Thesis

Insights and innovations in sarcoidosis-associated small fiber neuropathy Lisette Raasing

Insights and innovations in sarcoidosis- associated small fiber neuropathy Inzichten en innovaties in sarcoïdose-geassocieerde dunnevezelneuropathie (met een samenvatting in het Nederlands) PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. ir. W. Hazeleger, ingevolge het besluit van het College voor Promoties in het openbaar te verdedigen op Dinsdag 1 april 2025 des middags te 2.15 uur door Lisette Regina Maria Raasing Geboren op 8 augustus 1993 te Westervoort

Promotor: Prof. dr. J.C. Grutters Copromotoren: Dr. O.J.M. Vogels Dr. M. Veltkamp Beoordelingscommissie: Prof. dr. A. Dahan Prof. dr. H.G.M. Heijerman Prof. dr. N.C. Notermans (voorzitter) Prof. dr. M.C. Post Prof. dr. ir. G.T. Rijkers

Insights and innovations of sarcoidosis-associated small fiber neuropathy This thesis was accomplished with financial support by the Netherlands Organisation for Health Research and Development (ZonMw) under project agreement TopZorg 842002001. The printing of this thesis was financially supported by St. Antonius Ziekenhuis Raad van Bestuur, Sarcoïdose belangenvereniging Nederland (Sarcoidose.nl), Nederlandse Vereniging voor Klinische Neurofysiologie (NVKNF), Boehringer Ingelheim B.V. and Westfalen Medical. Thesis University of Utrecht Printing Ipskamp printing Cover design and lay-out: Lisette Raasing ISBN/EAN 978-94-6473-746-2 © Lisette Raasing, 2025 All rights reserved. No part of this book may be reproduced, distributed, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author.

“It’s not the strongest of species that survive, nor the most intelligent, but the one most responsive to change” - Charles Darwin -

Table of Contents Chapter 1 General introduction and outline of the thesis Chapter 2 Current view of diagnosing small fiber neuropathy Chapter 3 Sarcoidosis-related small fiber neuropathy: focus on fatigue, pain, restless legs syndrome, and cognitive function Chapter 4 High prevalence of patient reported skin, muscle and joints pain in sarcoidosis Chapter 5 Thermal threshold testing: call for a balance between the number of measurements and abnormalities in the diagnosis of sarcoidosis-associated small fiber neuropathy Chapter 6 Fully automated, semiautomated and manual corneal nerve fiber analysis in patients with sarcoidosis Chapter 7 New patient-reported outcome measures are relevant to interpret the diagnosis of sarcoidosis-associated small fiber neuropathy Chapter 8 Infliximab decreases inflammatory activity but has no effect on small fiber neuropathy related symptoms in Dutch patients with sarcoidosis Chapter 9 [123I]-Meta-odinebenzylguanidine scintigraphy in sarcoidosis: bridging the gap between autonomic dysfunction and cardiac disease Chapter 10 Summary, general discussion and directions for future research Appendices Nederlandse samenvatting (Dutch Summary) Author affiliations List of Publications Dankwoord Curriculum Vitae Abbreviations Appendix 1 - Additional results Appendix 2 - Questionnaire (SFNPQ)

General introduction and outline of the thesis 1

7 General introduction Sarcoidosis is a granulomatous, multisystem disorder of unknown cause, mainly affecting the lungs and lymph nodes.1 Small fiber neuropathy (SFN) is a significant problem in sarcoidosis patients, with an estimated prevalence between 40-86%.2,3 This thesis focuses on SFN and cardiac autonomic dysfunction that is associated with sarcoidosis. Diagnosis, symptoms, measurement of patientreported outcomes and treatment of SFN and cardiac autonomic dysfunction in sarcoidosis patients will be subject of study. In this chapter, these topics will be explained, after first introducing the different types of fibers of the nervous system and stages of neuropathy. Neurophysiology and neuropathology of small nerve fibers SFN can affect small sensory fibers, autonomic fibers or both, resulting in sensory changes, autonomic dysfunction, or combined symptoms.4 The peripheral nervous system is classified into different types of nerves based on diameter, myelin sheet, and conduction velocity, as depicted in Figure 1A.5,6 Aα- and Aβ-fibers are classified as large nerve fibers, while Aδ- and C-fibers are classified as small nerve fibers. SFN is caused by damaged sensory and/or autonomic Aδ- and C-fibers. Small myelinated Aδfibers are characterized by faster conduction velocities (4-36 m/s)7,8 compared to unmyelinated Cfibers (0.4-2.8 m/s),8–10 due to larger diameter and myelin.11 Due to the difference in conduction velocity, Aδ-fibers are responsible for the sharp, pricking or “first” pain response, and C-fibers for the burning or “second” pain response, as shown in Figure 1B.5 Aδ-fibers that respond to heat are divided into type I and II A mechano-heat (AMH) units. AMH type I fibers are responsible for first pain sensation of mechanical stimuli, and AMH type II fibers are involved in first pain sensation of heat pain stimuli.5,11 C-fibers can be polymodal, responsive to noxious, thermal and mechanical stimuli. Additionally, they may respond to a specific stimulus, multiple stimuli or non-specific stimuli.12 Figure 1 A) Overview of nerve fiber sizes, conduction velocities and other characteristics. Aα and Aβ fibers are large and myelinated nerve fibers, Aδ nerve fibers are small myelinated nerve fibers, and C-fibers are small unmyelinated nerve fibers. B) Corresponding pain response. Large nerve fibers show a fast response with high amplitude. The smaller the nerve fiber, the lower the amplitude and the slower conduction velocities. 1 8 1

