Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy

Fabry disease (FD) is an X-linked lysosomal storage disorder which may lead to impaired peripheral nerve function, mostly affecting small nerve fibers, and to neuropathic pain. Characteristics of the neuropathy associated with FD and the covariates for its development and temporal course have not been described in a large cohort. We studied small fiber function and morphology in 120 Fabry patients at baseline and in subgroups of these until 4-year follow-up. Baseline neurological (89/120) and electrophysiological (106/120) examination was mostly normal. Quantitative sensory testing revealed impaired cold detection thresholds in 84% of men and 39% of women. Lower leg intraepidermal nerve fiber density (IENFD) was reduced to 46% in Fabry patients compared to controls and to 12.5% in men with impaired renal function. Patients with abnormal IENFD more often had pain. Group means for IENFD did not improve under enzyme replacement therapy (ERT), but IENFD in the back increased under ERT in 4/15 patients with good renal function and clinical improvement. Cutaneous cytokine gene expression did not differ from controls. We conclude that ERT may improve proximal skin innervation in patients with good renal function, but does not protect small fiber function in men with impaired renal function.     

Determinants of epidermal nerve fiber density in normal individuals

We studied the relationship between epidermal innervation and age, gender, height, and weight. Intraepidermal nerve fiber density (IENFD) of skin biopsies obtained from the proximal thigh and ankle of 84 normal individuals was quantified. A linear regression model was performed using IENFD at the thigh, IENFD at the ankle, and the thigh IENFD/ankle IENFD ratio, with age, gender, and height-weight interaction as predictors. An independent, negative correlation was found between age and IENFD at the ankle. No correlation was found between age and IENFD at the thigh. With increasing age the thigh IENFD/ankle IENFD ratio, a measure of the length-dependent distal-to-proximal gradient of epidermal nerve density, increased significantly. Gender, height, and body weight did not independently influence IENFD at either site. In normal individuals, distal epidermal innervation decreases in a length-dependent manner with advancing age. This must be considered when interpreting IENFD in disease states.     

Intraepidermal nerve fiber density as a marker of early diabetic neuropathy

The purpose of the study was to reliably identify an early stage of diabetic polyneuropathy (DPN) by measuring injury to epidermal nerve fibers. We compared intraepidermal nerve fiber density (IENFD) at the ankle and thigh of 29 diabetic subjects who had no clinical or electrophysiological evidence of small- or large-fiber neuropathy to that of 84 healthy controls. The mean ankle IENFD of diabetic subjects was 9.1+/-5.0 mm and that of controls, 13.0+/-4.8 mm (P<0.001). The thigh IENFD did not differ significantly. The IENFD ratio (thigh IENFD divided by ankle IENFD) was 2.39+/-1.30 in diabetic subjects and 1.77+/-0.58 in controls (P<0.001), indicating a length-dependent reduction of IENFD in diabetics. Ankle IENFD remained significantly lower and the IENFD ratio higher in diabetic subjects after adjusting for age. Two subjects had parasympathetic dysfunction, two had retinopathy, and two early nephropathy. Age, height, weight, duration of diabetes, and average HbA1c did not influence IENFD among diabetic subjects. We used receiver operating characteristic (ROC) curves to describe and compare the utility of various threshold values of ankle IENFD and IENFD ratio for the diagnosis of early DPN. The sensitivity and specificity of diagnosing DPN using ankle IENFD of less than 10 mm were 72.4% and 76.2%, respectively. Thus, asymptomatic diabetics have a measurable, length-dependent reduction of distal epidermal nerves. Analogous to microalbuminuria in diabetic nephropathy, reliable identification and quantitation of nascent diabetic neuropathy may have potential therapeutic implications.     

