Gigi J. Ebenezer, Ryan O'Donnell, Peter Hauer, Nicholas P. Cimino, Justin C. McArthur, and Michael Polydefkis
Diabetic complications and vascular disease are closely intertwined. Diabetes mellitus is a well-established risk factor for both large and small vessel vascular changes, and conversely other vascular risk factors confer increased risk for diabetic complications such as peripheral neuropathy, nephropathy and retinopathy. Furthermore, axons and blood vessels share molecular signals for purposes of navigation, regeneration and terminal arborizations. We examined blood vessel, Schwann cell and axonal regeneration using validated axotomy models to study and compare patterns and the relationship of regeneration among these different structures. Ten subjects with diabetes mellitus complicated by neuropathy and 10 healthy controls underwent 3 mm distal thigh punch skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy at different time points. Serial sections were immunostained against a pan-axonal marker (PGP9.5), an axonal regenerative marker (GAP43), Schwann cells (p75) and blood vessels (CD31) to visualize regenerating structures in the dermis and epidermis. The regenerative and collateral axonal sprouting rates, blood vessel growth rate and Schwann cell density were quantified using established stereology techniques. Subjects also underwent a chemical ‘axotomy’ through the topical application of capsaicin, and regenerative sprouting was assessed by the return of intraepidermal nerve fibre density through regenerative regrowth. In the healed 3 mm biopsy sites, collateral and dermal regenerative axonal sprouts grew into the central denervated area in a stereotypic pattern with collateral sprouts growing along the dermal–epidermal junction while regenerative dermal axons, blood vessels and Schwann cells grew from their transected proximal stumps into the deep dermis. Vessel growth preceded axon and Schwann cell migration into the denervated region, perhaps acting as scaffolding for axon and Schwann cell growth. In control subjects, Schwann cell growth was more robust and extended into the superficial dermis, while among subjects with diabetes mellitus, Schwann tubes appeared atrophic and were limited to the mid-dermis. Rates of collateral (P = 0.0001), dermal axonal regenerative sprouting (P = 0.02), Schwann cell migration (P < 0.05) and blood vessel growth (P = 0.002) were slower among subjects with diabetes mellitus compared with control subjects. Regenerative deficits are a common theme in diabetes mellitus and may underlie the development of neuropathy. We observed that blood vessel growth recapitulated the pattern seen in ontogeny and preceded regenerating nerve fibres, suggesting that enhancement of blood vessel growth might facilitate axonal regeneration. These models are useful tools for the efficient investigation of neurotrophic and regenerative drugs, and also to explore factors that may differentially affect axonal regeneration.