8 A healthy nerve has a small diameter and normal nerve fiber length. In SFN, several stages are defined. In an early pathological stage, the small nerve swells, increasing the nerve fiber area (NFA) while keeping the nerve fiber length (NFL) and density (NFD) within normal ranges.13 Advanced degeneration results in a severely decreased intraepidermal nerve fiber density (IENFD). After degeneration, sprouting results in new nerve structures, see Figure 2A. Figure 2B shows a schematic course of NFA and NFL during the stages of a healthy nerve, nerve swelling and nerve degeneration. Nerve degeneration might be followed by regeneration due to nerve sprouting, as illustrated in Figure 2.14 Figure 2 A) Several stages of small nerve fiber neuropathy in the epidermis. B) Change of nerve fiber area and nerve fiber length during different stages of SFN. Diagnosis The lack of a gold standard makes diagnosing SFN challenging in daily clinical practice.15 Common nerve conduction tests assess only large myelinated nerve fibers, providing no information on small fiber function.6,16 Furthermore, the heterogeneous presentation of SFN requires multimodal testing. Diagnostic criteria suggest diagnosis based on different levels of certainty. A diagnosis of possible SFN can be made if symptoms (explained in the next paragraph) are present and at least two clinical signs of SFN, such as pinprick, thermal sensory loss, allodynia or hyperalgesia. A diagnosis of probable SFN can be made when symptoms, at least 2 clinical signs and normal nerve conduction studies are present. Finally, a diagnosis of definite SFN can be established if symptoms, at least 2 clinical signs, normal nerve conduction studies and abnormal quantitative sensory testing (QST) or decreased IENFD are present, as shown in Table 1.17 Table 1 Diagnostic criteria for small fiber neuropathy according to the Besta criteria Diagnosis Criteria Possible SFN Symptoms ≥2 clinical signs Probable SFN Symptoms ≥2 clinical signs Normal nerve conduction studies Definite SFN Symptoms ≥2 clinical signs Normal nerve conduction studies Abnormal QST or Decreased IENFD SFN = small fiber neuropathy; QST = quantitative sensory testing; IENFD = intra-epidermal nerve fiber density 1 9 1

9 Until now, SFN has generally been assessed by skin biopsy and QST, following diagnostic criteria.17 Currently, however, up to 25 methods have been developed to assess specific small nerve fiber functions. This paragraph will be limited to those used during our prospective studies. The other methods are described in Chapter 2. Skin biopsy Decreased IENFD, as mentioned in the diagnostic criteria, can be established through skin biopsy. Assessment of IENFD is recommended using a manual staining technique with 50 µm of skin biopsy slides, which is available only in specialized centers due to the need for adequate training and its laborintensive nature.18 Due to the lack of automatic staining protocols for assessing IENFD, this specific method for diagnosing SFN is unavailable in most hospitals in the Netherlands. Another limitation of IENFD is the low sensitivity, between 28-38%,19–21 for diagnosing SFN in patients with sarcoidosis. Thermal threshold testing QST assesses both small and large nerve fibers.22 Since performing the full test is time-consuming, a subset of parameters has been selected to specifically assess small nerve fiber function through thermal threshold testing (TTT).23–25 Additionally, nerve conduction studies are recommended to assess large nerve fibers.26 TTT measures the cold detection threshold (CDT), warm detection threshold (WDT), thermal sensory limen (TSL), paroxysmal heat sensation (PHS), cold pain threshold (CPT) and heat pain threshold (HPT). CDT and WDT can be measured using the method of limits (MLi) and the method of levels (MLe). The MLi is time-dependent and requires the participant to respond as soon as they feel a change in temperature. MLe applies standardized temperature changes and requires feedback with a yes or no button to determine whether the next stimulus is higher (after “no”) or lower (after “yes”). No clear agreement has been reached on the superiority of either method, and results are conflicting.17,25 For example, the German Research Network on Neuropathic Pain (DFNS) protocol,27 the most widely recognized and standardized protocol for QST procedures, recommends the MLi. In contrast, the Besta criteria, the most widely recognized and standardized diagnostic criteria for SFN, recommend the MLe.17 Known disadvantages of the MLe are high time consumption and desensitization due to the numerous repeated measurements.28 Corneal Confocal Microscopy Corneal confocal microscopy (CCM) has been explored as a novel and minimally invasive alternative method to detect SFN.29,30 CCM generates in vivo images of the corneal subbasal nerve plexus with resolutions comparable to ex vivo histochemical methods.31 The cornea harbors a high nerve fiber density, up to 400 times higher compared with skin.32 Morphologic changes in the subbasal nerve plexus such as nerve fiber beading, length, branching, and tortuosity, are related to the presence of small fiber neuropathy.31,33 Multiple different quantification methods are available to analyze corneal nerve fibers,34 varying from manual analysis35 to semi-automatic analysis36 and automatic analysis.37,38 In patients with and without diabetes, good agreement is found between manual, semi-automatic, and automatic analysis of corneal nerve fiber length (CNFL).39 Parameters such as corneal nerve fiber density (CNFD), CNFL, corneal nerve branch density (CNBD) and nerve fiber area (NFA) can be identified with these techniques. CNFD counts the number of main nerves in the image (no./mm2), CNBD counts the number of branches (no./mm2) and CNFL counts the total length of both main nerves and branches (mm/mm2). Compared to CNFL, NFA is defined by the sum of total length of both main nerves and branches and the variation of nerve fiber width (µm2/mm2). Consequently, NFA has a nonlinear relation with CNFL and provides additional information when the structure, but not the length of the small fibers change. In the early stage of small fiber neuropathy nerves tend to swell, whereas degeneration of nerves can be observed in more advanced stages of SFN (see Figure 1A).40 Based on the fact that nerve fiber 1 10 1

