Imaging Skin Models With CRS

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5.1 Introduction

The skin is the largest organ of the mammalian body with an estimated total weight of 5 kg and a surface around 2 m2 for adult humans [151]. Being most exposed to the environment, it represents a major physical and immunological protection against injury and infection.  Similar to the mucosal immune system, a skin immune system (SIS) has been described representing a coordinated system in which epithelial cells, resident immune cells, and a local microenvironment including locally produced vitamins control immunity and tolerance to self and foreign antigens [152 – 154]. In addition, recent work indicates a major role for the skin microbiome, which is composed of up to 1012 microorganism/m2, mostly localized in the intercorneocytic spaces [157]. In addition to physical and immunological protection, the skin plays an important role in thermoregulation, transmission of stimuli, storage/synthesis, and absorption [151, 155, 156]. [150]

Much dermal research relies on mouse skin models as an analogy for human skin[1-36].  From the 1940s, 1950s, 1960s and beyond, extensive inquiry has been done regarding carcinogenesis, epidermal regeneration, cell mitosis[2, 3, 20, 37-46], melanocytes[47-51], among others[23][6, 101-114].  One particular area of interest has been topical pharmaceuticals[115-136].  Indeed, skin-penetration studies play an essential role in the selection of drugs for dermal or transdermal application. Therefore, the choice of predictive ex-vivo penetration models is highly important. Ideally, one would like to use human skin to evaluate penetration properties of candidate drugs.  However, specimens of human skin of sufficient size and quality for penetration experiments are not readily accessible to most investigators and in any case, are only available in limited amounts.

Part of the attraction to rodent dermal models over those of ‘higher’ mammals are their easy availability, low cost, and small size.  Additionally, the comparatively thin dermis present in rats and mice make for easier transdermal imaging.  In spite of this, numerous studies have suggested that pig skin is a more accurate approximation to human [137-146], and therefore more appropriate where human medical science is the desired beneficiary of research.  One of the initial goals in this project has been to identify differences, using coherent Raman scattering, between porcine and mouse skin, and to demonstrate that porcine is a more realistic model for the human epidermis.

This chapter demonstrates the use of F-CARS, epi-CARS and SRS in determining those structural differences between pig & mouse skin, with partial assistance given from second harmonic generation to display the differences in density and structure of collagen.  The attractive non-invasiveness of epi-CARS is highlighted in this chapter, as well as consideration given to answering questions of the effective depth penetration of CARS & SRS, and how to measure depth as the skin expands (or contracts).  CARS & SRS are discussed with respect to their comparative suitability for structural imaging.  The findings are then used to reinforce prior work which concludes that pig skin is a better model than mouse for human skin.  Figures 1 & 2 show each skin type, with comparison images at six different depths.

5.2 Pig skin vs mouse skin

Small mammals, such as the rabbit, guinea pig, rat and mouse, are frequently used in wound healing studies as they are inexpensive and easy to handle. Despite these advantages, small mammals differ from humans in many anatomical and physiological ways. For example, these mammals have a dense layer of body hair, thin epidermis and dermis and, more significantly, they heal primarily through wound contraction as opposed to reepithelialization. Anatomically and physiologically, pig skin is more like human skin. Both pig and man have a thick epidermis. Human epidermis ranges from 50 to 120 µm and the pigs from 30 to 140 µm, but because in both animals the epidermal thickness varies considerably based on body site, an alternative measure is the dermal-epidermal thickness ratio [1].  It has been reported that this ratio ranges from 10:1 to 13:1 in the pig and is comparable in measurements of human skin [2]. Both man and pig show well-developed rete-ridges and dermal papillary bodies, with abundant subdermal adipose tissue.

Porcine dermal collagen is similar to human dermal collagen biochemically, accounting for its use in a number of wound healing products [6]. Although pig dermis has a relatively high elastic content as compared to other mammals, it is still less than that found in human skin [7]. Neither pig nor man has a panniculus carnosus as is found in small (loose skinned) animals [5]. The size, orientation, and distribution of blood vessels in the dermis of the pig are similar to blood vessels in human skin; however, the sub epidermal plexus, which supplies adenexal structures, is somewhat less developed in the pig [8–10]. The number and distribution of adenexal structures in swine and man are similar but not identical [1,11,12]. Both pig and man have sparse body hair which, unlike many animals, progresses through the hair cycle independently of neighbouring follicles [5]. This is important as adenexal structures, including hair follicles, play an important role in reepithelialization.

Adenexal differences between pig and man are that pig skin contains no eccrine glands, and unlike man, apocrine glands are distributed through the skin surface [1]. Functionally, pig and man are similar in terms of epidermal turnover time, type of keratinous proteins, and lipid composition of the stratum corneum [12–14]. Immunohistochemical staining of porcine and human skin shows similar staining patterns for several antigens including keratins 16 and 10, filaggrin, collagen IV, fibronectin, and vimentin [15].  Man and pig heal through physiologically similar processes [16]. Most small animals have a panniculus carnosus and rely on wound contraction for wound closure. Conversely, man and swine close partial-thickness wounds largely though reepithelialization. Additionally, the pig’s overall physiology is close to human physiology, with most key organ systems being similar in anatomy and function [17,18].

