Sung-Tsang Hsieh

Cutaneous innervation in sensory neuropathies Evaluation by skin biopsy

Objective: To use punch skin biopsies to evaluate the loss of intra-epidermal nerve fibers in sensory neuropathies. Background: Previous assessments of epidermal nerve fibers have been constrained by relatively insensitive staining techniques and variability in quantification. Methods: Punch skin biopsies were performed on the heel and leg of HIV-seronegative controls, HIV-seropositive individuals without neuropathy, and patients with sensory neuropathies, including HIV-seronegative and HIV-positive individuals. After formalin fixation, 50-micro meter free-floating sections were stained with a monoclonal antibody to neuron-specific ubiquitin hydrolase, PGP9.5. The number of intraepidermal fibers/mm in at least three sections from each patient was counted by one observer blinded to site and clinical status. Results: Dermal and epidermal nerve fibers were readily identified and quantified. The immunostaining technique reliably demonstrated a dermal plexus of myelinated and unmyelinated fibers parallel to the surface of the skin. In the epidermis, unmyelinated fibers ascended vertically between the keratinocytes to reach the stratum corneum. The number of intra-epidermal fibers/mm in the distal leg (mean plus minus SEM) was 17.84 plus minus 3.03 in seven HIV-seronegative controls. Epidermal fiber number was significantly reduced (p equals 0.01) in five HIV-infected patients with sensory neuropathies associated with didanosine or zalcitabine therapy (1.07 plus minus 0.40) and in eight HIV-seronegative patients with sensory neuropathies (3.1 plus minus 3.1). Four of five neurologically normal HIV-seropositive subjects had reduced numbers of epidermal fibers, suggesting a subclinical neuropathy. Serial biopsies in one individual demonstrated the evolution of degenerating epidermal fibers after development of zalcitabine-induced sensory neuropathy. Conclusion: Skin biopsies stained with the sensitive panaxonal marker anti-PGP9.5 demonstrated significant reduction in intraepidermal fibers in sensory neuropathies. This simple and repeatable technique is a reliable method for quantitation of small cutaneous sensory fibers. In addition, skin biopsies may be useful in assessing the course and spatial distribution of involvement in peripheral nerve disease. NEUROLOGY 1995;45: 1848-1855

Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barré syndrome

SummaryThe axonal patterns of Guillain-Barré syndrome, associated in many cases with antecedentCampylobacter jejuni infection, are now recognized as frequent causes of acute flaccid paralysis in some regions of the world. This study examined ultrastructurally the PNS of seven cases of the acute motor axonal neuropathy form of Guillain-Barré syndrome. In this disorder previous studies of advanced cases have found Wallerian-like degeneration of motor fibres in the spinal roots and peripheral nerves, with little lymphocytic inflammation or demyelination. The present study was focused on identifying early changes and establishing the sequence of changes. By electron microscopy the earliest and mildest changes consisted of lengthening of the node of Ranvier with distortion of the paranodal myelin, and in some instances with breakdown of the outermost myelin terminal loops. At this stage many nodes had overlying macrophages which extended their processes through the Schwann cell basal lamina covering the node and apposed the axolemma. Macrophage processes then extended beneath the myelin terminal loops, and the whole macrophage entered the periaxonal space at the paranode. Macrophage processes dissected the axon from the adaxonal Schwann cell plasmalemma and the macrophages advanced into the internodal periaxonal space, where they typically surrounded a condensed-appearing axon. At this stage the adaxonal Schwann cell cytoplasm regularly degenerated and disappeared, so that the periaxonal space was bounded by the innermost myelin lamella, and the axolemma of many fibres could not be seen. The internodal myelin sheath and the abaxonal Schwann cell cytoplasm remained normal. This arrangement appeared to be stable for some time, but in many fibres the axon subsequently underwent Wallerian-like degeneration. By interfering with impulse conduction, these nodal and periaxonal changes may explain paralysis in some pathologically mild cases. In addition, at early stages, these changes may be reversible, thus explaining the rapid recovery of some patients who become paralysed with acute motor axonal neuropathy. These observations, taken together with previous studies, suggest that acute motor axonal neuropathy is an antibody- and complement-mediated disorder in which the relevant epitopes are present on the nodal and internodal axolemma.