10 length remains stable in the early stage of SFN, it is suggested that using NFA increases diagnostic sensitivity for SFN.38 Although research has been conducted over the past 20 years to support its clinical use, CCM is still primarily used for research purposes. CCM was explored in this thesis due to its benefits over skin biopsy and its potential as a diagnostic method for SFN in the future. Autonomic function testing Besides IENFD, TTT and CCM, multiple other tests have been developed to assess small fiber autonomic dysfunction, including Sudoscan,41 blood pressure variability (BPV)42 and heart rate (HR)43 after postural change, and water immersion skin wrinkling (WISW).44 Sudoscan measures the electrochemical skin conductance, enabling the detection of hypo- or hyperhidrosis in patients with SFN. BPV and HR after postural change can detect orthostatic hypotension caused by autonomic dysfunction. Sympathetic nerve dysfunction prevents vasoconstriction, resulting in absent skin wrinkling after exposure to warm water. Currently, the prevalence of SFN is likely underestimated due to lack of a gold standard and limited awareness among clinical physicians.45,46 Improving diagnostic methods is crucial to enhance the recognition of SFN symptoms, deepen insights into its pathophysiology, and facilitate future drug trials. Symptoms Because a gold standard for diagnosing SFN is still lacking, it is important to evaluate the symptoms associated with SFN. This section describes a selection of SFN-associated symptoms and their relation to various small nerve fiber functions. The potential symptoms are numerous and often significantly impacts quality of life.47 More general and nonspecific SFN-related symptoms include fatigue, cognitive impairment, widespread musculoskeletal pain, headache, and temporomandibular malfunction.46,48 Sensory dysfunction result in symptoms of neuropathic pain, burning sensations, or itching.6,16,46 Autonomic dysfunction results in sweating abnormalities, cardiovascular dysfunction, gastrointestinal dysfunction, urogenital dysfunction, or other autonomic functions.4,46 Table 2 summarizes some of the best-known SFN-related symptoms. Table 2 Symptoms related to small fiber neuropathy General Symptoms Sensory disturbances Autonomic dysfunction Fatigue Neuropathic pain Skin changes Cognitive disturbances Burning sensations Sweating abnormalities Widespread musculoskeletal pain Numbness Dry eyes Headache Tingling Dry mouth Itching Gastrointestinal dysmotility Frostbite-like sensations Orthostasis Bedsheet intolerance Palpitations or arrhythmias Stocking-glove / random / migratory and/or intermittent Bowel or bladder changes Sexual dysfunction SFN pathophysiology and phenotypes SFN is associated with a wide variety of diseases as underlying mechanisms, but can also present as idiopathic.46,49 Although the pathogenesis of SFN remains unknown, the possible underlying disease seems to influence its presentation. For example, in patients with diabetes mellitus (DM) typical length-dependent symptoms are described. Moreover, studies confirm that distal axonal loss, measured in skin biopsies, predicts the progression of distal SFN to proximal large fiber neuropathy.13 In contrast, studies examining patients with diseases other than DM reveal that SFN rarely progresses 1 11 1

11 and tends to be a stable disorder. These studies suggest that a demyelinating process is unlikely to cause SFN, as it would also affect large nerve fibers, which are not involved in SFN. Therefore, distal axonal loss or extraordinarily neuronal degeneration is more likely to be the underlying cause.4 The heterogeneous presentation within SFN has led to conceptual clinical phenotyping subsets of SFN that are presented in Figure 3. These clinical phenotypes may contribute to a better understanding of the pathophysiology and potential treatments. Four clinical phenotypes are suggested and distinguish:  Small fiber sodium channel dysfunction  Small fiber mediated painful neuropathy  Small fiber mediated widespread pain  Small fiber mediated autonomic dysfunction16 Figure 3 Roadmap: from symptoms to phenotypes and from phenotype to diagnostic method. Small fiber sodium channel dysfunction is caused by pathogenic mutations in the SCN9A, SCN10A and SCN11A genes, so genotyping is indicated. Skin biopsy, quantitative sensory testing (QST) and corneal confocal microscopy (CCM) can identify small fiber-mediated painful neuropathy and widespread pain. Autonomic dysfunction can be diagnosed with tests such as Sudoscan or the water immersion skin wrinkling test (WISW). 1 12 1

