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.

To visualize sensory innervation of the cornea, each mouse was anesthetized with 4% isoflurane in oxygen before transfer to the platform of a corneal confocal microscope (Heidelberg Retina Tomograph 3 with Rostock Cornea Module). Eye gel (GenTealTM, Novartis) was applied to both eyes to prevent drying and to connect the eyes to the microscope lens. The microscope objective was positioned close to the center of the apex of cornea and the depth adjusted to the beginning of the sub-basal nerve plexus. Using the Rostock Imaging Software, 40 sequential images, with 2 μm spacing, were collected encompassing the corneal epithelium, sub-basal nerve plexus and stroma. After identifying the sub-basal nerve plexus:stromal junction, five consecutive images of the sub-basal nerve layer moving outwards toward the epidermis and the first 10 images of the stromal layer were quantified. Corneal nerves in each image were traced using a digitizing tablet connected to a computer running Image J software (Image Processing Analysis in Java, National Institutes of Health). Corneal nerve density in each image was reported as number of pixels per area.

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