Cutaneous innervation in ross syndrome: a functional and morphological study in 8 patients

Evaluation of epidermal nerve fiber (ENF) density by skin biopsy is nowadays considered as the gold standard for the diagnosis of small fiber neuropathies. ENFs show in normal subjects quite a regular distribution between the keratinocytes while in patients with small fiber neuropathies we can find, besides a loss of ENFs, evident anomalies of their distribution. Different patterns of nerve fiber dispersion can express different pathophysiological processes. The aim of this study was to define a tool capable of describing the distribution of ENF in the epidermis. For this purpose, we measured the distance between subsequent epidermal fibers on skin samples of healthy and diabetic subjects. We calculated the standard deviation of the interfiber length as a parameter (the dispersion index – DI) to evaluate the variability of the distance between consecutive ENFs. We selected thigh skin samples from five healthy subjects (age range 30–50 years) and five age and sex matched diabetic patients. Samples were processed using immunohistochemical techniques. Sections were PGP 9.5 and Collagen IV double stained to show nerve fibers and basement membrane. One randomly selected section for each subject was acquired for its entire length (usually seven 20x confocal images obtained from a z‐stack of 16 two micron optical sections) using a confocal microscope (CARV, Atto Biosciences, Rockville MD, USA). We measured the distance between consecutive ENFs along the entire epidermal length, using dedicated software (ScionImage, Scion Corporation Frederick, Maryland USA). In diabetic patients the DI resulted significantly higher than in normal subjects. We suggest that DI along with the ENF density can be useful to better characterize epidermal innervation.     

Epidermal innervation in healthy children and adolescents

Introduction: Epidermal nerve fiber (ENF) density, morphology, and epidermal innervation patterns were examined in children using 2 different techniques, punch biopsy and suction blister. Methods: Healthy children without symptoms or history of peripheral neuropathy and normal by neurologic examination were studied. Punch biopsy and suction blister specimens were collected from the lateral thigh and distal leg. ENFs were traced from confocal images of immunohistochemically stained samples. Statistical methods included repeated‐measures analysis of covariance. Results: Blister and biopsy nerve counts were associated. ENF density in children was dense, lower for older children (P < 0.01) and with no difference between boys and girls (P = 0.92). Many ENFs appeared multibranched and elongated. Conclusions: Epidermal innervation in the pediatric population is dense and age‐dependent. Blister specimens are less invasive and may provide an alternative to punch biopsy for determining ENF density in children at risk for neuropathy. Muscle Nerve 51 : 378–384, 2015     

Skin biopsy and quantitative sensory testing do not predict response to lidocaine patch in painful neuropathies

Predictors of response to neuropathic pain treatment in patients with painful distal sensory neuropathies are lacking. The 5% lidocaine patch is believed to exert its effects on neuropathic pain via a local stabilizing effect on cutaneous sensory afferents. As such, it provides a model to assess whether the status of epidermal innervation as determined by skin biopsy or quantitative sensory testing (QST) of small- and large-diameter sensory afferents might serve as predictors of response to topical, locally active treatment. In this study we assessed associations between epidermal nerve fiber (ENF) densities, sensory nerve conduction studies (NCS), QST, and response to a 5% lidocaine patch in patients with painful distal sensory neuropathies. We observed no association between distal leg epidermal and subepidermal innervation and response to the lidocaine patch. Several patients with complete loss of distal leg ENF showed a response to the lidocaine patch. Similarly we observed no consistent association between treatment response and QST for vibration, cooling, warm, heat-pain, and cold-pain thresholds, or distal sensory NCS. Thus, distal-leg skin biopsy, QST, and sensory NCS cannot be used to identify patients with painful polyneuropathy likely to respond to a lidocaine patch in clinical practice. Further studies are required to clarify precisely the mechanism and site of action of the lidocaine patch in patients with peripheral neuropathic pain.     

Small-fiber neuropathy/neuronopathy associated with celiac disease: skin biopsy findings