Corneal confocal microscopy: A novel noninvasive means to diagnose neuropathy in patients with fabry disease

Neuropathy is a cause of significant disability in patients with Fabry disease, yet its diagnosis is difficult. In this study we compared the novel noninvasive techniques of corneal confocal microscopy (CCM) to quantify small‐fiber pathology, and non‐contact corneal aesthesiometry (NCCA) to quantify loss of corneal sensation, with established tests of neuropathy in patients with Fabry disease. Ten heterozygous females with Fabry disease not on enzyme replacement therapy (ERT), 6 heterozygous females, 6 hemizygous males on ERT, and 14 age‐matched, healthy volunteers underwent detailed quantification of neuropathic symptoms, neurological deficits, neurophysiology, quantitative sensory testing (QST), NCCA, and CCM. All patients with Fabry disease had significant neuropathic symptoms and an elevated Mainz score. Peroneal nerve amplitude was reduced in all patients and vibration perception threshold was elevated in both male and female patients on ERT. Cold sensation (CS) threshold was significantly reduced in both male and female patients on ERT (P < 0.02), but warm sensation (WS) and heat‐induced pain (HIP) were only significantly increased in males on ERT (P < 0.01). However, corneal sensation assessed with NCCA was significantly reduced in female (P < 0.02) and male (P < 0.04) patients on ERT compared with control subjects. According to CCM, corneal nerve fiber and branch density was significantly reduced in female (P < 0.03) and male (P < 0.02) patients on ERT compared with control subjects. Furthermore, the severity of neuropathic symptoms and the neurological component of the Mainz Severity Score Index correlated significantly with QST and CCM. This study shows that CCM and NCCA provide a novel means to detect early nerve fiber damage and dysfunction, respectively, in patients with Fabry disease. Muscle Nerve, 2009     

Neuropathic symptoms and findings in women with Fabry disease

ObjectiveTo examine the neurologic and neurophysiologic findings and neurologic symptoms in 12 women with Fabry disease and to study the relationship between the subjective symptoms and the findings on the various tests done.MethodsNeurography, vibratory and thermal quantitative sensory testing (QST), skin biopsy for measuring intraepidermal nerve fiber density (IENFD). Heart rate variability (HRV) and sympathetic skin response (SSR) tests for detecting autonomic dysfunction, pain-, depression- and somatic symptom questionnaires and clinical examination.ResultsOnly two women had no persistent symptoms or signs of polyneuropathy, 10 had symptoms of small fiber neuropathy. Neurological examination was normal in most patients. Five patients had decreased IENFD or thermal hypoesthesia in QST. In QST, Aδ-fiber function for innocuous cold was more often impaired than C-fiber function. Conventional nerve conduction studies were mostly normal. Carpal tunnel syndrome (CTS) incidence was increased, 25% had symptomatic CTS.ConclusionsHeterozygous women carrying the gene for Fabry disease have symptoms and findings of small-fiber polyneuropathy more often than has previously been considered. The prevalence of CTS is also increased.SignificanceWhile the clinical diagnosis of small-fiber neuropathy is difficult, the diagnostic yield can be increased using a combination of thermal QST and IENFD measurements.     

Skin biopsy in assessing meralgia paresthetica

Introduction: Meralgia paresthetica is a focal neuropathy caused by compression of the lateral femoral cutaneous nerve (LFCN). The disease can be difficult to assess by neurophysiological or imaging studies. Methods: We studied 5 patients who presented to our neuromuscular clinic from April 2012 to December 2014 with a clinical suspicion of meralgia paresthetica and had skin biopsies with intraepidermal nerve fiber density (IENFD) evaluation. Results: The mean age at onset was 37.2 (range 21–59) years. There were 4 women and 1 man. Two were obese, 2 wore tight jeans, and 1 had mild diabetes mellitus. IENFD was reduced in the symptomatic proximal thigh in all 5 patients and was also reduced in the asymptomatic thigh in 2 patients. It was normal in the distal leg in 4 patients. Conclusion: Meralgia paresthetica is associated with loss of small intraepidermal nerve fibers. Skin biopsy with IENFD evaluation may be a useful diagnostic tool for this disease. Muscle Nerve 53 : 641–643, 2016     