Motor nerve terminal degeneration provides a potential mechanism for rapid recovery in acute motor axonal neuropathy after Campylobacter infection

Article abstract-We investigated the possible mechanisms of paralysis and recovery in a patient with the acute motor axonal neuropathy (AMAN) pattern of the Guillain-Barre syndrome. The AMAN pattern of GBS is characterized clinically by acute paralysis without sensory involvement and electrodiagnostically by low compound motor action potential amplitudes, suggesting axonal damage, without evidence of demyelination. Many AMAN patients have serologic or culture evidence of recent Campylobacter jejuni infection. Pathologically, the most severe cases are characterized by wallerian-like degeneration of motor axons affecting the ventral roots as well as peripheral nerves, but some fatal cases have only minor changes in the roots and peripheral nerves, and some paralyzed patients with the characteristic electrodiagnostic findings of AMAN recover rapidly. The mechanism of paralysis and recovery in such cases has been uncertain. A 64-year-old woman with culture-proven Campylobacter upsaliensis diarrhea developed typical features of AMAN. She improved quickly following plasmapheresis. Her serum contained IgG anti-GM1 antibodies. The lipopolysaccharide of the organism bound peanut agglutinin. This binding was blocked by cholera toxin, suggesting that the organism contained the Gal(beta 1-3)GalNAc epitope of GM1 in its lipopolysaccharide. Motor-point biopsy showed denervated neuromuscular junctions and reduced fiber numbers in intramuscular nerves. In contrast, the sural nerve biopsy was normal and skin biopsy showed normal dermal and epidermal innervation. In AMAN the paralysis may reflect degeneration of motor nerve terminals and intramuscular axons. In addition, the anti-GM1 antibodies, which can bind at nodes of Ranvier, might produce failure of conduction. These processes are potentially reversible and likely to underlie the capacity for rapid recovery that characterizes some cases of AMAN. NEUROLOGY 1997;48: 717-724

Influence of aging on thermal and vibratory thresholds of quantitative sensory testing

Abstract  Quantitative sensory testing has become a common approach to evaluate thermal and vibratory thresholds in various types of neuropathies. To understand the effect of aging on sensory perception, we measured warm, cold, and vibratory thresholds by performing quantitative sensory testing on a population of 484 normal subjects (175 males and 309 females), aged 48.61 ± 14.10 (range 20–86) years. Sensory thresholds of the hand and foot were measured with two algorithms: the method of limits (Limits) and the method of level (Level). Thresholds measured by Limits are reaction‐time‐dependent, while those measured by Level are independent of reaction time. In addition, we explored (1) the correlations of thresholds between these two algorithms, (2) the effect of age on differences in thresholds between algorithms, and (3) differences in sensory thresholds between the two test sites. Age was consistently and significantly correlated with sensory thresholds of all tested modalities measured by both algorithms on multivariate regression analysis compared with other factors, including gender, body height, body weight, and body mass index. When thresholds were plotted against age, slopes differed between sensory thresholds of the hand and those of the foot: for the foot, slopes were steeper compared with those for the hand for each sensory modality. Sensory thresholds of both test sites measured by Level were highly correlated with those measured by Limits, and thresholds measured by Limits were higher than those measured by Level. Differences in sensory thresholds between the two algorithms were also correlated with age: thresholds of the foot were higher than those of the hand for each sensory modality. This difference in thresholds (measured with both Level and Limits) between the hand and foot was also correlated with age. These findings suggest that age is the most significant factor in determining sensory thresholds compared with the other factors of gender and anthropometric parameters, and this provides a foundation for investigating the neurobiologic significance of aging on the processing of sensory stimuli.

Role of acid-sensing ion channel 3 in sub-acute-phase inflammation.