BACKGROUND: Celiac disease (CD) is increasingly recognized in North America and is associated with a peripheral neuropathy. OBJECTIVE: To report the clinical characteristics and skin biopsy results in patients with CD and small-fiber neuropathy symptoms. DESIGN: Case series. SETTING: Academic peripheral neuropathy clinic. PATIENTS: Eight patients with CD and neuropathy symptoms.Intervention Three-millimeter punch biopsy using the panaxonal marker protein gene product 9.5 to assess epidermal nerve fiber (ENF) density and a gluten-free diet. MAIN OUTCOME MEASURE: Clinical data and ENF density. RESULTS: All patients had asymmetric numbness and paresthesias. Three had more prominent involvement of hands than feet, and 3 had facial numbness. Celiac disease was diagnosed in 5 after their neuropathy began. The following serum antibody levels were elevated: tissue transglutaminase (n = 6), IgA gliadin (n = 4), and IgG gliadin (n = 7). Results of nerve conduction studies were normal in 7 patients. One patient had mildly reduced sural amplitudes. The ENF density was reduced in 5 patients. The ENF density was at the low limit of the normal range in 3 additional patients, 2 of whom had morphologic changes in axons. Three patients had decreased ENF density at the thigh or forearm, which was more severe than at the distal leg, compatible with a non-length-dependent process. Four reported improvement with a gluten-free diet. One had no improvement after 4 months. Symptoms developed in 2 while receiving a gluten-free diet. CONCLUSIONS: Patients with CD may have a neuropathy involving small fibers, demonstrated by results of skin biopsy. The pattern of symptoms, with frequent facial involvement and a non-length-dependent pattern on skin biopsy findings, suggests a sensory ganglionopathy or an immune-mediated neuropathy. Improvement of symptoms in some patients after initiating a gluten-free diet warrants further study.     

Re-organization of P2X3 receptor localization on epidermal nerve fibers in a murine model of cancer pain

To determine whether ATP and P2X3 receptors contribute to bone-cancer pain in a mouse model, immunohistochemical techniques were used to identify whether changes in the labeling of P2X3 receptors on epidermal nerve fibers (ENFs) occurred during tumor development. C3H mice were injected with osteolytic fibrosarcoma cells in and around the calcaneus bone. These mice exhibited mechanical hyperalgesia by day 10 post-implantation as assessed using von Frey monofilaments. Biopsies of the plantar skin overlying the tumor were obtained at days 10, 14, and 18 post-implantation. Confocal images were analyzed for the number of PGP 9.5, P2X3, and CGRP immunoreactive (ir) ENFs. The overall ENF population (PGP-ir) decreased progressively over time, whereas the subsets of P2X3-ir fibers demonstrated a modest increase and CGRP-ir nerve fibers remained fairly constant. Importantly, the proportion of CGRP-ir fibers that labeled for P2X3 increased from approximately 6% in control animals to nearly 30% at day 14 following tumor cell implantation. These studies demonstrate increased expression of P2X3 receptors on CGRP-ir ENFs during tumor growth and suggest a role for ATP in cancer-related pain.     

Small fibers involvement in Friedreich's ataxia.

Although the involvement of large myelinated sensory fibers in Friedreich's ataxia (FA) is well documented, an impairment of unmyelinated fibers has not been described. We demonstrate an involvement of cutaneous unmyelinated sensory and autonomic nerve fibers in FA patients. We performed a morphological and functional study of cutaneous nerve fibers in 14 FA patients and in a population of control subjects. We used immunohistochemical techniques and confocal microscopy applied to punch skin biopsies from thigh, distal leg, and fingertip, and compared the density of epidermal nerve fibers (ENFs) with the results of mechanical pain sensation and thermal and tactile thresholds performed on hand dorsum, thigh, distal leg, and foot dorsum. We observed in our patients a statistically significant loss of ENFs, a reduced innervation of sweat glands, arrector pilorum muscles and arterioles, and an impairment of thermal and tactile thresholds and mechanical pain detection.     

Evaluation of dermal myelinated nerve fibers in diabetes mellitus

Skin biopsies have primarily been used to study the non‐myelinated nerve fibers of the epidermis in a variety of neuropathies. In this study, we have expanded the skin biopsy technique to glabrous, non‐hairy skin to evaluate myelinated nerve fibers in the most highly prevalent peripheral nerve disease, diabetic polyneuropathy (DPN). Twenty patients with DPN (Type I, n = 9; Type II, n = 11) and 16 age‐matched healthy controls (age 29–73) underwent skin biopsy of the index finger, nerve conduction studies (NCS), and composite neuropathy scoring. In patients with DPN, we found a statistically significant reduction of both mechanoreceptive Meissner corpuscles (MCs) and their afferent myelinated nerve fibers (p = 0.01). This myelinated nerve fiber loss was correlated with the decreased amplitudes of sensory/motor responses in NCS. This study supports the utilization of skin biopsy to quantitatively evaluate axonal loss of myelinated nerve fibers in patients with DPN.     

