Analysis of small sensory fibers in the epidermis (intraepidermal nerve fibers, IENF) was performed as described in detail previously (37). Briefly, hind-paw skin was fixed in 4% buffered paraformaldehyde overnight at 4°C then stored in 0.1M sodium phosphate buffer at 4°C before embedding in paraffin blocks. 6 μm sections were cut using a rotary microtome (Leitz, model 1512) and mounted onto glass slides. Mounted tissues were immunostained with antibody against protein gene product (PGP) 9.5 (1:1,000; cat. #7863-0504, AbD Serotec). Quantification of IENF was done using bright field light microscopy at 40 × magnification. All nerve fragments in the epidermis were counted for detection of early IENF terminal loss prior to retraction as far as the dermis (39). The length of the paw skin was traced under a light microscope using Scion Image software and a tracing pad. IENF density was reported as IENF profiles/mm.

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