Quantitative analysis of epidermal innervation in Fabry disease

OBJECTIVE: To use skin biopsy specimens to quantitate the cutaneous innervation density of Fabry patients who had preserved renal function. BACKGROUND: The small fiber neuropathy of Fabry disease is difficult to detect and quantitate by conventional methods. Because this neuropathy is a common characteristic of Fabry disease, quantitating changes in this parameter would be helpful in demonstrating the effectiveness of enzyme or gene replacement therapy. METHODS: Patients underwent skin biopsy at the thigh and foot. Innervation density was determined by counting free nerve endings in the epidermis. These data were compared with nerve conduction studies, and in selected patients, fiber quantitation of sural nerve biopsy specimens. RESULTS: The Fabry patients had normal results of nerve conduction studies and large fiber quantitation by sural nerve biopsy. However, the involvement of small cutaneous fibers in these patients was easily demonstrable and quantifiable by skin biopsy. All patients showed severe loss of intraepidermal innervation at the ankle, but fiber loss at the distal thigh was proportionately less severe. CONCLUSIONS: The nerve damage in Fabry patients with preserved renal function involves exclusively small myelinated and unmyelinated fibers, and skin biopsy is a useful in detecting and quantitating such damage. Comparison of cutaneous innervation density with quantitation of sural nerve biopsy specimens demonstrated that skin biopsy specimens were as sensitive in detecting the presence of neuropathy as were the nerve specimens. It is speculated that analysis of cutaneous innervation may provide a useful marker of the nervous system's response to specific therapy for Fabry disease.     

Enzyme replacement therapy improves peripheral nerve and sweat function in Fabry disease

Fabry disease is an X-linked disorder caused by a deficiency of lysosomal alpha-galactosidase A resulting in accumulation of alpha-D-galatosyl conjugated glycosphingolipids. Clinical manifestations include a small-fiber neuropathy associated with debilitating pain and hypohidrosis. We report the effect of a 3-year open-label extension of a previously reported 6-month placebo-controlled enzyme replacement therapy (ERT) trial in which 26 hemizygous patients with Fabry disease received 0.2 mg/kg of alpha-galactosidase A every 2 weeks. The effect of ERT on neuropathic pain scores while off pain medications, quantitative sensory testing, quantitative sudomotor axon reflex test (QSART), and thermoregulatory sweat test (TST) is reported. In the patients who crossed-over from placebo to ERT (n = 10), mean pain-at-its-worst scores on a 0-10 scale decreased (from 6.9 to 4.5). There was a significant reduction in the threshold for cold and warm sensation in the foot. At the 3-year time-point, pre-ERT sweat excretion in 17 Fabry patients was 0.24 +/- 0.33 microl/mm(2) vs. 1.05 +/- 0.81 in concurrent controls (n = 38). Sweat function improved 24-72 h post-enzyme infusion (0.57 +/- 0.71 microl/mm(2)) and normalized in four anhidrotic patients. TST confirmed the QSART results. We conclude that prolonged ERT in Fabry disease leads to a modest but significant improvement in the clinical manifestations of the small-fiber neuropathy associated with this disorder. QSART may be useful to further optimize the dose and frequency of ERT.     

皮肤神经活体组织检查在周围神经病诊断中的应用

目的 探讨皮肤神经活体组织检查在周围神经病诊断中的作用,建立正常参考值范围,并比较临床表现、神经电生理检查与表皮神经纤维病变的一致性.方法 对51例有周围神经病症状和(或)体征的患者进行皮肤神经活体组织检查,计算表皮神经纤维密度(IENFD);同时收集10名健康志愿者作为对照.51例患者中,41例行常规肌电图及神经传导速度(NCV)检查.21例行皮肤交感反射(SSR),比较IENFD与NCV及SSR的一致性.结果 对照组与病例组相比,大腿IENFD(根/mm)分别为21.4±2.7及15.0±6.3(t=2.976,P=0.004);小腿IENFD分别为15.4±2.2及8.1±5.9(t=3.191,P=0.002).病例组与对照组相比大、小腿IENFD均有减少,差异有统计学意义.51例患者中,皮肤神经活体组织检查异常48例(94.1%),其中33例表现为长度依赖性周围神经病变;41例行常规肌电图检查,21例异常(51.2%);21例行SSR检查,异常17例(81.0%).仅表现为小纤维病变症状和(或)体征的29例患者中,27例(93.1%)皮肤神经活体组织检查异常;其中20例行NCV,异常6例(30.0%);14例行SSR,11例异常.结论 皮肤神经活体组织检查操作简单安全,对于以小神经纤维受累为主的周围神经病皮肤神经活体组织检查有较高的灵敏度.     