12 Measurement of patient-reported outcomes Patient-reported SFN can be evaluated with the SFN-screening list (SFNSL).50 The SFNSL is a generic questionnaire that can also be used in sarcoidosis-associated SFN. The SFNSL evaluates specific symptoms of SFN, however, it does not distinguish between different phenotypes of SFN. Both for clinical management as well as for research on improvement of treatment options there is need for fine-tuning in assessment of SFN symptoms in sarcoidosis patients. First, distinguishing between length-dependent, non-length-dependent, intermittent and continuous pain is important. Moreover, patients should be able to indicate the location of pain specific to pain on the skin, muscles, joints or other levels. Finally, assessment of either intermittent or continuous pain, is mandatory. This thesis investigates whether a new questionnaire is meeting these requirements. General treatment of SFN Treatment of SFN generally focusses on treating the underlying condition and providing symptom relief. In cases of persistent painful SFN symptoms, anti-depressants, serotonin-norepinephrine reuptake inhibitors (SNRI), anticonvulsants, or pain-relieving drugs can be considered as first-line treatment (Figure 4).46 Second-line treatment options include anticonvulsants and opioids. Finally, third line treatment is limited to anticonvulsants. Therapeutic strategies are primarily symptomatic, achieving about 50% pain relief in only half of patients.51 Figure 4 General symptomatic treatment of small fiber neuropathy (SFN) treatment via a step-up approach. When persistent painful SFN symptoms are present, first line, second line, or third line treatment may be considered including tricyclic anti-depressants (TCA), serotonin norepinephrine reuptake inhibitors (SNRI), anticonvulsants, paracetamol (PCM), non-steroid anti-inflammatory drugs (NSAIDs), or opioids. Specific disease management strategies are not shown in the figure. Sarcoidosis This thesis focuses on SFN associated with sarcoidosis. Therefore, this section describes the background of this disease. Sarcoidosis is a granulomatous, multisystem disorder of unknown cause, mainly affecting lungs and lymph nodes. However, virtually any organ can be affected leading to a heterogeneous clinical presentation. The disease resolves spontaneously within 2-5 years in most patients (60%), but in approximately 40% of cases, it persists, often requiring years of treatment and monitoring.52,53 Pharmacological treatment of sarcoidosis is primarily aimed at preventing specific organ damage or alleviating symptoms and includes corticosteroids, methotrexate, disease-modifying anti-rheumatic drugs (DMARDs), or anti-tumor necrosis factor alpha (anti-TNF-α) treatments like infliximab.3,4 Many symptoms in patients with sarcoidosis are not organ-specific and include fatigue, cognitive failure, or symptoms related to sarcoidosis-associated SFN.54 Of these symptoms, fatigue is the most frequently reported in patients with sarcoidosis, with an estimated prevalence between 6090%, and it has the greatest impact on daily life.54 1 13 1

13 Pathophysiology and treatment of sarcoidosis-associated SFN Although granulomas are the hallmark of sarcoidosis, SFN in sarcoidosis patients does not appear to be directly related to the granulomatous inflammation of small nerve fibers. Therefore, the pathophysiology of sarcoidosis-associated SFN is comparable to the pathophysiology of SFN in general, as described above. Both general symptomatic treatment of SFN, as described in Figure 4, and treatment aimed at suppressing the underlying disorder apply to sarcoidosis-associated SFN. For inflammation-mediated SFN, such as sarcoidosis-associated SFN, suppression of inflammation seems a logical step in the treatment strategy. Although randomized controlled trials on immunosuppressive treatment for SFN in patients with sarcoidosis are lacking, case reports and small case series suggest that anti-TNF-α treatments, such as infliximab, may be effective for symptomatic treatment of sarcoidosis-associated SFN.55–57 Furthermore, specific treatment for autonomic dysfunction can be administered depending on the affected organs. The autonomic nervous system innervates viscera, vascular smooth muscle, endocrine and exocrine glands, the immune system, and soft tissues. Associated symptoms, as described in Table 2, may occur as consequence of autonomic dysfunction. Patients with sarcoidosisassociated SFN show an increased prevalence of autonomic dysfunction.20 For example, cardiac autonomic dysfunction may occur in patients with sarcoidosis and SFN and is associated with increased mortaility.20,58 In addition, the beta-blocker carvedilol has been suggested for its potential to improve the parasympathetic activity of the cardiac sympathetic nervous system, which is more profound than for instance the more widely used beta-blocker metoprolol.59 In conclusion, there is need for further research to understand how to accurately diagnose and treat small fiber neuropathy in sarcoidosis patients, potentially leading to improved management and quality of life for affected individuals. 1 14 1

