Document Type

Honors Project

Abstract

Burn survivors frequently experience persistent sensory morbidity, including pain, itch, altered thermal sensitivity, and abnormal cutaneous sensation long after wound closure. These complications are especially important in deeper wounds and grafted skin, where structural recovery of cutaneous innervation may remain incomplete. Despite this clinical burden, reproducible structural methods for quantifying postinjury reinnervation in wound tissue remain limited. This thesis adapted and applied a Kennedy-derived, nerve optimized histologic protocol for a porcine model of burns to assess peripheral cutaneous innervation. The first study arm was a pilot deep partial-thickness thermal burn study in a Red Duroc pig designed to compare healing wound tissue with paired adjacent normal skin. The second arm was a longitudinal grafted full-thickness wound cohort in Yorkshire pigs, used to extend the nerve-analysis protocol across serial wound and scar time points. Three-millimeter punch biopsies were fixed in Zamboni’s fixative, cryoprotected in sucrose, embedded in optimal cutting temperature (OCT) compound, sectioned at 60 µm, and processed as free-floating sections for immunofluorescence. The pilot arm initially evaluated three antibody cocktails: mouse anti-PGP9.5 paired with rabbit anti-substance P, rabbit anti-CGRP, or rabbit anti-VIP, all with a DAPI counterstain. The graft arm then specifically applied the PGP9.5/VIP/DAPI protocol across all analyzed samples. Confocal imaging on a Zeiss LSM 800 and Neurolucida-based tracing were used to quantify epidermal nerve fiber counts and epidermal nerve fiber density (ENFD; ENFs/mm of epidermis), and VIP-associated signal was quantified as mean fluorescence intensity in Zeiss ZEN. The pilot cohort included one Red Duroc pig with six deep partial-thickness burns and paired adjacent normal biopsies collected on post-burn day 13. The graft cohort included two Yorkshire pigs with six full-thickness wounds per pig; the present nerve-analysis dataset ultimately included two wounds from each pig, analyzed longitudinally with side-matched baseline normal reference skin rather than time-matched adjacent normal controls at every wound-day. In the pilot cohort, wound tissue showed significantly lower raw ENF counts than paired normal skin (1.67 ± 0.82 vs 5.33 ± 1.75, p = 0.0019) and significantly lower ENFD (2.49 ± 1.64 vs 10.36 ± 4.15 ENFs/mm, p = 0.0028). Whole-image VIP mean fluorescence intensity was significantly higher in wound tissue than in paired normal skin (26.61 ± 14.44 vs 11.16 ± 3.00, p = 0.045). Compartment-specific analysis further showed that epidermal VIP mean fluorescence intensity was significantly higher in wound tissue (35.52 ± 20.49 vs 9.81 ± 4.66), whereas dermal VIP mean fluorescence intensity was increased in wounds (24.48 ± 12.74 vs 12.23 ± 5.87) but did not reach statistical significance in the current pilot dataset. No analyzed images or biopsies were excluded. All counters were blinded, and inter-rater reliability for ENF counting was 0.920. In the graft cohort, two wounds from each of two Yorkshire pigs were analyzed longitudinally. Early day 3, 6, and 10 biopsies were analyzed as distinct interstitial-space and graft site samples rather than as technical replicates. In the late wound/scar phase (days 29, 36, 43, 50, and 57), ENF counts remained sparse (0.50–1.75 per section) and ENFD remained low (0.14–0.53 ENFs/mm), representing only 1.2%–4.5% of the side-matched baseline normal reference ENFD. Raw and background-corrected VIP measures did not show significant temporal effects across the late graft time course. This thesis establishes a protocol for quantifying postinjury cutaneous innervation in healing and grafted skin from porcine studies. By pairing structural ENFD analysis with VIP-associated signal, it provides a framework for future studies of post-burn pain, itch, and neuroimmune remodeling.

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