Multimodal, in Situ Imaging of Ex Vivo Human Skin Reveals Decrease of Cholesterol Sulfate in the Neoepithelium during Acute Wound Healing

Abstract

Skin repair is a significant aspect of human health. While the makeup of healthy stratum corneum and epidermis is generally understood, the mobilization of molecular components during skin repair remains largely unknown. In the present work, we utilize multimodal, in situ, mass spectrometry, and immunofluorescence imaging for the characterization of newly formed epidermis, following an initial acute wound for the first 96 h of epithelization. In particular, TOF-SIMS and confirmatory MALDI FT-ICR MS (/MS) analysis permitted the mapping of several lipid classes, including phospholipids, neutral lipids, cholesterol, ceramides, and free fatty acids. Endogenous lipid species were localized in discrete epidermal skin layers, including the stratum corneum (SC), stratum granulosum (SG), stratum basale (SB), and dermis. Experiments revealed that healthy re-epithelializing skin is characterized by diminished cholesterol sulfate signal along the stratum corneum toward the migrating epithelial tongue. The spatial distribution and relative abundances of cholesterol sulfate are reported and correlated with the healing time. The multimodal imaging approach enabled in situ high-confidence chemical mapping based on accurate mass and fragmentation pattern of molecular components. The use of postanalysis immunofluorescence imaging from the same tissue confirmed the localization of endogenous lipid species and Filaggrin and Cav-1 proteins at high spatial resolution (approximately a few microns).

Figure 1.

(A) Tissue disks incubating in an air-liquid interface. (B) Schematic representation of biopsy punch and resulting disk. (C-E) H&E staining of parallel tissue sections. (F) Fluorescence microscopy using Filaggrin, Cav-1, and DAPI to delineate the stratum corneum, granulosum, spinosum, and basale of the epidermis.

Figure 2.

Phase contrast, fluorescence, and secondary ion images of an unwounded region of skin tissue. Negative ion images of cholesterol sulfate, adenine, α-Tocopherol, FA 24:0, 25:0, 26:0 are of the form [M-H]^-. The ion at 168.0 m/z is used here to represent the Sphingomyelin headgroup. A comparison between fluorescence and SIMS overlay demonstrates the fidelity of endogenous lipids as biomarkers for epidermal layers.

Figure 3.

TOF-SIMS imaging of unwounded skin tissue in positive mode detection. (A) Composite of the m/z channels shown in panels B-D. The m/z of 184 is shown to represent PC headgroup (B). For (C) cholesterol [M-H2O+H]⁺ ion is shown. Images D and E are constructed as the sum of various channels of the same lipid class, shown in Supplementary Figure S1 and listed in detail in Supplemental Table 1.

Figure 4.

Optical, SIMS imaging, and fluorescence imaging of re-epithelializing skin tissue. The ‘U’ and ‘R’ denote unwounded and re-epithelialized areas of 48h incubated skin, respectively. Individual secondary ion images comprising the SIMS overlay are shown in the middle row. Secondary ion images of sphingomyelin is represented as the of ion at 168.0 m/z. Here, signal of fatty acids 20:0–28:0 were combined and shown as a summed secondary ion image.

Figure 5.

Cholesterol sulfate progression in re-epithelializing tissue. The top row shows SIMS overlays of FA 20:0–28:0 (Red), cholesterol sulfate (yellow), adenine (blue), and sphingomyelin (grey). The red dashed line denotes the wound edge, as corroborated by microscopy. A typical linescan plot of the 96h re-epithelialized tissue (denoted with an *) in the lower left shows the integrated y-area intensity of cholesterol sulfate as a function of distance. Profiles were divided into quarters roughly equivalent to 24 h of re-epitheliazation, approximately 100 μm segments. In the lower right, a boxplot shows the minimum, maximum, lower quartile, upper quartile, median (line) and mean (circle) of observations pooled from 48h and 96h incubated tissues according to distance from the initial wound. N=7 for −100–200 μm; N=6 for 201–300; N=4 for 301–400 μm.