Quantification of pilomotor nerves: a new tool to evaluate autonomic involvement in diabetes

Quantification of the complex, autonomic networks in the skin is difficult. Although sporadic attempts focusing mainly on sudomotor plexus have been reported, an easy and reliable method of quantification has not yet been made available. We developed a method to quantify pilomotor nerve fibers (PNFs), which, compared to sudomotor nerves, have a less complex pattern. We used this procedure on a population of normal and diabetic subjects, and propose it as a new tool to study cutaneous autonomic nerves.

Skin biopsies were performed from thigh and distal leg in 20 diabetic patients and 20 age- and sex-matched controls. Samples were processed applying indirect immunofluorescence and using pan-neuronal and selective markers for cholinergic and noradrenergic fibers. Pilomotor nerve fiber density was blindly calculated on single 2-microm optical sections selected from confocal z-stacks. Interobserver and intraobserver reliability was evaluated. Results were compared with values obtained by 2 other methods that explored PNFs more extensively. Pilomotor nerve fibers density was compared to epidermal nerve fiber (ENF) density, to pilocarpine-activated sweat gland density, and to the severity of neuropathy as assessed by the modified total neuropathy score.

A significant loss of PNFs was found in diabetic subjects' thigh and leg. PNFs density did not correlate with ENF density, disease duration, or total neuropathy score. Noradrenergic PNFs correlated instead with sweating impairment.

A reliable assessment of PNF density is possible. When studying cutaneous innervation, PNF quantification should be done to gain information on autonomic nerves in addition to somatic nerves.