类淀粉变性多神经病

目的　探讨类淀粉性周围神经病的临床特点及病理改变。方法　报告 3例经腓肠神经和皮肤活检证实的病例 ,结合文献分析其临床特点、病理改变及治疗。结果　本病为慢性进行性周围神经病 ,可伴有心、胃肠、肾等损伤。病理改变示刚果红染色阳性 ,可见类淀粉物沉积于神经内膜、束膜及血管壁外膜 ,有髓及无髓纤维减少 ,节段性脱髓鞘。结论　根据临床特点及周围神经皮肤活检进行诊断 ,肝移植是治疗本病最有前途的方法     

Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2-3.4 Hz) in 34% of cutaneous C-fibers adjacent to the tumor (9-17 d after implantation). C-fibers in tumor-bearing mice exhibited a mean decrease in heat threshold of 3.5 +/− 0.10 °C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16-24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor-bearing mice. The development of spontaneous activity and sensitization to heat in C-fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C-fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.     

Symptom duration and clinical features in painful sensory neuropathy with and without nerve conduction abnormalities

BACKGROUND: The term "small fiber sensory neuropathy" (SFSN) refers to an axonal sensory polyneuropathy predominantly affecting cutaneous sensory modalities, often associated with pain and with no evidence of large fiber involvement. We hypothesized that, in most patients, SFSN is the earliest manifestation of a nonspecific axonal neuropathy and will usually progress to involve larger, heavily myelinated sensory and motor fibers. We sought indirect evidence of this through an analysis of the correlation between symptom duration and large fiber involvement in patients with painful sensory neuropathy (PSN). METHODS: A clinical diagnosis of PSN was supported by nerve conduction studies or measurement of epidermal nerve fiber (ENF) density in 43 patients. Symptom duration was correlated with the frequency of large fiber loss as measured by nerve conduction abnormalities. The severity and extent of clinical signs and symptoms were also evaluated in subjects with and without electrodiagnostic abnormalities. RESULTS: Patients with large sensory axon involvement had symptoms of longer duration than patients with SFSN. The frequency of electrodiagnostic abnormalities increased in direct proportion to disease duration. Patients with electrodiagnostic abnormalities also had more extensive pinprick sensory deficits, suggesting that small fiber loss was more advanced in this group as well. CONCLUSIONS: In PSN, the incidence of large fiber involvement appears to increase in proportion to symptom duration. This represents indirect evidence that SFSN usually progresses to involve both large and small fibers within 2-10 years.     

Aspects of peripheral nerve involvement in patients with treated hypothyroidism

Background:  We studied involvement of large and small nerve fibres in patients with hypothyroidism and symptoms and signs of polyneuropathy.
Methods:  Sixteen patients with established diagnosis of hypothyroidism were extracted from a patient population participating in a 'polyneuropathy study'. In addition, seven patients with other additional potential causes of polyneuropathy than hypothyroidism were investigated. The patients underwent neurological examination, routine blood tests, nerve conduction studies (NCS), quantitative sensory testing (QST) and skin biopsies with assessment of intraepidermal nerve fibre (IENF) density.
Results:  Sixty-three per cent of the patients with 'pure' hypothyroidism had abnormalities on NCS, 25% had reduced IENF density and 31% had abnormalities on QST. Four patients (25%) met criteria for small fibre polyneuropathy, the other (75%) were classified as having mixed fibre polyneuropathy. There were no differences in the amount of abnormalities on NCS, QST and skin biopsy between patients with hypothyroidism and those with hypothyroidism and other potential causes of polyneuropathy.
Conclusions:  The majority of patients with hypothyroidism had involvement of both large and small nerve fibres. However, some patients had isolated small fibre polyneuropathy. Patients with 'pure' hypothyroidism had essentially the same degree of peripheral nerve fibre involvement as those with other additional causes of polyneuropathy.     

Is Ross syndrome a dysautonomic disorder only? An electrophysiologic and histologic study.

OBJECTIVE: To define the involvement of peripheral nerve fibers in Ross syndrome. METHODS: Mechanical pain perception, tactile and thermal thresholds on hand, foot dorsum, thigh, median nerve orthodromic sensory conduction velocity (SCV) and motor conduction velocity (MCV), sural nerve antidromic SCV, peroneal nerve MCV, H-reflex, F-wave, median, tibial nerve somatosensory evoked potentials (SSEPs), perioral, hand CO(2) laser late (LEPs) and ultralate evoked potentials, sympathetic skin response (SSRs), cardiovascular, Minor sweat, silastic imprint, histamine, photopletysmographic and pupil pilocarpine tests, cutaneous innervation immunohistochemical techniques were studied in 3 patients with Ross syndrome. RESULTS: Quantitative sensory testing showed altered results in patients 1 and 2, and patient 3 had a slight impairment of mechanical pain perception. Nerve conduction, except for a median nerve distal reduction of sensory conduction in patient 1, F-wave and SSEP findings were normal; H-reflex was absent at rest in all patients. Hand LEPs were absent in patient 2, ultralate potentials were absent in patients 1 and 2. Skin biopsy showed a disease duration related reduction of unmyelinated and myelinated sensory fibers and a lack of unmyelinated autonomic fibers in all patients. CONCLUSIONS: Our data suggest that Ross syndrome is a degenerative disorder involving progressive sudomotor fibers, and then epidermal sensory unmyelinated and myelinated fibers.     

