An unbiased method for estimation of total epidermal nerve fibre length

It is of interest to quantify accurately lineal biological features such as nerve fibres, capillaries, and tubules for studies of development and diseases such as sensory neuropathy, and for evaluation of therapeutic regimens in humans and animal models. An unbiased stereological method to sample and estimate total length of immunostained epidermal nerve fibres by using vertical sections of punch skin biopsies from two human volunteers is presented. The essential steps in the procedure are as follows: (1) serially section the skin punch in a random plane perpendicular to the cutaneous surface; (2) immunostain a known fraction of total sections with antibody specific for nerve fibres; (3) orient a test line-grid over the epidermis; and (4) count intersections between test lines and immunostained epidermal nerve fibres. The optical fractionator method is employed to estimate total length of immunostained epidermal nerve fibres in the biopsy. By using these techniques the total length of nerve fibre in a defined region can be determined without methodological bias, assumptions or correction factors.