Background

Small fiber neuropathy (SFN) is a disorder characterized by dysfunction of small sensory fibers, including thinly myelinated Aδ fibers and unmyelinated C fibers. Clinically, it manifests as a variable combination of sensory symptoms, such as pain, tingling, and burning sensations in the extremities, and may also involve autonomic dysfunction [1,2]. These symptoms typically present in a length-dependent, symmetric fashion, i.e., starting on the feet and progressing proximally to involve the knees and hands. Patients frequently report severe symptoms that can greatly affect their quality of life, highlighting the need for effective management of this condition.

SFN is regarded as a common disease, with an estimated prevalence of over 100 cases per 100,000 [3] and may result from multiple conditions. These include metabolic disorders (commonly diabetes mellitus and pre-diabetic hyperglycemia as well as vitamin deficiencies), immune-mediated diseases (such as Sjögren syndrome, celiac disease, and sarcoidosis), infections (such as hepatitis and HIV), exposure to toxins or drugs (including chemotherapy and some antibiotics), and hereditary diseases (such as amyloidosis, Fabry disease, and sodium channel subunit variants) [4–8].

Diagnosis of SFN is challenging, as the conventional electrodiagnostic examination of neuropathy with nerve conduction tests (NCT) and electromyography (EMG) assesses large fibers, and therefore remains normal in patients with SFN [9,10]. The current gold standard for diagnosing SFN in humans is a punch skin biopsy to identify skin denervation. Although sensory symptoms of polyneuropathy typically begin at the most distal sensory site, at the glabrous skin of the toes, and evaluation of this anatomical location may have the best sensitivity for identifying early axonal changes, digit skin biopsy is not performed in routine clinical practice due to poor healing and the absence of normative data. Commonly, a hairy skin punch biopsy from the lateral distal leg, 10 cm proximal to the lateral malleolus, is employed as a practical compromise, with available normative data. However, Meissner corpuscles, mechanoreceptors responsible for pressure and touch sensation, are found in glabrous but not in hairy skin [11]. This is performed using a circular 3-mm diameter blade, which removes a conical skin sample [5,10]. The skin is fixed and sectioned, nerve fibers are immunolabeled with antibodies against the pan-neuronal marker protein gene product 9.5 (PGP9.5), and the intraepidermal nerve fiber density (IENFD) is quantified by counting the nerve fragments that cross the dermal-epidermal junction, which is highlighted by the basal keratinocyte cell layer [12]. In humans, a diagnosis of SFN is confirmed when the measured IENFD is below the fifth percentile of age- and sex-matched healthy controls. This method was endorsed by the Peripheral Nerve Society and the American and European Academies of Neurology and is supported by consensus in the medical literature [12,13]. However, in preclinical research, a reliable, simple, and reproducible method for assessing IENFD in mice using anti-PGP9.5 monoclonal antibodies has not yet been established.

Most rodent studies have examined glabrous (hairless) skin from the hind paw, but often without specifying the exact anatomical location (e.g., metatarsal pads or flat skin regions), distinguishing between distal and proximal pads, or reporting the sample dimensions. Some studies employ the whole hind limb immersed in fixation, while more commonly, the skin is removed before fixation. Zamboni’s or paraformaldehyde fixative is variably used, and skin size collection may include either a dissected region of varying size or 2- to 3-mm skin punches [11]. Skin section thicknesses ranging from 5 to 50 μm are employed, using either free-floating or slide-mounted methods, with varying anti-PGP9.5 antibodies. These antibodies are labeled and visualized using chromogenic or fluorescent markers. Multiple groups used manual IENFD quantification counting rules, which are similar to those described for human patients [14]. However, others included free intraepidermal fragments that do not cross the dermal–epidermal junction as part of the total count or measured the length of fragments in the epidermis [15]. Some published methods used confocal microscopy to produce high-resolution images [16], which is time-consuming, expensive, and less practical for routine, large-scale assessment.

In our previous study [12], IENFD quantification was performed under suboptimal conditions, including sampling from the central paw rather than the distal footpads, suboptimal tissue preservation, thicker sections, and different antibody and permeabilization settings. These factors led to weak PGP9.5 staining, low image resolution, and inconsistent fiber identification, necessitating larger sample sizes to reach statistical significance. These limitations motivated the development of the improved protocol presented in this work. Other published studies used section thicknesses between 30 and 50 μm [17] and applied different markers such as PGP9.5, TRPV1, or thy1-YFP [14] to visualize either total or subtype-specific nerve fibers. Reported IENFD values in control animals also vary widely, ranging from approximately 32–39 fibers/mm in dietary studies [18] to around 15 fibers/mm in other reports [19]. In the present work, we analyzed male mice aged 9–12 weeks using 20 μm cryosections and PGP9.5 immunostaining as a pan-axonal marker, yielding control values of about 15 fibers/mm. This alignment with lower-range control values likely reflects the use of thinner sections and standardized quantification criteria. Together, these methodological differences across studies highlight the importance of consistent sectioning and staining parameters for reliable comparison of IENFD measurements in experimental models.

Furthermore, recent studies have introduced 3D cleared-tissue imaging methods for IENFD quantification, which typically yield lower fiber densities than traditional 2D section-based counting [20].

Here, we present a protocol that enables skin processing and IENFD quantification within 72 h. We employed fluorescence immunolabeling to offer rapid application and co-labeling with additional molecular markers. This method is described in a step-by-step, detailed manner, providing researchers with a practical and reproducible tool to study SFN in experimental rodent models.