Sensory action potentials and biopsy of the sural nerve in the neuropathy of nonmalignant IgMk plasma cell dyscrasia

Electrophysiological findings in 10 patients with polyneuropathy and nonmalignant IgMk plasma cell dyscrasia are reported. The amplitude of sensory action potentials and maximum conduction velocity along the sural and median nerves are related to the structural changes found at the biopsy and to the immunologic results. Quantitative analysis of the sural nerve showed a loss of large myelinated fibers in all 8 patients, in whom the nerve could be examined. The sural action potential as well as conduction velocity were undetectable in 4 cases, diminished in 5 and normal in 1. The primary mechanism underlying nerve damage in these neuropathies is discussed. It is suggested that the IgM paraprotein may act heterogeneously on the peripheral nerve's component.     

A pediatric case of critical illness polyneuropathy: clinical and pathological findings

Critical illness polyneuropathy (CIP) is a sensorimotor polyneuropathy recognized in adult intensive care patients with sepsis and multiple organ dysfunction and only a few cases have been reported in children. Here we report a 13-year-old Japanese boy with CIP that developed during the course of encephalopathy. Two months after the onset of encephalopathy, he developed tetraplegia although consciousness had already recovered. Deep tendon reflex was absent. MRI of the brain and spinal cord was normal and no abnormality in the cerebrospinal fluid was detected. Motor and sensory nerve conduction velocities of the lower limbs and somatosensory evoked potential could not be detected. The motor activity subsequently showed gradual recovery, although standing and walking could not be achieved. Sural nerve biopsy performed 3 years after the onset showed severe reduction of the number of myelinated large-diameter fibers, thin myelin in almost all fibers and cluster formation of myelinated small-diameter fibers, indicating primary axonal degeneration with regeneration. We report here for the first time the neuropathological changes in peripheral nerves during the chronic stage of CIP in children.     

Painful small-fiber neuropathy in Sjogren syndrome

Of 20 consecutive patients with Sj?gren neuropathy, 16 (80%) presented with burning feet and 12 (60%) with non-length-dependent sensory symptoms. Leg and thigh skin biopsies, performed in 13 patients, including 7 with normal electrophysiology, showed either reduced epidermal nerve fiber (ENF) density or abnormal morphology. ENF loss was frequently non length dependent, suggesting that patients with this disorder commonly have a small-fiber sensory neuronopathy rather than a "dying-back" axonopathy.     

The value of skin biopsy with recording of intraepidermal nerve fiber density and quantitative sensory testing in the assessment of small fiber involvement in patients with different causes of polyneuropathy

The primary aim of our study was to demonstrate how the diagnostic characteristics of skin biopsy used to evaluate small fiber involvement in patients with different causes of polyneuropathy are intrinsically related to the method used to establish the reference values (cut-off values). We also investigated intraepidermal nerve fiber (IENF) density and abnormalities in quantitative sensory testing (QST) in patients with different causes of polyneuropathy and signs of small fiber involvement. A total of 210 patients with symptoms and signs of polyneuropathy were entered into the study. All patients underwent neurological examination, nerve conduction studies, QST on the thigh and distal part of the calf with detection of warm and cold perception thresholds, and skin biopsy with assessment of IENF density. Cut-off values for IENF density were established from our reference material using Z-scores (calculated from multiple regression analysis), fifth percentile, and receiver operating characteristic (ROC) analysis. Of the patients participating in the study, 65 had an established diagnosis of diabetes mellitus, 70 were classified with idiopathic polyneuropathy, and 75 had other possible causes of polyneuropathy. Forty-five patients met the criteria for small fiber polyneuropathy (SFN), and the remaining 165 had also involvement of large nerve fibers. Of the total patient cohort, 84 (40%) had reduced IENF density based on the Z-score, and 106 patients (50%) had at least one abnormality based on QST. In the SFN group, skin biopsy showed a sensitivity of 31% and a specificity of 98% when reference values were presented with Z-scores. When the fifth percentile was used as the cut-off value (6.7 fibers/mm), sensitivity was 35% and specificity 95%. Applying the ROC analysis with a chosen sensitivity of 78% and specificity of 64%, we had a cut-off point of 10.3 fibers/mm. We conclude that skin biopsy with assessment of IENF is a useful method for investigating patients with SFN. The diagnostic value of the test, however, depends upon on the approach used to estimate the reference values. An erratum to this article can be found at     

