Immunohistochemestry for nerve morphometry?
Authors: Giorgetto Carolina , Simoes Ana Leda Bertoncini , Zanon Renata Graciele , Fazan Valeria Paula Sassoli 
Published: June 2021

Peripheral nerve biopsy is an invaluable aid to diagnosis carefully selected patients. The nerves histological evaluation gives clues for investigating diseases mechanisms and causes, as well as guides the therapeutic planning of inflammatory, infectious, demyelinating or degenerative lesions [1]. Nerves histological processing method that provides the best image quality is the epoxy resin embedding. Thus, nerve sections can be observed by light and/or transmission electron microscopy (TEM) and classes of nerve fibers can be morphometrically identified and studied. Epoxy resin embedding and TEM is considered the gold standard method for unmyelinated fibers identification and quantification. More recently, the immunohistochemistry technique (IHC) is being added to the inflammatory neuropathy investigation and the most important of the panel of antibodies employed are those needed to differentiate lymphocytes into T (CD3þve) and B cells (CD20þve). In addition, antibodies against macrophages, myelin basic protein, neurofilaments, and epithelial membrane antigen (EMA) (for perineurial cells) are included [1]. Neurotransmitters and their fiber “carrier” are much less explored. We suggest that it would be possible to use the IHC to identify nerve fiber classes as an alternative to TEM and morphometry, with the gain on functional information. To investigate this hypothesis we used IHQ and immunofluorescence (IF) to immunolabel small myelinated and unmyelinated fibers in sural nerves of rats. The nerves were surgically removed, immersed in paraformaldehyde 4% for 18 hours, and cryoprotected in increasing concentrations of sucrose solutions, before transversal cryosectioning (12μm). For IHC, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:5.000, 18h) followed by anti‐rabbit IgG (1:500; 2h) and Extravidin‐HRP (1:1500, 2h). A reaction with diaminobenzidine intensified with nickel revealed immunoreactivity, observed by light microscopy. For IF, sections were incubated in rabbit polyclonal antibody anti‐substance P (1:1.000, 18h) followed by anti‐rabbit IgG (Alexa Fluor® 594) (1:200, 2h). The immunoreactivity was observed by fluorescence microscopy. Both protocols worked on transverse sections to identify substance P immunolabeled small myelinated and unmyelinated fibers. Results were compared with transverse sections prepared for TEM and the IHC results showed labeled small myelinated and unmyelinated axons intermingled with large unlabeled myelinated axons, with preservation of their myelin sheath. The IHC transverse sections were comparable to the epoxy resin embedded for small fiber localization in the endoneural space. Further studies on quantification of these fibers will be performed comparing both methods (IHC and IF) with the TEM images in order to show that IHC might also be a reliable method for quantifying axons.Support or Funding InformationFinancial support: FAPESP, CNPq, CAPES and FAEPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.