BackgroundInflammation-mediated hyperalgesia involves tissue acidosis and sensitization of nociceptors. Many studies have reported increased expression of acid-sensing ion channel 3 (ASIC3) in inflammation and enhanced ASIC3 channel activity with pro-inflammatory mediators. However, the role of ASIC3 in inflammation remains inconclusive because of conflicting results generated from studies of ASIC3 knockout (ASIC3-/-) or dominant-negative mutant mice, which have shown normal, decreased or increased hyperalgesia during inflammation.ResultsHere, we tested whether ASIC3 plays an important role in inflammation of subcutaneous tissue of paw and muscle in ASIC3-/- mice induced by complete Freunds adjuvant (CFA) or carrageenan by investigating behavioral and pathological responses, as well as the expression profile of ion channels. Compared with the ASIC3+/+ controls, ASIC3-/- mice showed normal thermal and mechanical hyperalgesia with acute (4-h) intraplantar CFA- or carrageenan-induced inflammation, but the hyperalgesic effects in the sub-acute phase (1–2 days) were milder in all paradigms except for thermal hyperalgesia with CFA-induced inflammation. Interestingly, carrageenan-induced primary hyperalgesia was accompanied by an ASIC3-dependent Nav1.9 up-regulation and increase of tetrodotoxin (TTX)-resistant sodium currents. CFA-inflamed muscle did not evoke hyperalgesia in ASIC3-/- or ASIC3+/+ mice, whereas carrageenan-induced inflammation in muscle abolished mechanical hyperalgesia in ASIC3-/- mice, as previously described. However, ASIC3-/- mice showed attenuated pathological features such as less CFA-induced granulomas and milder carrageenan-evoked vasculitis as compared with ASIC3+/+ mice.ConclusionWe provide a novel finding that ASIC3 participates in the maintenance of sub-acute-phase primary hyperalgesia in subcutaneous inflammation and mediates the process of granuloma formation and vasculitis in intramuscular inflammation.

Epidermal denervation and its effects on keratinocytes and Langerhans cells

SummarySkin innervation has been considered to subserve sensory perception only, but several lines of evidence suggest that there are ‘effector’ influences of skin innervation on the immune system and keratotinocytes. In this study, we transected the sciatic nerves of rats and examined the effects of denervation on the epidermis. In normal skin, the epidermis was densely innervated by fine axons that were immunostained with several axonal markers, including neuronal ubiquitin carboxyl terminal hydrolase (protein gene product 9.5). All of the epidermal axons in the regions innervated by sciatic nerve disappeared within 24–48 h after transection of sciatic nerve, and remained absent as long as subsequent reinnervation by regenerating axonal sprouts was prevented. Denervation produced changes in both the keratinocytes and the Langerhans cells, the bone marrowderived antigen-presenting cells of the epidermis. The thickness of epidermis decreased within 7 days. By 48h after transection, the Langerhans cells and their dendritic processes became intensely immunoreactive for protein gene product. Protein gene product 9.5 expression on Langerhans cells remained prominent as long as skin was denervated, but disappeared with reinnervation. By reverse transcription-polymerase chain reaction, we demonstrated the presence of the transcripts for protein gene product 9.5 in epidermis, consistent with the synthesis of the protein by the Langerhans cells. We conclude that epidermal sensory fibres have novel influences on both keratinocytes and Langerhans cells of the epidermis.

Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: Cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single‐cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three‐dimensional (3D) and two‐dimensional (2D) T2‐weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions. Magn Reson Med 58:717–724, 2007.

Cutaneous innervation in chronic inflammatory demyelinating polyneuropathy

Abstract—The authors evaluated epidermal nerve density (END) and thermal thresholds in 18 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). END of patients with CIDP were lower than those of controls (4.5 ± 2.9 vs 10.5 ± 3.9 fibers/mm, p < 0.001). Reduced END were associated with autonomic symptoms. Thermal thresholds of patients with CIDP were elevated (88.2% for warm stimuli and 70.6% for cold stimuli). Patients with CIDP have small-fiber sensory and autonomic neuropathies.

