3.1. Intraepidermal Skin Biopsy (IESB)

Skin biopsy can be used for measurements of intraepidermal nerve fiber density (IENFD) of C-fibers (unmyelinated nerves) [21]. Using protein gene product (PGP) antibody labeling, which binds to unmyelinated nerve fibers, the density of these nociceptive fibers can be evaluated. Intraepidermal skin biopsy (IESB) is widely available and provides an objective assay of changes in distal nerve fiber integrity:

Data Collection: IESB is a relatively simple in approach, can be performed in clinical outpatient offices, and has commercial kits available for the procedure. Punch biopsies of the skin are taken from the region of interest, and the linear density of IENFD is quantified. The procedure, as defined previously, includes examining “at least three 50-µm-thick sections per biopsy, fixed in 2% paraformaldehyde lysine periodate or Zamboni’s solution, by bright-field immunohistochemistry or immunofluorescence, with anti-PGP 9.5 antibodies” [22]. The antibody PGP9.5 (protein gene product 9.5) is a neuron-specific protein that allows labeling of neurons and nerve fibers at all levels of the nervous system. Currently, a standard battery of antibodies to differentiate nerve fibers, such as C vs. A-delta vs. A-beta, has not yet been established. Such a test would be helpful for differential diagnoses of pathologies that differentially affect these nerve fibers, such as SFN.

Data Analysis: Analysis involves quantification of fibers that intersect the dermal–epidermal basement membrane. A number of laboratories can now provide quantification of the skin biopsy. Details are provided elsewhere [21].

Sensitivity and Specificity: IESB is considered to be reliable and reproducible [23]. Although the state or stage of the disease is difficult to define, multiple skin biopsies may be taken, allowing for longitudinal measurement of relative changes in a particular patient, as well as evaluation of clinical correlations, including disease etiology. Reduced IENFD is both sensitive and specific for well-defined clinical syndromes, but clinical phenotyping can be nonspecific. For example, in a retrospective report, IENFD abnormalities were detected in 88% of patients with symptoms suggestive of SFN, which was significantly better compared with a clinical exam or quantitative sensory testing (QST) [24]. In addition, IENFD was abnormal in approximately 80% of clinically defined mixed large- and small-fiber neuropathies, with no changes observed in large-fiber neuropathy.

Disease Evaluation: Skin biopsy for small-fiber abnormalities has been shown across well-described conditions that have SFN (e.g., HIV, diabetes, chemotherapy-induced neuropathy), mixed peripheral neuropathies, and also in conditions having less defined pathophysiologies (e.g., Parkinson’s or fibromyalgia) and autonomic neuropathies. The European Federation of Neurological Societies has provided guidelines for the use of skin biopsy in the diagnosis of peripheral neuropathies [22]. Diagnostic specificity and sensitivity using IESB is high. Normative data has been provided in healthy controls and neuropathic patients [25]. These authors reported the number of intraepidermal fibers in normal controls to be 21.1 ± 10.4 per mm (mean ± SD) in the thigh (fifth percentile, 5.2 per mm) and 13.8 ± 6.7 per mm at the distal part of the leg (fifth percentile, 3.8 per mm). Using a cutoff taken from the fifth percentile of healthy data, they concluded that the approach had “a positive predictive value of 75%, a negative predictive value of 90%, and a diagnostic efficiency of 88%”.

Data interpretation: IESB has been useful in evaluating small-fiber neuropathies across multiple small-fiber neuropathies and mixed-fiber neuropathies (see Table 1). IEDNF is reduced in patients with small-fiber neuropathy and mixed-fiber neuropathy, but not in large-fiber neuropathies. Because C-fibers function for thermal and pain transduction, thermal testing for noxious heat can be a useful or confirmatory adjunct [16].

Reports of cornea or skin-based nerve metrics on corneal confocal microscopy (CCM) or intraepidermal skin biopsy (IESB) measures in different conditions/disease states in humans.

Corneal nerve branch density (CNBD), corneal nerve fiber area (CNFA), corneal nerve fiber branches (CNFB), corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), dendritic cells (DC), intraepidermal nerve fiber density (IENFD).

Static evaluation: Single nerve biopsies may be useful in diagnosis, but evaluation across gender and race/ethnicity has not been fully researched. For example, skin biopsy values in a Chinese population for diagnosing SFN have shown low sensitivity [86].

Dynamic evaluation: Despite the capacity for repeated measurements, few longitudinal studies have evaluated the utility of IESB. Some studies have evaluated the effects of age on epidermal sensory innervation [87]. Their findings noted an age-associated decrease in the facial biopsies, but not in biopsies derived from the abdomen. Evaluation of SFN in three clinical groups (idiopathic, impaired glucose tolerance, and diabetes) showed similar decreases over three time points at three sites (proximal thigh, distal thigh, and distal leg), noting similar rates of decrease in the different clinical categories over time [7]. Thus, axon loss may not be length-dependent over time, and the process inducing these changes may be local rather than at the level of the cell bodies [88]. However, IESB measures of SFN have been associated with central nervous system changes in patients following the development of central post-stroke pain [6]. It was unclear whether there was a true correlation/causality of the disease or whether the age of the patients in central post-stroke pain played a role (all were >50 years old), although none of the patients had a diagnosis consistent with coexistent SFN and the extent of reduced fiber density observed in the calf (<3.4 fibers per mm) was well below normal, even when accounting for age. Of note, a study in Parkinson’s patients detected no changes in IEDNF, although decreases in corneal nerve-fiber density and length were detected using CCM [89].

A complex relationship exists between epidermal fibers and autonomic function. Changes in IENFD have been observed in conditions, such as amyotrophic lateral sclerosis (ALS)—not previously thought to have a pathophysiology affecting epidermal nerves [90]. In ALS, one mechanism of alteration in nerve fibers may relate to the deposition of transactive response DNA-binding protein (TDP)-43/pTDP-43 [91]