Sullivan et al., when comparing results of wounds healing studies performed in humans, swine, small mammals, and in vitro, identify a concordance of 78 between human and pig; 53 between human and small mammal; and 57 between human in vivo and in vitro.  They conclude that the body of research presents a clear result: the porcine model is an excellent tool for the evaluation of therapeutic agents destined for used in human wounds [147].

Investigators could study the biochemistry of human skin surface lipids more conveniently if they we able to discover experimental animals that produced the same lipids. Human skin surface lipids differ markedly from those of the animals previously studied such as the sheep [1], various rodents (rat, mouse, guinea pig, and rabbit) [2], and birds [3].

To define the details of similarities and differences, Montagna and Yun [138] compared, by thin layer chromatography (TLC), the skin surface lipids of adult humans, vernix caseosa, and eighteen species of animals. They found additional differences, but more significantly, that only man produces a “triglyceride type” of sebum. None of the other animals have significant amounts of triglycerides or free fatty acids in their skin surface lipid except the pig.

Furthermore, the amount of lipid obtained from the pig was only of the order of one tenth the amount per unit area obtained from other furrier animals. Additionally, pig surface lipid showed the same lipid classes as those obtained from the nonpolar epidermal lipid samples of different human sources, namely, sterol esters, triglycerides, free fatty acids, and free sterols for sole epidermis [6] for the living layer of leg epidermis [6] and for the wall and sac contents of epidermal cysts [10]. [138]

Porcine skin has a gross resemblance to that of man, particularly after the bristles have been removed. Like man, the pig has a sparse cover of hair; the epidermis has a well-differentiated under sculpture, the dermis has a thick papillary body and a rich population of elastic fibres.  In both animals the pelage is sparse, resulting in a relatively thick epidermis. The surface of both skins is grooved by intersecting lines which form characteristic geometric patterns, and the dermis has a well-differentiated papillary body in both skins.  One of the most striking resemblances between these two skins is the large content of elastic tissue in the dermis. [139]

5.2.1.  Imaging mouse vs pig skin

The ears of adult follicular white mice which had been frozen at -18oC for less than six months were excised, washed, and mounted between two glass cover slips prior to imaging.  Abdominal pig skin to be used, was dermatomed to a nominal thickness of 300 µm, trimmed and washed, prior to being placed in frozen storage (-18oC) for less than six months.  Images were taken using a 60x 1.2NA water-immersion lens (OlympusUK), at the strong lipid C-H stretching resonance at 2845 cm-1, using an incident power of ≈1.5 mW, and 512 x 512 pixels per image, with a scan period of 54 seconds per frame (Fig. 1).

Hair

Cell nuclei

60 micron depth

Stratum basale

15 micron depth

Stratum spinosum

30 micron depth

Stratum basale

Sebaceous gland

 

Figure 1: The physiology of mouse skin at six different depths, taken with SRS at 2845 cm-1.  Identifiable layers are labelled, along with the darker, cell nuclei.  The scale bar represents 20 µm.

In both sets of skin, nucleated cells of the granular and spinous layers are clearly discernible, though the visual quality of both layers appears to be superior in the mouse skin.  The fat-rich sebaceous gland and comparatively large basal cells are clearly visible in the mouse ear.  In contrast, the basal layer in the porcine skin cannot be seen through the relatively dense spinous cells.  The spinous layer in pigs is know from histology in the literature [144] to be substantially thicker than that encountered in rodents.  This may be the cause of structural definition loss encountered at 60 microns in Fig. 2.  The anucleated stratum corneum is also suspected to be significantly thicker in pig skin as compared with mouse, since it is clearly visible in the two uppermost imaging regions of Fig. 2, but so thin as to be difficult to visualise in Fig. 1.

60 micron depth

30 micron depth

Stratum spinosum

15 micron depth

Stratum granulosum

Cell nuclei

 

Figure 2: The topology of pig skin at six different depths, taken with SRS at 2845 cm-1.  Identifiable layers are labelled.  The scale bar represents 20 µm.

Gray and Yardley [143] found that total lipids accounted for approximately 8% of the pig, 10% of the human, and 20% of the rat epidermal cell (dryweight). Phospholipids in pig, human, and rat cells accounted for, respectively, 62%, 53%, and 35% of the total lipids. Phosphatidylcholine (34-38%), phosphatidylethanolamine (18-23%), and sphingomyelin (17-21%) were major compounds in all species.  The major neutral lipids were sterols (mostly cholesterol) and triglycerides.  Free fatty acids were a major lipid class in pig and human cells, whereas wax esters were a major component in rat epidermal cells. Nearly half (45%) of the sterols in rat cells but less than 10% of those in pig and human cells were esterified.  Cholest-7-ene-3/2-01 accounted for 20% of the total sterols in rat cells. Cholesteryl sulfate and ceramide were minor lipids in the three species. The predominant glycosphingolipid (>99%) was glucosylceramide, which accounted for 7% and 9%, respectively, of the total lipids in pig and human cells. A significant proportion (pig, 17%; human, 11%) of the fatty acids in the glucosylceramides were C26:0 and C28:0.

