Using Infrared and Raman Microspectroscopies to Compare Ex Vivo Involved Psoriatic Skin and Normal Human Skin

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1 Using Infrared and Raman Microspectroscopies to Compare Ex Vivo Involved Psoriatic Skin and Normal Human Skin Leroy, Marie (Laboratoire d'ingénierie de Surface (LIS)) Lefèvre, Thierry (Groupe de recherche en biophysique et RMN des solides) Pouliot, Roxane (Centre LOEX de l'université Laval, Génie Tissulaire et Régénération) Auger, Michèle (Groupe de recherche en biophysique et RMN des solides) Laroche, Gaétan (Laboratoire d'ingénierie de Surface (LIS)) Introduction Psoriasis is a chronic dermatitis which affects 3% of the world's population (80 millions of people)^1. Unfortunately, the etiology of this autoimmune pathology is still incomplete. Skin consists in three principal layers which are the hypodermis, dermis, and epidermis. The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum (SC), which is the outermost layer of the skin. In fact the SC has a particular "bricks and mortar" structure, where the cells are embedded in an extracellular matrix of lipids organized in a tightly packed bilayer structure that ensures an efficient barrier function. Vibrational spectroscopies (infrared (IR) and

2 Raman) are non-invasive and non-destructive optical techniques that can both provide direct molecular-level information on biological tissues. These analytical methods are based on the detection of chemical group frequencies that are in turn, sensitive to molecule structure, organization, and conformation. Both IR and Raman spectroscopies have been previously demonstrated to be powerful complementary techniques for the determination of lipid organization and protein secondary structure. The IR spectra exhibit features due to the stretching vibrational modes of methyl and methylene groups in lipid acyl chains. The symmetric stretching mode of the lipid methylene groups (2850 cm^-1) is sensitive to the acyl chain overall order. Moreover the frequencies of the IR components underneath the envelop of the amide I band provide information about the overall protein secondary structure. IR imaging allows getting firsthand visual information on sample composition and its molecular organization. On the other hand, Raman microspectroscopy provides specific information about the carbon skeleton of lipid acyl chains and amino acid side chains.^2 In the present work IR and Raman microspectroscopy analyses

3 were used to get in-depth molecular-scale characterization of ex vivo involved psoriatic human skin (PHS) compared to normal human skin (NHS). Materials and Methods This study was conducted in agreement with the Helsinki declaration and performed under the guidelines of the research ethics committee of the "CHU de Québec". Samples come from ex vivo psoriatic plaques from 3 deceased patients. Biopsies were embedded in OCT (Optimal Cutting Temperature) gel for further analyses. In IR microspectroscopy, 10 µm-thick cryosections of the biopsies were placed on reflective-coated microscope slides (Low-e MirrIR, Kevley Technologies, Chesterland, OH) for spectroscopic analyses. IR microscopy images were recorded with an Agilent 620 IR microscope (Agilent Technologies, Australia) equipped with a liquid-nitrogen cooled 32 x 32 focal plane array detector and a motorized stage. Each of these 1024 detector elements measured 5.5 µm Á 5.5 µm, which therefore corresponds to the pixel size in the infrared images. Raman microspectroscopy experiments were

4 performed on 20 µm-thick transverse sections of skin samples put in place on glass microscope slides. Raman spectra were recorded using a LabRam HR-800 spectrometer (Horiba Jobin-Yvon, Villeneuve d'ascq, France) coupled to an Olympus BX-30 fixed stage microscope, equipped with an open electrode Peltier-cooled CCD detector (1024 x 256 pixels) (Andor Technology, Belfast, Northern Ireland). An internal He-Ne laser set at 633 nm (red) was used for the acquisition of all the spectra. Ten spectra were taken in each characteristic layer of the samples (SC, LE, and dermis). Results In IR microspectroscopy, the symmetric stretching mode of the lipid methylene groups (2850 cm^-1) is sensitive to the acyl chain overall order. The frequency of this mode in the outermost layer of the PHS is higher than in the SC of NHS (data not shown). In Raman microspectroscopy, the C-C stretching modes at 1061 and 1128 cm^-1 are characteristic of trans conformers while gauche conformers give rise to a broad band at 1081 cm^1. The intensity ratio of the bands at 1061 and at

5 1081 cm^-1 shows that a higher amount of gauche conformers is observed in the outermost layer of the PHS compared to NHS (data not shown). Therefore, both IR and Raman analyses reveal a decrease of the lipid matrix organization in the outermost layer of the PHS, despite a similar distribution of lipids and collagen for both NHS and PHS (Figure 1C, 1D, 1H, 1I). Histology pictures of NHS and PHS show that PHS displays a less regular organization. In fact, some large inclusions seem to infiltrate the epidermis (Figure 1F), contrary to NHS, which is nicely organized in three distinct layers. These inclusions are also visible in the infrared images (Figure 1H and 1I) and are likely composed of proteins (Figure 1I). Discussion and Conclusion In previous studies on the comparison of NHS and human skin substitutes, the samples were described in terms of their different layers^3,4. Clearly, such an analytical approach cannot be taken for PHS as the organization of this specimen is clearly erratic, with the presence of large protein inclusions. IR and Raman microspectroscopy are powerful techniques

6 that allowed characterizing the molecular organization of PHS compared to that of NHS. Vibrational microspectroscopies analyses show that the lipids are more ordered in the outermost layer of the NHS than in that of PHS. Further experiments will take advantage of both IR and Raman microspectroscopies to identify the preferential pathways and evaluate the diffusion rate of exogenous molecules through human skin. Figure 1: (A and F) histologies (scale bar 100µm), (B and G) microscopic views (15X objective), infrared images of (C and H) the lipid and (D and I) collagen distribution, (E and J) frequency of the symmetric stretching mode of the lipid CH2 groups (1 pixel =

7 5.5µm), for NHS and PHS respectively. Acknowledgements The authors acknowledge the financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes for Health Research (CIHR) through their joint Collaborative Health Research program, and from the Fonds de recherche du Québec Nature et technologies (FRQ-NT). References 1.Schon, M. P.; Boehncke, W. H., Psoriasis. N Engl J Med 2005, 352, Flach, C. R.; Moore, D. J., Infrared and Raman imaging spectroscopy of ex vivo skin. Int J Cosmetic Sci 2013, 35, Leroy, M.; Labbe, J. F.; Ouellet, M.; Jean, J.; Lefevre, T.; Laroche, G.; Auger, M.; Pouliot, R., A comparative study between human skin substitutes and normal human skin using Raman microspectroscopy. Acta Biomater 2014, DOI: /j.actbio Leroy, M.; Lafleur, M.; Auger, M.; Laroche, G.; Pouliot, R., Characterization of the structure of human skin substitutes by infrared microspectroscopy. Anal Bioanal Chem 2013, 405,

Vibrational Microspectroscopy of Skin: Applications to Pharmacology and Biochemistry. Rich Mendelsohn Rutgers University

Vibrational Microspectroscopy of Skin: Applications to Pharmacology and Biochemistry Rich Mendelsohn Rutgers University Scientific Students/Associates R. Mendelsohn Dr. G. (Jin) Zhang* C. R. Flach Dr.