Thermal thresholds and motor sensory conduction measurements in Guillain Barré syndrome: 12-month follow-up study.

We performed serial neurophysiological tests in 22 patients with confirmed Guillain Barré syndrome (GBS) for 12 months following onset of the disease. Twenty-five age- and sex-matched healthy controls were also tested. Tests included nerve conduction and automated quantitative thermal threshold measurements. The commonest early abnormality was delayed distal motor latency, F wave abnormality and abnormal thermal threshold measurements in 1 or more nerves. Further abnormalities of all measurements were observed during maximum disability. After 12 months, all measured parameters returned to normal levels in the majority of patients except the thermal thresholds which remained abnormal in a large proportion of asymptomatic patients. The findings are suggestive of an early pathological involvement of the smaller nerve fibres with slow recovery in GBS.     

Peripheral nerve conduction in Fabry disease

Peripheral nerve conduction was studied in 34 members, aged 3 to 54 years, from three separate kindreds with Fabry disease. Nerve conduction velocity was prolonged in one-third (11 of 34) of the study group. Of the 9 control family members with normal plasma alpha-galactosidase levels, 8 had normal velocity. A 6-year-old girl with normal plasma and leukocyte alpha-galactosidase levels and absent corneal deposits had slightly lower than normal nerve conduction velocity. One of the 3 obligate female carriers and 1 of 10 suspected carriers of Fabry disease had prolonged conduction velocity but normal distal latency. On the other hand, 8 of 12 males affected with Fabry disease demonstrated slow nerve conduction velocity while 4 of the 8 had prolonged distal latency. Frequently, only one of the two nerves studied showed abnormalities. These abnormalities were not related to patient age. Interfamilial variations in the abnormalities were present.     

Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies

In this prospective study, intraepidermal nerve fiber densities (IENFD) and subepidermal nerve plexus densities (SENPD) were quantified by immunostaining in skin punch biopsies from the distal calf in 99 patients with clinical symptoms of painful sensory neuropathy and from 37 age-matched healthy volunteers. The clinical diagnosis was based on history and abnormal thermal thresholds on quantitative sensory testing (QST). In patients with neuropathy, IENFD and SENPD were reduced to about 50% of controls. Elevated warm detection thresholds on QST correlated with IENFD but not with SENPD. Using receiver-operating characteristic (ROC) curve analysis of IENFD values, the diagnostic sensitivity for detecting neuropathy was 0.80 and the specificity 0.82. For SENPD, sensitivity was 0.81 and specificity 0.88. With ROC analysis of both IENFD and SENPD together, the diagnostic sensitivity was further improved to 0.92. The combined examination of IENFD and SENPD is a highly sensitive and specific diagnostic tool in patients suspected to suffer from painful sensory neuropathies but with normal values on clinical neurophysiological studies.     

Acute intermittent porphyria with peripheral neuropathy complicated by small‐fiber neuropathy

We describe a 60‐year‐old male patient with acute intermittent porphyria (AIP) who presented with initial abdominal pain and subsequent quadriplegia and respiratory failure. Small fiber neuropathy was demonstrated by measuring intra‐epidermal nerve fiber density (IENFD) using protein gene product 9.5 (PGP 9.5) immunostaining on three consecutive skin punch biopsies of the distal lower limb. The biopsy findings demonstrated some correlation with progression of the patient’s clinical condition. Neuropathy in AIP can have a small‐fiber component rather than being solely a large‐fiber neuropathy.     

Temperature threshold testing: a systematic review

The diagnosis of small fiber neuropathy (SFN) has been recently defined as typical symptoms due to small nerve fiber dysfunction accompanied by reduced intra‐epidermal nerve fiber density (IENFD) or abnormal temperature threshold testing (TTT). Guidelines have been published for the assessment of IENFD. However, international guidelines for TTT are lacking. This paper presents a systematic literature review on reported TTT methods and provides recommendations for its future use in studies evaluating patients. A total of 164 papers fulfilled pre‐defined requirements and were selected for review. Over 15 types of instruments are currently being used with a variety of methodological approaches for location, stimulus application, and sensation qualities examined. Consensus is needed to standardize the use of TTT as a diagnostic and follow‐up tool in patients.     

