolabeled images of green peripherin (A), red NCAM (B), with yellow colocalization (C), and DAPI (D). Peripherin stained small axons surrounding both arteries (full arrowhead) and less prominently veins (empty arrowhead). NCAM stained nonmyelinating Schwann cells that embed perivascular small axons and co-stain with peripherin. Smooth muscle cells of vessels were often stained with peripherin. Arteries showed thicker walls compared with veins with DAPI staining. Scale bar 5 50 mm. NCAM, neural cell adhesion molecule. 202
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Patients with a history of bariatric surgery had marked predominant loss of myelinated axons. Our measures of unmyelinated endoneurial axon loss had a better correlation with vascular denervation. Patients with predominant loss of small axons generally had small fiber neuropathy involving several tissues. Morphometry may play an important role in interpretation of sural nerve biopsies, particularly for early detection of predominant large or small axon loss. However, quantitation of axon loss is commonly based on general categorization during visual inspection of sural nerve cross-sections.1 This commonly focuses on myelinated axon parameters (number, density, diameter, myelin sheath thickness, and presence of regenerated myelinated axonal clusters) and not on unmyelinated axons or a comparison of their relative loss. Computer-based quantitation methods were developed to increase sensitivity and accuracy of axon counts, but these also focus on myelinated axon parameters,1,2 not on unmyelinated axons. Endoneurial unmyelinated axon quantitation is commonly based on axon counts in electron micrographs.12–17 Accordingly, this technique covers a limited area of endoneurium, rendering this technique susceptible to sampling errors. Counting of immune-stained PGP 9.5 unmyelinated axons shows a significant correlation with that obtained by electron microscopy analysis,18 but both methods are used uncommonly. We routinely stain the sural nerve with a combination of antibodies against multiple neurofilaments19,20 for qualitative assessment of myelinated and unmyelinated axon loss with light microscopy. This includes anti-neurofilament 200 kDa (NF200), anti-SMI32R, and anti-peripherin. While this combination maximizes visualization of axons, our initial observation identified NF200 and SMI32R to be more abundant in large axons, while peripherin stained both large (myelinated) and small (unmyelinated) axons. In this study, we used only anti-peripherin antibodies to stain similar neurofilaments in both large and small axons. Axon quantitation was based on the abundance of this neurofilament, extrapolating its spatial distribution to persistence of axons. Peripherin is thought to play an important role in maintenance of the neuronal cytoskeleton through heteropolymerization with other neurofilaments21–25 and regulating their diameter.26 Use of a single neurofilament stain like peripherin, which stains both myelinated and unmyelinated axons, provides reproducible and comparable quantitation of axon loss. Peripherin staining could underestimate the density of large axons if peripherin is selectively reduced, perhaps early in the disease course, while other neurofilaEndoneurial Axon Quantitation
ments or axonal structures remain intact. Whether such pathologies occur requires further study. Early ultrastructural changes in unmyelinated axons in chronic neuropathies may include an increased number of nonmyelinating Schwann cell cytoplasmic processes followed by increased numbers of “empty” Schwann processes.14,27,28 This suggests that detection of axon loss from nonmyelinating Schwann cells may be more sensitive than axon counting.28 Our quantitation of UAS ratios takes advantage of this change and makes it a sensitive tool for detecting loss of unmyelinated axons from their embedding Schwann cells. The UAS ratio measuring loss of unmyelinated axons from their embedding Schwann cells also allows quantification of epineurial vasa nervorum innervation. Denervation of the vasa nervorum in the epineurium was long thought to cause endoneurial ischemia that results with focal axonal loss as occurs in some neuropathies29–32 including diabetes.33–36 Small axon loss occurs in the early stages of diabetes,37,38 and microangiopathy may be detected before axon loss.39,40 In our patients, vasa nervorum denervation correlated with loss of small endoneurial axons. This is similar to our previous demonstration that peripherin-labeled perivascular nerves in muscles are proportional to the IENFD in small fiber neuropathies and controls. In patients with