Effect of Age and Anatomical Site on Density of Sensory Innervation in Human Epidermis

Confocal microscopy micrograph showing innervation of human eyelid skin. Nerve fibers (NF) were immunolabeled with a protein gene product 9.5 antibody with Cy-3 fluorophore. Basal membranes (BM) of the dermoepidermal junction and blood vessels (BV) were immunostained using collagen type IV antibody with rhodol green fluorophore. Bundles of nerve fibers (labeled in red) are located close to the microvasculature in the dermis (D) and branch out into the superficial part and penetrate the basal membrane. In the superficial part of the dermis, the microcirculation (labeled in green) and nerve fibers establish fine contacts together (labeled in yellow) and terminate as free nerve endings in the epidermis (E) just beneath the stratum corneum (SC). Bar = 50 µm. Inset shows details of the epidermal innervation. Bar = 30 µm.

Illustration of the computer-assisted structure recognition and the different steps of the morphometric analysis for intraepidermal nerve fiber quantification. A, Step 1: detection of the lower part of the epidermis using collagen type IV immunostaining. B, Step 2: computer-generated image assigning a green color to all areas not stained with collagen type IV antibodies. C, Step 3: computer-generated image assigning yellow color to the epidermis used to calculate total epidermal area. D, Step 4: detection of the intraepidermal nerve fibers, using protein gene product 9.5 immunostaining. Measurement of nerve fiber surface was performed by automatic delineation of the stained areas. E, Step 5: the stratum corneum was delineated manually and epidermal length was measured. Bar = 30 µm.

Histograms showing the mean epidermal innervation density of 4 anatomical sites measured by morphometric analysis and expressed as a ratio between the nerve fiber surface and epidermal surfaces. Error bars show SD.

Intraepidermal nerve fiber density: comparison between 2 truncal (A) and 2 facial (B) body areas. In both facial areas, regression lines display similar decrease with age. The preauricular region shows significantly lower innervation density compared with the eyelid area. Conversely, both sun-protected areas demonstrated a slight increase in epidermal innervation with aging reflected by similar regression line slopes. Also, innervation density was comparable between abdominal and mammary skin.

Variations of epidermal nerve density according to age groups in the 4 anatomical sites analyzed: eyelid (A), preauricular area (B), abdomen (C), and mammary area (D). Samples are distributed by 10-year range. Results are expressed as ratio between nerve fibers and epidermal surfaces.

Micrographs showing representative immunofluorescence labeling of different anatomical sites evaluated at 2 ages. Eyelid at ages 37 (A) and 76 (B) years; preauricular area at ages 48 (C) and 75 (D) years; mammary area at ages 27 (E) and 49 (F) years; and abdomen at ages 22 (G) and 56 (H) years. Bar = 25 µm.