14 Aims and outline of the thesis The aim of this thesis is to gain more insight into the diagnosis, symptoms, measurement of patientreported outcomes, and treatment of SFN and cardiac autonomic dysfunction in patients with sarcoidosis. Chapter 2 will provide an overview of the current literature regarding small fiber neuropathy. First, diagnostic methods currently in use will be evaluated concerning their corresponding physiologic mechanisms. Additionally, a systematic literature review will be performed to identify diagnostic accuracy of all methods. Chapter 3 will highlight the high rates of fatigue, restless legs syndrome, pain and cognitive impairment in patients with sarcoidosis-associated small fiber neuropathy compared with patients with sarcoidosis without small fiber neuropathy. Additionally, the correlation between these nonorgan specific symptoms will be established. Chapter 4 will use the new SFNPQ questionnaire to assess pain at cutaneous, muscular and joint pain in patients with sarcoidosis. A distinction will be made between pain in the head, thorax, arms, hands, back/abdomen, legs, and feet. Chapter 5 will indicate the diagnostic performance of thermal threshold testing (TTT). The strengths and limitations of this method will be evaluated, and a proposal is made to select the best parameters and measuring sites. Chapter 6 will compare multiple analysis methods to assess corneal nerve fiber length (CNFL) from corneal confocal microscopy images. Additionally, CNFL will be compared among healthy controls, sarcoidosis patients without SFN and sarcoidosis patients with established SFN. Chapter 7 will use the new SFNPQ questionnaire to identify non-length-dependent, length-dependent, intermittent or continuous SFN symptoms in sarcoidosis patients. Additionally, the association between symptom phenotype and different diagnostic modalities will be investigated. Chapter 8 will investigate the effects of treating the underlying disease of SFN. In this study the effects of infliximab on sarcoidosis activity and small fiber neuropathy-associated symptoms will be assessed. Patients with sarcoidosis, at least 3 months on infliximab treatment and high scores on the small fiber neuropathy screening list (SFNSL) will be included. Chapter 9 will reveal the clinical characteristics of patients with sarcoidosis referred for assessment of cardiac involvement and further analysis with an Iodine-123 meta-iodobenzylguanidine (123I-MIBG) scintigraphy to rule out autonomic dysfunction of the heart. Furthermore, the effectiveness of the beta-blocker carvedilol on patients with impaired parasympathetic activity will be determined. Chapter 10 will provide a summary, a general discussion on the results of this thesis and directions for future research. 1 15 1

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17 40. Lauria G, Morbin M, Lombardi R, et al. Axonal swellings predict the degeneration of epidermal nerve fibers in painful neuropathies. Neurology. 2003;61(5):631-636. doi:10.1212/01.WNL.0000070781.92512.A4 41. Casellini CM, Parson HK, Richardson MS, Nevoret ML, Vinik AI. Sudoscan, a Noninvasive Tool for Detecting Diabetic Small Fiber Neuropathy and Autonomic Dysfunction. Diabetes Technol Ther. 2013;15(11):948-953. doi:10.1089/dia.2013.0129 42. Low PA, Benarroch EE. Clinical Autonomic Disorders. third. (DeStefano F, McMillan L, Larkin J, eds.). Lippincott Williams & Wilkins; 2008. 43. Freeman R. Assessment of cardiovascular autonomic function. Clin Neurophysiol. 2006;117(4):716-730. doi:10.1016/j.clinph.2005.09.027 44. Wilder-Smith EP, Guo Y, Chow A. Stimulated skin wrinkling for predicting intraepidermal nerve fibre density. Clin Neurophysiol. 2009;120(5):953-958. doi:10.1016/j.clinph.2009.03.011 45. Sène D. Small fiber neuropathy: Diagnosis, causes, and treatment. Jt bone spine. 2018;85(5):553-559. doi:10.1016/j.jbspin.2017.11.002 46. Voortman M, Fritz D, Vogels OJM, et al. Small fiber neuropathy: A disabling and underrecognized syndrome. Curr Opin Pulm Med. 2017;23(5):447-457. doi:10.1097/MCP.0000000000000413 47. Bakkers M, Faber CG, Hoeijmakers JGJ, Lauria G, Merkies ISJ. Small fibers, large impact: Quality of life in small-fiber neuropathy. Muscle and Nerve. 2014;49(3):329-336. doi:10.1002/mus.23910 48. Themistocleous AC, Ramirez JD, Shillo PR, Lees JG, Selvarajah D. The Pain in Neuropathy Study ( PiNS ): a cross-sectional observational study determining the somatosensory phenotype of painful and painless diabetic neuropathy. Pain. 2016;157:1132-1145. 49. Hovaguimian A, Gibbons CH. Diagnosis and treatment of pain in small-fiber neuropathy. Curr Pain Headache Rep. 2011;15(3):193-200. doi:10.1007/s11916-011-0181-7 50. Hoitsma E, De Vries J, Drent M. The small fiber neuropathy screening list: Construction and cross-validation in sarcoidosis. Respir Med. 2011;105(1):95-100. doi:10.1016/j.rmed.2010.09.014 51. Sopacua M, Hoeijmakers JGJ, Merkies ISJ, Lauria G, Waxman SG, Faber CG. Small-fiber neuropathy: Expanding the clinical pain universe. J Peripher Nerv Syst. 2019;24(1):19-33. doi:10.1111/jns.12298 52. Baughman RP, Barriuso R, Beyer K, et al. Sarcoidosis: patient treatment priorities. ERJ Open Res. 2018;4(4):00141-02018. doi:10.1183/23120541.00141-2018 53. Judson MA. The Clinical Features of Sarcoidosis: A Comprehensive Review. Clin Rev Allergy Immunol. 2015;49(1):63-78. doi:10.1007/s12016-014-8450-y 54. Drent M, Strookappe B, Hoitsma E, De Vries J. Consequences of Sarcoidosis. Clin Chest Med. 2015;36(4):727-737. doi:10.1016/j.ccm.2015.08.013 55. Elfferich MD, Nelemans PJ, Ponds RW, De Vries J, Wijnen PA, Drent M. Everyday cognitive failure in sarcoidosis: The prevalence and the effect of anti-TNF-α treatment. Respiration. 2010;80(3):212-219. doi:10.1159/000314225 56. Parambil JG, Tavee JO, Zhou L, Pearson KS, Culver DA. Efficacy of intravenous immunoglobulin for small fiber neuropathy associated with sarcoidosis. Respir Med. 2011;105(1):101-105. doi:10.1016/j.rmed.2010.09.015 57. Tavee JO, Karwa K, Ahmed Z, Thompson N, Parambil J, Culver DA. Sarcoidosis-Associated Small Fiber Neuropathy in a Large Cohort: Clinical Aspects and Response to IVIG and Anti-TNF Alpha Treatment. Vol 126. Elsevier Ltd; 2017. doi:10.1016/j.rmed.2017.03.011 58. Gawałko M, Balsam P, Lodziński P, et al. Cardiac arrhythmias in autoimmune diseases. Circ J. 2020;84(5):685-694. doi:10.1253/circj.CJ-19-0705 59. Bloom HL, Vinik AI, Colombo J. Differential effects of adrenergic antagonists (Carvedilol vs Metoprolol) on parasympathetic and sympathetic activity: a comparison of clinical results. Heart Int. 2014;9(1):15-21. 1 18 1