neuropathy it appears that there is usually multi-organ denervation with a general loss of unmyelinated axons in skin, muscle, endoneurium, and epineurium. This study is limited by the lack of absolutely normal sural nerve biopsies from patients with absence of clinical complaints. Incorporation of possibly normal nerves from autopsies was abandoned due to low quality of axon stains. Sural nerves from patients with selective loss of large myelinated or small unmyelinated axons due to well defined genetic disorders such as spinocerebellar degeneration or Fabry disease may have strengthened our conclusions but were unavailable. Age-dependent changes in unmyelinated axons were also not evaluated here, although sural nerves with high and low endoneurial UAS ratios were detected in similar age groups. Additionally, while regional axon loss in the endoneurium is easy to see with our staining method, we have currently not determined its quantitation. In conclusion, we show that endoneurial myelinated and unmyelinated axon loss is quantifiable by peripherin/NCAM staining and digital analysis. Identification of selective or predominant loss of myelinated or unmyelinated axons can provide important information about the etiology of neuropathies. Selective loss of large axons may be common with nutritional deficiencies. Our MUSCLE & NERVE
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peripherin staining techniques enables uniform quantitation of small axons in epineurial and perimysial vascular innervation as well as in the endoneurium. Use of additional neurofilament or axon markers should allow quantitation of subpopulations of sensory or autonomic axons in different neuropathies. The study was supported by the Washington University Neuromuscular Research Fund. REFERENCES 1. Pamphlett R, Sjarif A. Is quantitation necessary for assessment of sural nerve biopsies? Muscle Nerve 2003;27:562–569. 2. Vonturkovich MA, Wadsworth MP, Pendlebury WW, Taatjes DJ. A novel combined imaging/morphometrical method for the analysis of human sural nerve biopsies for clinical diagnosis. Methods Mol Biol 2013;931:391–411. 3. Dori A, Lopate G, Keeling R, Pestronk A. Myovascular innervation: axon loss in small-fiber neuropathies. Muscle Nerve 2015;51:514–521. 4. Mozaffar T, Pestronk A. Myopathy with anti-Jo-1 antibodies: pathology in perimysium and neighbouring muscle fibres. J Neurol Neurosurg Psychiatry 2000;68:472–478. 5. Pestronk A. Testing at Washington University Neuromuscular Laboratory. http://neuromuscular.wustl.edu/over/labdis.html#labwu2015. 6. Pestronk A, Schmidt RE, Choksi R. Vascular pathology in dermatomyositis and anatomic relations to myopathology. Muscle Nerve 2010;42:53–61. 7. Manousakis G, Koch J, Sommerville RB, El-Dokla A, Harms MB, AlLozi MT, et al. Multifocal radiculoneuropathy during ipilimumab treatment of melanoma. Muscle Nerve 2013;48:440–444. 8. Lauria G, Hsieh ST, Johansson O, Kennedy WR, Leger JM, Mellgren SI, et al. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. Eur J Neurol 2010;17:903–912; e944–e909. 9. Lopate G, Streif E, Harms M, Weihl C, Pestronk A. Cramps and small-fiber neuropathy. Muscle Nerve 2013;48:252–255. 10. Lauria G, Bakkers M, Schmitz C, Lombardi R, Penza P, Devigili G, et al. Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study. J Peripher Nerv Syst 2010;15:202–207. 11. Le Forestier N, Lescs MC, Gherardi RK. Anti-NKH-1 antibody specifically stains unmyelinated fibres and non-myelinating Schwann cell columns in humans. Neuropathol Appl Neurobiol 1993;19:500–506. 12. Ochoa J, Mair WG. The normal sural nerve in man. I. Ultrastructure and numbers of fibres and cells. Acta Neuropathol 1969;13:197–216. 13. Ochoa J, Mair WG. The normal sural nerve in man. II. Changes in the axons and Schwann cells due to ageing. Acta Neuropathol 1969; 13:217–239. 14. Behse F, Buchthal F, Carlsen F, Knappeis GG. Unmyelinated fibres and Schwann cells of sural nerve in neuropathy. Brain 1975;98:493– 510. 15. Pollock M, Nukada H, Allpress S, Calder C, Mackinnon M. Peripheral nerve morphometry in stroke patients. J Neurol Sci 1984;65:341– 352. 16. Jacobs JM, Love S. Qualitative and quantitative morphology of human sural nerve at different ages. Brain 1985;108(Pt 4):897–924. 17. Kanda T, Tsukagoshi H, Oda M, Miyamoto K, Tanabe H. Morphological changes in unmyelinated nerve fibres in the sural nerve with age. Brain 1991;114(Pt 1B):585–599. 18. Johnson PC, Beggs JL, Olafsen AG, Watkins CJ. Unmyelinated nerve fiber estimation by immunocytochemistry. Correlation with electron microscopy. J Neuropathol Exp Neurol 1994;53:176–183.
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