Lipid changes within the epidermis of living skin equivalents observed across a time-course by MALDI-MS imaging and profiling

Abstract

Background: Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. Here, its application to the study of the distribution of lipids in sections of reconstructed living skin equivalents during their development and maturation is described.

Methods: Living skin equivalent (LSE) samples were obtained at 14 days development, re-suspended in maintenance medium and incubated for 24 h after delivery. The medium was then changed, the LSE re-incubated and samples taken at 4, 6 and 24 h time points. Mass spectra and mass spectral images were recorded from 12 μm sections of the LSE taken at each time point for comparison using matrix assisted laser desorption ionisation mass spectrometry.

Results: A large number of lipid species were identified in the LSE via accurate mass-measurement MS and MSMS experiments carried out directly on the tissue sections. MS images acquired at a spatial resolution of 50 μm × 50 μm showed the distribution of identified lipids within the developing LSE and changes in their distribution with time. In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species.

Conclusions: MSI can be used to study changes in lipid composition in LSE. Determination of the changes in lipid distribution during the maturation of the LSE will assist in the identification of treatment responses in future investigations.

Figures

Fig. 1

Positive ion MALDI Mass spectrum of a LSE (24 h incubation) across the full thickness of the tissue, using α-CHCA/ANI as a matrix

Fig. 2

Principal component analysis of MALDI-MS spectra acquired from LSE. Score plots were generated showing groupings and variability between the 4, 6 and 24 h group spectra from the a) epidermis or c) dermis regions of the skin. The loading plots show a distribution of m/z spectra ion species which are contributors of grouping and variability in the b) epidermis or d) dermis between the time-course groups; the quadrant space in corresponding between the plots

Fig. 3

a MALDI-MSI-IMS image mapping the in-source fragment m/z ion species for SM (d18:1/16:0) in tissue and within a SM (d18:1/16:0) lipid standard. Particularly m/z 520.5024 is likely to be the N-paltimitoylsphingosine unit of the structure following neutral loss of cyclophosphane [H (HO)P (O) (OCH2CH2O)] m/z 183. The ion m/z 644.4309 correspond to a loss of trimethylamineN (Ch3) m/z 59 from the M + H adduct. Ion map for m/z 256.2599 represents the d18 side chain of the SM lipid structure. The approximate fragment conformations are depicted in the structural diagram. b. MALDI-MSI-IMS of commonly occurring fragment ions found within tissue and lipid standards representative of fatty acyl side chains

Fig. 4

MALDI-IMS-MSI images mapping the protonated and sodiated ions for ceramides and PC species across skin equivalent tissue sections representing the three time-stages within the time-course study (4, 6 or 24 h incubation period [from left to right]) and across lipid standards. Images are at a spatial resolution of 50 μm × 50 μm and are normalised against the total ion count

Fig. 5

MALDI-IMS-MS images mapping the ions m/z 759.56, 757.54, 732.54 and 703.56 in skin equivalent tissue, at the different stages of a time course (4, 6 and 24 h respectively). a - (c) m/z 732.54 at 4,6, and 24 h, (d)-(f) m/z 757.54 (g)-(i) m/z 759.56 at 4,6 and 24 h and (j)-(l) m/z 703.56 at 4,6, and 24 h . These signals were identified from the PCA data as being significant contributors of change in the spectra between the epidermis and dermis. Images are at a spatial resolution of 50 μm × 50 μm and are normalised against the total ion count. Optical images of Oil Red O stained sections of the tissue at the different stages of a time course (4, 6 and 24 h respectively) are also shown (m)-(o)

Fig. 6

Haematoxylin & eosin stained skin section against a MALDI-MS map of skin sections focusing on the 24 h time-point, showing the potential of the 50 μm × 50 μm resolution to discern epidermal ultrastructural region