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

Mitchell CA, Long H, Donaldson M, Francese S, Clench MR - Lipids Health Dis (2015)

Bottom Line: In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species.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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield, S1 1WB, UK. c.mitchell@surrey.ac.uk.

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.

No MeSH data available.


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)
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Fig5: 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)

Mentions: The PCA plots show ions which are major contributors to variances in the epidermis (Fig. 2a, c). Figure 5 shows MALDI-MS images for the distribution of the ion m/z 759.5592 which was projected to vary significantly between the dermis and epidermis from the PCA loadings plot. From these images some changes can be visualised: the distribution of the ionm/z 759.5592 changes with time; in the 24 h sample it is located primarily at the edge of the epidermis whereas in the 4 and 6 h tissue sections the signal is observed throughout the sections. This is also the case for the ions m/z 757.5411 and m/z 732.5531. These species have been tentatively identified via accurate mass measurement (Additional file 1: Table S1) to an error of 5 ppm. Repeat studies carried out on a different instrument gave the same findings; a distinct epidermal band can be seen when mapping select ions, particularly for the 24 h sample section. This is particularly shown for the ions m/z 759.5 and 703.5 ions. When directly comparing the Oil Red O stained sections across the time-course, noticeably some basal and granular epidermal layer changes can be seen in the later time-point. The epidermal region appears more compressed and dense in the 24 h sample, whereas the 4 h sample is thicker in appearance and less compact. Some lipid droplets can be found in the 24 h sample epidermis. Intriguingly the 24 h stratum corneum appears darker indicative of being a richer neutral lipid layer.In all cases a stratified epidermis could be visualised as a red band across the outer region of the skin. This stratified feature can also be shown as a dark purple edge in the haematoxylin and eosin stained sections across the time-course (Fig. 6).Fig. 5


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

Mitchell CA, Long H, Donaldson M, Francese S, Clench MR - Lipids Health Dis (2015)

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)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4525729&req=5

Fig5: 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)
Mentions: The PCA plots show ions which are major contributors to variances in the epidermis (Fig. 2a, c). Figure 5 shows MALDI-MS images for the distribution of the ion m/z 759.5592 which was projected to vary significantly between the dermis and epidermis from the PCA loadings plot. From these images some changes can be visualised: the distribution of the ionm/z 759.5592 changes with time; in the 24 h sample it is located primarily at the edge of the epidermis whereas in the 4 and 6 h tissue sections the signal is observed throughout the sections. This is also the case for the ions m/z 757.5411 and m/z 732.5531. These species have been tentatively identified via accurate mass measurement (Additional file 1: Table S1) to an error of 5 ppm. Repeat studies carried out on a different instrument gave the same findings; a distinct epidermal band can be seen when mapping select ions, particularly for the 24 h sample section. This is particularly shown for the ions m/z 759.5 and 703.5 ions. When directly comparing the Oil Red O stained sections across the time-course, noticeably some basal and granular epidermal layer changes can be seen in the later time-point. The epidermal region appears more compressed and dense in the 24 h sample, whereas the 4 h sample is thicker in appearance and less compact. Some lipid droplets can be found in the 24 h sample epidermis. Intriguingly the 24 h stratum corneum appears darker indicative of being a richer neutral lipid layer.In all cases a stratified epidermis could be visualised as a red band across the outer region of the skin. This stratified feature can also be shown as a dark purple edge in the haematoxylin and eosin stained sections across the time-course (Fig. 6).Fig. 5

Bottom Line: In particular development of an epidermal layer was observable as a compaction of the distribution of phosphatidylcholine species.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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield, S1 1WB, UK. c.mitchell@surrey.ac.uk.

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.

No MeSH data available.