Prostaglandin E 2, Interleukin-1α, and Potassium Release from Artificial Human Skin after Freeze-Thaw Injury

Abstract

Living skin equivalent (LSE) was used to identify nonvascular aspects of freeze-thaw injury to human skin cells. Pairs of transwell cell culture inserts, containing LSE, were washed twice for 30 min in maintenance media at 37°C in a CO 2 incubator and placed in two wells of a six-well assay tray (containing assay media). One specimen was maintained at room temperature. The other was cooled at 1°C/min to - 15°C and rapidly rewarmed. Both were incubated for 24 h on fresh maintenance media at 37°C in a CO 2 incubator. This process was repeated producing 12 per group. Prostaglandin E 2 (PGE 2), interleukin-1α (IL-1α), and K + were measured (mean ± SE) in the assay medium, after the rewarming interval, and in the maintenance media after the 24-h incubation. Specimens were then processed for electron microscopy. After the rewarming interval, PGE 2 (164.0 ± 23.2 pg/0.1 ml), IL-1α (24.3 ± 2.2 pg/0.1 ml), and K + (6.47 ± 0.41 m M) released from frozen LSE were significantly increased compared to controls (PGE 2 = 22.2 ± 4.6 pg/0.1ml, IL-1α = 5.7 ± 1.0 pg/0.1 ml, K + = 4.32 ± 0.02 m M). After the 24-h incubation, PGE 2 and IL-1α released by frozen LSE (PGE 2 = 1126 ± 208 pg/0.1 ml; IL-1α = 80.6 ± 6.8 pg/0.1 ml) remained significantly higher than controls (PGE 2 = 229.0 ± 45 pg/0.1 ml; IL-1α = 4.9 α 0.7 pg/0.1 ml). At this time, K + leakage from frozen LSE (4.39 ± 0.03 m M) had returned to a normal range (control = 4.65 ± 0.02 m M). Keratinocytes and, to a lesser extent, fibroblasts showed ultrastructural freeze-thaw damage. These data indicate that the process of freezing and rewarming LSE, at rates consistent with frostbite, produces significant release of PGE 2- and IL-1α from these cell types, which could influence the outcome of frostbite in vivo.