Concordance between epidermal nerve fiber density and sensory examination in patients with symptoms of idiopathic small fiber neuropathy

Quantitation of epidermal nerve fiber (ENF) density is an objective diagnostic test of small fiber neuropathy (SFN). For a diagnostic test to be clinically useful it should correspond well with clinically meaningful physical findings. We performed a retrospective analysis of the concordance between foot ENF density and clinical findings in all patients seen at our institution with possible idiopathic SFN who underwent skin biopsy for ENF density determination. We found a high concordance between reduced foot ENF density and loss of pinprick sensitivity in this patient population. Our findings indicate that ENF density determination is a clinically relevant objective test in patients undergoing evaluation for possible SFN.     

Collateral sprouting of human epidermal nerve fibers following intracutaneous axotomy

Despite the clinical need, there are no therapeutic compounds available to promote peripheral nerve regeneration. In part, this may be due to a lack of sensitive measures of nerve growth. Here, we describe a novel approach of measuring collateral sprouting of epidermal nerve fibers (ENF) in human subjects and describe the effect of the neuroimmunophilin ligand timcodar dimesylate on collateral nerve sprouting. The objective of this study was to describe a model of intracutaneous axotomy and evaluate the ability of timcodar dimesylate to accelerate human cutaneous nerve regeneration through collateral sprouting. Subjects were randomized to receive placebo, 12.5 or 50 mg/day timcodar dimesylate in a prospective, two-center, double-blind, placebo-controlled trial. A 3-mm distal thigh punch skin biopsy was performed at baseline, and a 4-mm overlapping concentric biopsy was taken after 56 days of treatment. Biopsies were processed to visualize ENF, and the collateral sprouting distance (CSD) was measured. Sixty-two subjects completed the trial, and the CSD was measurable in 52. The CSD (mean +/- SEM) was 474.5 microm +/- 38.3, 473.4 microm +/- 28.4, and 450.8 microm +/- 26.5 for the placebo, low and high dose groups, respectively (p = 0.84). The baseline ENF density was associated with the CSD (p = 0.02). Collateral sprouting was efficiently measured using an intracutaneous axotomy model and suggests a collateral sprouting rate of 8.5 microm/day in healthy subjects. The model was consistent across treatment groups and had a low coefficient of variation. Timcodar dimesylate treatment was safe over an 8-week period but did not improve collateral sprouting among healthy subjects.     

Diagnosis and management of diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most commonly occurring major complications of diabetes. The disease may manifest in several clinical patterns: most frequently as distal symmetrical sensory polyneuropathy. Guidelines are available for the diagnosis of DPN by the primary care physician. These recommend that a review of diabetic patients, including a questionnaire and inspection and neurological examination of the feet, is undertaken annually. Techniques used for studying the disease process in clinical trials may include nerve conduction and quantitative sensory function tests, autonomic nervous system testing, post-ganglionic sudomotor function and skin biopsy. Current therapies for managing DPN are strict glycaemic control, palliative treatments and foot ulcer prevention. Future treatments aim to beneficially affect the underlying disease pathology and putative agents are currently being investigated.     

Epidermal nerve fiber density, axonal swellings and QST as predictors of HIV distal sensory neuropathy

We investigated the associations of baseline epidermal nerve fiber (ENF) densities and morphology (percent ENF swellings) and quantitative sensory testing (QST) with clinically defined human immunodeficiency virus (HIV)-associated distal polyneuropathy (DSP) and whether these measures are predictive of development of symptomatic DSP over time. Fifty-seven HIV-infected subjects with and without DSP and 19 controls participated. Mean ENF densities were lower at the distal leg and proximal thigh in asymptomatic or symptomatic DSP than in controls. Mean ENF densities did not differ significantly among the HIV groups. Percent ENF swellings was higher in patients with symptomatic DSP than controls at the distal leg, and was also greater at the proximal thigh in patients with asymptomatic or symptomatic DSP than in controls. The percent ENF swellings at the distal leg correlated with the thresholds for both minimal (HP 0.5) and intermediate (HP 5.0) heat pain (HP) intensity. A higher percent ENF swellings in the distal leg [hazard ratio (HR) 1.16, 95% CI 1.02-1.31] and HP 0.5 thresholds (HR 1.03, 95% CI 1.01-1.05) were the only two measures associated with a shorter time to development of symptomatic DSP. Quantitation of ENF swellings and heat pain thresholds deserve further study as predictors of symptomatic neuropathy.