Current view of diagnosing small fiber neuropathy 2 Lisette R.M. Raasing, Oscar J.M. Vogels, Marcel Veltkamp, Christiaan F.P. van Swol and Jan C. Grutters Journal of Neuromuscular Diseases 2021; 8(2):185–207

20 Abstract Small fiber neuropathy (SFN) is a disorder of the small myelinated Aδ-fibers and unmyelinated Cfibers.1,2 SFN might affect small sensory fibers, autonomic fibers or both, resulting in sensory changes, autonomic dysfunction, or combined symptoms.3 As a consequence, the symptoms are potentially numerous and have a large impact on quality of life.4 Since diagnostic methods for SFN are numerous and its pathophysiology complex, this extensive review focuses on categorizing all aspects of SFN as disease and its diagnosis. In this review, sensitivity in combination with specificity of different diagnostic methods are described using the areas under the curve. In the end, a diagnostic workflow is suggested based on different phenotypes of SFN. Introduction Etiology Small fiber neuropathy (SFN) is a disorder of the small myelinated Aδ-fibers and unmyelinated Cfibers.1 Incidence and prevalence are estimated to be 12/100,000 and 53/100,000, respectively, and are expected to rise with increasing awareness of SFN worldwide.2 SFN can affect either small sensory fibers, autonomic fibers, or both, resulting in sensory changes, autonomic dysfunction or combined symptoms.3 As a consequence, the potential symptoms are numerous and show a large impact on quality of life.4 General symptoms are fatigue, cognitive disturbances, widespread musculoskeletal pain, headache and temporomandibular disorder.5,6 Somatic small nerve fibers transmit information about temperature, pain and itch.5,7,8 The autonomic small nerve fibers are responsible for thermoregulatory, sudomotor, cardiovascular, gastrointestinal, urogenital and other autonomic functions.3,5 SFN is associated with a great variety of diseases as underlying mechanisms but can also present idiopathic.1 Table 1 shows an overview of some underlying disorders.1,5 Common nerve conduction tests only assess large myelinated nerve fibers. As a consequence, SFN is difficult to diagnose following the regular procedures.7,8 Currently, the prevalence of SFN is probably highly underestimated due to lack of a gold standard and awareness among clinical physicians.5 Improving diagnostic methods is important to improve recognition of symptoms in SFN patients, it can improve insight of pathophysiology and will facilitate future drug trials. 2 22 2

