Skin Punch as a Potential Diagnostic Tool for Peripheral Neuropathies of Dogs: Set up of an Indirect Immunofluorescence Protocol on Formalin-Fixed Paraffin-Embedded (FFPE) Biopsy.

In veterinary medicine, the diagnosis of peripheral neuropathies is currently performed using semithin sections or nerve fiber teasing from nerve biopsy. However, these methods actually fail to identify more specific length-dependent and somatosensitive neuropathies. In humans, skin punch biopsy is used to diagnose the latter, through the identification and count of intraepidermal nerve fibers (IENFs) crossing the dermal-epidermal junction, with indirect immunofluorescence (IIF). However, the current need for frozen samples for this technique limits its routine application in clinical practice. In this study, we set up an IIF protocol to identify IENFs in dogs' skin punch biopsies. Six tests were performed on canine formalin-fixed paraffin-embedded (FFPE) 8 mm skin punches, using an antibody anti-PGP9.5, also known as ubiquitin carboxyl-terminal hydrolase-1. Three parameters were checked: (1) the effectiveness of the co-localization immunoreaction, (2) the thickness of sections, and (3) the magnification for image acquisition. The best IIF results in terms of the sharpness of fiber visualization and the possibility to count them were obtained with 10 µm sections, with a high-power field (×40), without co-localization for nuclei and epithelial structures. Reference data concerning the IENF density of different skin regions in healthy animals of different ages remain to be defined for future diagnostic applications.