Intraepidermal nerve fiber density of healthy human

Abstract

Objectives:

To analyze intraepidermal nerve fiber density (IEFND) by skin biopsy, evaluate the effect of age, anatomical sites, and ethnic origin on IEFND and develop a reference range of IENFD at the distal leg of healthy human.

Methods:

Seventy skin biopsy specimens from surgical procedures involving 70 patients were analyzed. Specimens were fixed routinely in formalin and thereafter embedded in paraffin. Nerve fibers of 10-μm-thick sections were observed using immunoperoxidase staining with panaxonal antibody protein gene product 9·5 (PGP 9·5). The morphology of intraepidermal nerve fibers (IENFs) and the IENFD was determined using light microscope. The statistical analysis was performed with SPSS 16·0 software.

Results:

No significant correlation was observed between IENFD and age (P_wrist = 0·830, P_{distal leg} = 0·478). The significant correlation was observed between IENFD and anatomic site (P = 0·001), the IENFD of upper arm and proximal thigh were significantly higher than that of wrist and distal leg. The reference range for IENFD of distal leg in normal Chinese humans was 40·55 fibers/mm². The IEFND of Chinese healthy human was significantly lower than that of Finnish (62·87 ± 15·25 vs 114·617 ± 32·322 fibers/mm², P < 0·05).

Discussion:

Skin biopsy may be a useful tool in sensory neuropathies. IENFD is independent of age, but varies in different parts of the body. The proximal sites have a higher IENFD, but no significant difference is found between the wrist and distal leg.     

The reliability of skin biopsy with measurement of intraepidermal nerve fiber density

Intraepidermal nerve fiber density (IENFD) is a sensitive measure of small fiber injury, and holds promise as a clinical trial endpoint measure. A total of 48 punch biopsies were obtained from 22 patients. Tissue was sectioned and stained with PGP9.5. The relative intertrial variability (RIV) of IENFD measurements for each section and punch made by two different observers was determined (interobserver variability). Intraobserver variability (same observer measuring twice) was determined for 50% of the sections and punches. Sections from 12 punch biopsies were also stained at a second laboratory. The effect of the number of sections counted and processing site on reproducibility was investigated. A total of 223 sections were analyzed. The mean IENFD was 6.7 fibers/mm. Mean (+/-standard deviation) interobserver variability was 9.6%+/-9.4 for each biopsy site and 10.2%+/-11.9 for individual sections. Mean intraobserver variability was 9.6%+/-8.9 for biopsies, and 8.8%+/-9.0 for sections. There was no significant difference in IENFD for tissue stained at different laboratories. Intraclass correlation coefficients were greater than 0.98 for each comparison. There was no relationship between absolute IENFD and reproducibility. Reproducibility was highest when four sections were counted. IENFD measurement is highly reproducible. At least four sections should be analyzed. Reliability does not vary with severity of disease. These findings suggest IENFD may be a useful endpoint measure in future neuropathy treatment trials.     

帕金森病患者皮肤神经磷酸化α-突触核蛋白研究

目的探讨帕金森病患者磷酸化α-突触核蛋白(phosphorylated α synuclein,p-α-syn)在皮肤神经中的沉积特点,以及其作为帕金森病外周生物标志物的可能性。方法纳入2017年6月1日至2018年8月31日就诊于郑州大学第一附属医院神经内科的15例帕金森病患者及同期年龄匹配的健康志愿者31名,进行13项小纤维神经病和症状问卷(SFN-SIQ)评分。将帕金森病患者按照主要临床表现分为运动迟缓(n=7)和静止性震颤(n=8)2个亚组,并以Hoehn-Yahr分级评价病情的严重程度,其中0~2.5级为早期组(n=11),3.0~5.0级为中晚期(n=4)。采用环形钻孔器取帕金森病患者小腿和颈部皮肤,健康对照组只取小腿部位皮肤,进行免疫组织化学和免疫荧光染色,观察p-α-syn在皮肤神经中的沉积情况并计数穿过单位长度基底膜的神经纤维数量即表皮内神经纤维密度(intraepidermal nerve fiber density,IENFD)。结果12/15的帕金森病患者表皮下神经丛、真皮神经束、汗腺、立毛肌、血管或毛囊周围神经中可见点状或线状p-α-syn沉积,健康对照组皮肤中未见沉积。p-α-syn在单个部位沉积的阳性率分别为小腿6/15、颈部7/15,总阳性率为12/15。帕金森病组的IENFD为(6.85±1.94)根/mm,较对照组[(10.45±3.70)根/mm]明显下降(t=-3.303,P=0.002),与SFN-SIQ评分呈负相关(r=-0.561,P=0.046)。疾病早期与中晚期患者之间,以及以震颤和以运动迟缓为主要表现的患者之间比较,p-α-syn沉积阳性率和IENFD差异均无统计学意义。结论p-α-syn在帕金森病患者皮肤神经纤维中沉积,伴随IENFD明显下降。提示皮肤神经中p-α-syn沉积可能是帕金森病固有的外周病理改变,有作为帕金森病患者诊断外周生物标志物的可行性。     