21 Table 1 Underlying diseases associated with SFN Pathophysiology The peripheral nervous system is classified into different types of nerves, based on diameter, myelin sheath, and conduction velocity, see Figure 1A.7,33 Aα- and Aβ-fibers are classified as large nerve fibers and Aδ- and C-fibers are classified as small nerve fibers. Small myelinated Aδ-fibers show faster conduction velocities (4-36 m/s)34,35 compared to unmyelinated C-fibers (0.4-2.8 m/s),35–37 due to larger diameter and myelin.38 SFN is described as dysfunction of the small nerve fibers. The exact pathophysiology of isolated SFN is unknown. However, since demyelinating processes do not solely affect small nerve fibers, it is unlikely that this would be the underlying pathogenesis. Distal axonal loss or perhaps extraordinary neuronal degeneration are therefore more likely to be the underlying cause of SFN.3 Five decades ago, four stages of neuropathy pathology in unmyelinated nerve fibers were defined.39 1) Mild proliferation: This stage is characterized by merely an increase in number of isolated, small Schwann cell projections. As consequence, these Schwann cells show a more irregular shape. 2) Fiber loss: in a more advanced stage, a decreased amount of fibers in combination with increased amount of empty Schwann cells are established. 3) Regeneration: Subsequently, regeneration of unmyelinated fibers associated with signs of fiber loss have been identified. An increment can be noted from the total number of unmyelinated fibers as well as small fibers with a diameter below 0.8 µm and empty Schwann cell sub-units. 4) Advanced regeneration: Finally, the amount of empty Schwann cells will return to a normal level. During this stage, only an increase of small nerve fibers with a diameter below 0.8 µm, and of small isolated projection of Schwann cells can be distinguished.39 Patients with diabetic-mediated SFN might show a different pathophysiology compared with other underlying etiologies. For example, in diabetic patients, axon swelling seen in skin biopsies can predict progression of distal SFN to proximal large fiber or polyneuropathy.40–42 Conversely, recent research which included no diabetic patients, claims that SFN is a stable disorder and rarely progresses.43 Although symptoms typically are length-dependent, resulting in symptoms in Associated diseases of small fiber neuropathy5,9  Idiopathic Hereditary  Fabry’s disease10  Mutation in sodium channels11  Wilsons disease12  Familial amyloidosis13 Metabolic  Diabetes mellitus14  Impaired glucose intolerance15  Vitamin B12 deficiency16  Copper deficiency17  Abnormal thyroid function18 Infectious  HIV19  Lyme20  Hepatitis C21 Toxic  Alcohol22  Chemotherapy23  Neurotoxic drugs24  Vaccine-associated21,25 Immune-mediated  Fibromyalgia26  Monoclonal gammopathy27  Ehlers-Danlos28  Sarcoidosis29  Rheumatic diseases (undifferentiated connective tissue disorders, rheumatoid arthritis, psoriasic arthropathy)24  Sjögren syndrome30  Primary systemic amyloidosis27  Acute inflammatory small fiber neuropathy24  Lupus31  Connective tissue disease24  Chronic inflammatory demyelinating polyneuropathy32 2 23 2

22 distal extremities,6 it also commonly presents with a non-length-dependent character.15 Non-length dependent SFN is likely to be associated with immune-mediated conditions and it presents more often in women at younger age.44 Somatic system The somatic nervous system is responsible for voluntary muscle control and sensory function. Sensory function, is broadly divided into special senses and general senses. Special senses include olfaction, vision, hearing, balance, and taste. General senses are divided into exteroceptors, (present in skin: nociception (pain, temperature, touch, pressure)), interoceptors (present in viscera: mechanical and chemical stimuli) and proprioceptors (present in muscles, joints and tendons: awareness of posture and movement).45 SFN mainly results in symptoms caused by damage of the nociceptive system. As a result, patients complain about pain, burning, tingling, prickling, shooting pain or numbness. Due to difference in conduction velocity, Aδ-fibers are responsible for the sharp, pricking or first pain response and C-fibers for the burning or second pain response, see Figure 1B.33 Aδ-fibers which respond to heat, are divided into type I and II A mechano-heat (AMH) units. AMH type I nerve fibers have a high response threshold (>53 0C), and their discharge rate increase during a prolonged stimulus. Typically type I AMH fibers show a higher sensitivity for mechanical stimuli compared to AMH type II fibers. AMH type II fibers have a short-latency adapting response, they have a lower threshold for heat stimuli (43-47 0C) and exhibit slower conduction velocities.46 As consequence, AMH type I fibers are responsible for first pain sensation of mechanical stimuli and AMH type II fibers are involved in first pain sensation of heat pain stimuli.33,38 C-fibers can be polymodal; responsive for noxious, thermal and mechanical stimuli. In addition, they can be responsive for a specific stimulus, but also for multiple stimuli or for non-specific stimuli.47 Table 2 shows an overview of functions from specific small fiber types. Also, specific temperature thresholds are shown, which are used for Quantitative Sensory Testing (QST) measurements. Figure 1 A) Overview of nerve fiber sizes, conduction velocities and other characteristics. Aα and Aβ fibers are large and myelinated nerve fibers, Aδ nerve fibers are small myelinated nerve fibers and C-fibers are small unmyelinated nerve fibers. B) Corresponding pain response. Large nerve fibers show a fast response with high amplitude. The smaller the nerve fiber, the lower the amplitude and the slower conduction velocities. 2 24 2

23 Autonomic system The autonomic system differs from the somatic system in that the somatic nervous system connects to its target organ via a single neuron, whereas the autonomic nervous system consists of two neurons. The autonomic ganglion forms the synaptic connection between the preganglionic and postganglionic neuron. The efferent autonomic nervous system can be divided into the sympathetic system (stress response), parasympathetic system (rest response) and enteric nervous system (digestive system).45 Preganglionic fibers are myelinated and use acetylcholine as neurotransmitters. Postganglionic nerve fibers are smaller compared to preganglionic fibers, are unmyelinated and use norepinephrine as neurotransmitter. An exception are sweat glands, which use cholinergic nerves (Figure 2).45 Table 2 Overview of different nociception receptors with corresponding small nerve fiber type35 Receptor type Fiber group Modality Cutaneous and subcutaneous mechanoreceptors Hair down Thermal receptors Cold receptors Warm receptors Heat nociceptors Cold nociceptor Nociceptors Mechanical Thermal-mechanical Thermal-mechanical Polymodal Muscle and skeletal mechanoreceptors Stretch-sensitive free endings Aδ Aδ C Aδ C Aδ Aδ C C Aδ Touch Light stroking Temperature Skin cooling (250C) Skin warming (410C) Hot temperatures (>450C) Cold temperatures (<50C) Pain Sharp, pricking pain Burning pain Freezing pain Slow, burning pain Limb proprioception Excess stretch or force 2 25 2