European Federation of Neurological Societies/Peripheral Nerve Society guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society

Revision of the guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy, published in 2005, has become appropriate due to publication of more relevant papers. Most of the new studies focused on small fiber neuropathy (SFN), a subtype of neuropathy for which the diagnosis was first developed through skin biopsy examination. This revision focuses on the use of this technique to diagnose SFN. Task force members searched the Medline database from 2005, the year of the publication of the first EFNS guideline, to June 30th, 2009. All pertinent papers were rated according to the EFNS and PNS guidance. After a consensus meeting, the task force members created a manuscript that was subsequently revised by two experts (JML and JVS) in the field of peripheral neuropathy and clinical neurophysiology, who were not previously involved in the use of skin biopsy. Distal leg skin biopsy with quantification of the linear density of intraepidermal nerve fibers (IENF), using generally agreed upon counting rules, is a reliable and efficient technique to assess the diagnosis of SFN (level A recommendation). Normative reference values are available for bright‐field immunohistochemistry (level A recommendation) but not yet for confocal immunofluorescence or the blister technique. The morphometric analysis of IENF density, either performed with bright‐field or immunofluorescence microscopy, should always refer to normative values matched for age (level A recommendation). Newly established laboratories should undergo adequate training in a well established skin biopsy laboratory and provide their own stratified age and gender‐matched normative values, intra‐ and interobserver reliability, and interlaboratory agreement. Quality control of the procedure at all levels is mandatory (Good Practice Point). Procedures to quantify subepidermal nerve fibers and autonomic innervated structures, including erector pili muscles, and skin vessels are under development but need to be confirmed by further studies. Sweat gland innervation can be examined using an unbiased stereologic technique recently proposed (level B recommendation). A reduced IENF density is associated with the risk of developing neuropathic pain (level B recommendation), but it does not correlate with its intensity. Serial skin biopsies might be useful for detecting early changes of IENF density, which predict the progression of neuropathy, and to assess degeneration and regeneration of IENF (level C recommendation). However, further studies are warranted to confirm the potential usefulness of skin biopsy with measurement of IENF density as an outcome measure in clinical practice and research. Skin biopsy has not so far been useful for identifying the etiology of SFN. Finally, we emphasize that 3‐mm skin biopsy at the ankle is a safe procedure based on the experience of 10 laboratories reporting absence of serious side effects in approximately 35,000 biopsies and a mere 0.19% incidence of non‐serious side effects in about 15 years of practice (Good Practice Point).

Patterns of contact heat evoked potentials (CHEP) in neuropathy with skin denervation: Correlation of CHEP amplitude with intraepidermal nerve fiber density

OBJECTIVE Contact heat evoked potentials (CHEPs) provide an objective approach to investigate cerebral responses to thermal stimuli mediated by Adelta fibers. Skin denervation is often associated with reduced thermal sensibilities. We aimed to investigate the influences of skin denervation on CHEPs in neuropathic patients. METHODS CHEPs were recorded at the vertex area by applying contact heat stimuli of 51 degrees C on the distal leg of neuropathic patients with sensory symptoms and pathological evidence of skin denervation in the distal leg. Patterns and parameters of CHEPs in the neuropathic group were compared with those in the control group of age- and gender-matched subjects. RESULTS There were 25 neuropathic patients with reduced intraepidermal fiber (IENF) density (1.46+/-1.70fibers/mm, range: 0-5.32). In the control group, well-defined averaged tracings of CHEPs with an initial negative peak (N-wave) followed by a positive peak (P-wave) were consistently recorded in all 25 subjects. The peripheral conduction velocities of CHEPs were 9.92+/-4.06m/s (range: 6.06-16.60), in the range of Adelta fibers. The group of neuropathic patients had markedly reduced N-P amplitudes (p<0.0001) and prolonged N-wave latencies (p=0.049) compared to the control group. IENF density was the only neuropathic parameter correlated with N-P amplitude on multiple linear regression analysis (p=0.010) compared to large-fiber parameters. CONCLUSIONS In neuropathic patients with pathological evidence of skin denervation, there were reduced amplitude and prolonged latencies in CHEPs mediated by Adelta fibers. The reduction of CHEP amplitude corresponded to the degree of skin denervation. SIGNIFICANCE CHEP offers electrophysiological evidence of thermal responses and provides an objective, non-invasive approach to assess the physiological counterparts of skin denervation in neuropathic patients.