Cramps and small‐fiber neuropathy

Introduction: Muscle cramps are a common complaint and are thought to arise from spontaneous discharges of the motor nerve terminal. Polyneuropathy is often causative, but small‐fiber neuropathy (SFN) has not been assessed. Methods: We performed skin biopsies on consecutive patients with cramps but without neuropathic complaints. Twelve patients were biopsied, 8 with normal small‐fiber sensation. Results: Seven patients had decreased intraepidermal nerve fiber density (IENFD), 2 with non–length‐dependent loss. A cause for neuropathy was found in 1 patient with cramp–fasciculation syndrome. Creatine kinase was elevated in 8 patients, 4 with decreased IENFD. Muscle biopsy, performed in 8 patients, but was diagnostic in only 1, with McArdle disease. Conclusions: Our data show that 60% of patients with muscle cramps who lack neuropathic complaints have SFN, as documented by decreased IENFD. Cramps may originate as local mediators of inflammation released by damaged small nerve that excite intramuscular nerves. Muscle Nerve, 48: 252–255, 2013     

健康皮肤标本表皮神经纤维密度研究

目的 探讨健康皮肤标本表皮神经纤维密度的参考值范围,并分析年龄、解剖部位以及种族对表皮神经分布的影响.方法 对70例皮肤标本进行皮肤神经活体组织检查.标本经10%福尔马林常规固定后切成10μm厚石蜡切片,以PGP9.5为特异性轴突标记物进行免疫组织化学染色.观察皮肤神经纤维的形态学特点,并测定表皮神经纤维密度,进行统计学分析.结果 不同年龄组小腿远端及腕部表皮神经纤维密度差异无统计学意义,但随年龄增加,各部位表皮神经分布有减少的趋势.上臂(91.8±21.1)、大腿近端(89.2±21.4)的表皮神经分布较腕部(64.5±22.5)、小腿远端(62.9±15.3)密集.健康人小腿远端的表皮神经纤维密度正常参考值范围>40.6纤维/m2.结论 通过皮肤活体组织检查技术可清晰显示神经纤维形态,便于对表皮神经纤维进行定量研究,为周围神经病的诊断和研究提供了一个可靠的平台.     

Small‐nerve‐fiber pathology in critical illness documented by serial skin biopsies

Introduction: Small‐fiber pathology can develop in the acute phase of critical illness and may explain chronic sensory impairment and pain in critical care survivors. Methods: Eleven adult ischemic stroke patients in a neurocritical care unit were enrolled in an observational cohort study. Intraepidermal nerve fiber density (IENFD) in the distal leg was assessed on admission to the intensive care unit and 10–14 days later, together with electrophysiological testing. Results: Of the 11 patients recruited, 9 (82%) had sepsis or multiple‐organ failure. Median IENFD on admission (5.05 fibers/mm) decreased significantly to 2.18 fibers/mm (P < 0.001), and abnormal IENFD was found in 6 patients (54.5%). Electrodiagnostic signs of large‐fiber neuropathy and/or myopathy were found in 6 patients (54.5%), and autonomic dysfunction was found in 2 patients (18.2%). Conclusion: Serial IENFD measurements confirmed the development of small‐fiber sensory involvement in the acute phase of critical illness. Muscle Nerve 52 : 28–33, 2015