24 Figure 2 Complete overview of the nervous system, showing anatomical differences between the somatic and autonomic system and differences in nerve anatomy and use of neurotransmitters. In addition, all diagnostic methods are presented at their corresponding measuring area. Different font styles are used to discriminate between methods based on NFD (bold), small nerve fiber function (italic) and imaging (normal). Moreover, for some functional tests, an additional mark is established to discriminate between tests based on thermal and/or mechanical nociceptors. Abbreviations: EPs, evoked potentials; fMRI, functional magnetic resonance imaging; TST, thermoregulatory sweat testing; US, ultrasound; CCM, corneal confocal microscopy; IENFD, intra-epidermal nerve fiber density; QST, quantitative sensory testing; TTT, temperature threshold testing; HR, heartrate; EMG, electromyography; MIBG, 123I-meta-iodobenzylguadine; QPART, quantitative pilomotor axon-reflex test; BP, blood pressure; SGNFD, sweat gland nerve fiber density; LDIflare, laser Doppler imaging flare; QSART, quantitative sudomotor axon reflex test; QDIRT, quantitative direct and indirect reflex test; SSR, sympathetic skin response. Diagnostic methods Various methods have been described to diagnose SFN. Diagnostic methods can be categorized into questionnaires, genetic analysis, quantification of small nerve fiber density (NFD), sensory function tests, autonomic function tests and imaging techniques to quantify small nerve fibers.48 Questionnaires are rather subjective and imaging techniques are still in the early stages of assessing their diagnostic utility for SFN. Quantification of small sensory nerve fibers through NFD measurements, as well as functional assessments of sensory and autonomic small nerve fibers, have been frequently studied and compared. It is important to keep in mind that NFD or functional outcomes are very different measures for SFN.49 Diagnosing SFN remains challenging and a gold standard is not yet available. The presence of at least two abnormal findings at clinical, QST and skin biopsy examination have been suggested as best diagnostic criteria for SFN.24,50 However, there remains some controversy on this suggestion.51 Moreover, the clinical utility of skin biopsy is limited by labor intensity, availability in few centers, high costs and impracticality for longitudinal studies. Therefore, another research group suggests the presence of at least two abnormal findings at clinical, QST and Quantitative Sudomotor Axon-Reflex Test (QSART) examinations for a definite diagnosis.52 2 26 2

25 Since no single method is sensitive enough to confirm or exclude SFN, a combination of multiple methods seems to be the best alternative. The more abnormal test results, the more secure the diagnosis will be. A recent study investigated six different methods and suggested even a combination of four methods (skin biopsy, Electrochemical Skin Conductance (ESC), Laser Evoked Potentials (LEP) and QST) for a definite diagnosis.53 In order to classify SFN, the following definitions are preferably used.54–56 1. Possible SFN: symptoms or clinical signs of small fiber damage 2. Probable SFN: symptoms or clinical signs of small fiber damage and normal sural nerve conduction studies 3. Definite SFN: symptoms or clinical signs of SFN-damage, normal sural nerve conduction studies and decreased intra-epidermal nerve fiber density (IENFD) and/or abnormal quantitative sensory testing (QST) thermal thresholds Diagnosis To diagnose isolated SFN, physicians should be aware of the broad range of symptoms that may exhibit. During physical examination, tendon reflexes should be normal, and there should be no signs of muscle weakness. Vibration sense and proprioception may vary, appearing either normal or abnormal.57 QST or skin biopsy are recommended for a definite diagnosis.50,55 However, these methods are time-consuming and are not widely available in every hospital. Objectives  Clarify which diagnostic methods are available  Define diagnostic accuracy of each method  Present a clinical diagnostic workflow Methods First, the huge amount of available diagnostic methods will be clarified to show an organized overview. Next, a systematic search is performed to develop an overview of diagnostic accuracy (AUC-values) for each method. The results of a systematic literature search between 2000-2019 are presented focusing on the sensitivity and specificity of all diagnostic methods. It is important to state that since no gold standard is available, the AUC-values are relative measures, based on an imperfect standard. Literature search is performed on 19 August 2019 in PubMed and Embase. Exclusion criteria were:  Publication date <2000  Case reports  Language other than English  Animal study  Large fiber neuropathy AUC-values are calculated based on published sensitivity and specificity values. Articles were included when sensitivity and specificity were clearly published or could be calculated if an overview of test results for all participants was available. The classification of definite SFN is used, in order to determine sensitivity and specificity. Only articles with isolated SFN were included. Figure 3 shows the search results and exclusion criteria. Review articles, animal models and case reports are labeled as “wrong study design”. In the end, several phenotypes of SFN are described based on different symptoms. Depending on the phenotypes, optimal diagnostic methods are suggested. 2 27 2

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