Though like pig and human epidermal cells in phospholipid composition, rat epidermal cells contain, respectively, approximately three and six times the amounts, as a percentage of the cell dry weight, of non-polar lipids. Nearly half (45%) of the sterols in rat cells are esterified, and approximately 20% of the sterols was cholest-7-ene-3&01. The wide variation in the amount of sterols present in each preparation of cells was possibly related to differences in age, weight, and sex of each batch of rats. On the other hand, values for both polar and nonpolar lipids from different batches of pig and human epidermis showed little variation. It has also been noted that the lipid compositions of cells from human leg and breast are very similar, as are those from pig tail and ear [143].

Directional collagen fibres

a

b

Figure 3: Typical collagen structures in a) pig skin, and b) mouse skin at 20 µm depth, as imaged by SHG performed with an 800 nm signal.  Scale bar represents 10 µm.

Second harmonic generation offers an additional insight into epidermal structure.  Highly directional collagen fibres form clusters known as fibrils that generate a strong SHG response at the 800 nm excitation wavelength (Fig. 3), particularly type I collagen, which is the most widespread structural protein in mammals and is the main component of connective tissues [145].  SHG does not, however, generate a response from structures/tissues which lack parallel clustering, such as cells, lipids, etc.  These require complimentary microscopy techniques to image. Illustrating the advantages of combined non-linear imaging methods.

Kong and Bhargava [149] employed Fourier transform infrared (FT-IR) spectroscopic imaging to holistically measure chemical species as well as spatial structure as a function of time to characterize porcine skin as a model for human skin. Porcine skin was found to resemble human skin spectroscopically and differences are elucidated below.

The results indicate that porcine skin is likely to be an attractive tool for studying diffusion dynamics of materials in human skin.

Stratum corneum

Absorbance

Epidermis

Dermis

1500

3000

2000

1000

3500

4000

2500

Wavenumber (cm-1)

Figure Q: Overlay of human & pig skin spectra [T].

Porcine samples differed in both intensity and position of some spectral features, indicating the abundance and environment of some biomolecules are likely different. There might be other sources for the observed differences, most notably the different sample preparations as we discussed earlier. The variations might also arise from the larger scattering related spectral distortions [41,42]. Figure Q show that, spectroscopically, porcine skin is very close to tissue derived from humans and is reasonably a substitute of the spectral properties of human skin.

Kong also concludes that porcine skin is very close to the human counterpart both structurally and chemically. The chemical properties of porcine skin are very stable over time at room temperature, and are relatively consistent among different samples [149].

Another useful tool in the biomedical researcher’s arsenal is that of epi-CARS, the backwards scattered CARS process outlined in chapter 2.  From the biophysicist prototyper’s point of view, epi-CARS can provide invaluable information and guidance when used in conjunction with, and during, novel use/development of, the relatively new modality SRS.  In a clinical setting, other methods of CRS data gathering cannot compare:  While the likes of F-CARS and SRS need a thin sample – usually involving in vitro work – epi-CARS can be performed in-vivo.  The advantages of such non-invasiveness during epidermal investigation are obvious:

  • Ethics:  there is no need to sacrifice and dissect a living organism in order to obtain skin samples.
  • Speed: finding, killing, excising and mounting dermal tissue takes significant time.
  • Versatility: skin damage caused by burns, punctures, disease, etc can be quickly imaged on any part of the body, and without the need for substantial preparation time.

When imaging pig skin, it has been observed that epidermal structures can be resolved down to a depth of approximately 60 µm.  Beyond this, image legibility begins to drop off significantly for both CARS and SRS at a similar point.  In contrast, structural information is clearly visible in mouse skin down to at least 90 µm (Fig. 4).

  

Figure 4: SRS image of lipid-rich cells at 89 µm depth within mouse skin.  Taken at the 2845 cm-1 C-H stretching resonance.  Scale bar represents 10 µm.

Figure 5: 3D projections of a 260 micron scan through pig skin, comparing three CRS modalities at the 2854cm-1 C-H stretching resonance. Side view: a) CARS stack, b) SRS stack, c) epi-CARS stack. ImageJ used.

An important factor to consider during transdermal imaging studies is the expansion and contraction undergone by skin whilst under the influence of unavoidable experimental pressures.  Fig. 6 illustrates the dramatic temporary volumisation experienced when the epidermal structure absorbs a solute.

Figure 6: An XZ SRS 2854 cm-1 stack of pig skin, taken at a) ten minutes post-dosing with undeuterated propylene glycol, and b) ninety-six minutes post-dosing.  The scale bar represents 20 µm.

The local swelling increases radial depth by approximately a factor of three, prior to subsidence.  A contrary effect is seen when heating of the skin is promoted via illumination with non-trivial pulse-laser powers – around 1.5 mW incident power in our studies.  The consequent shrinkage observed in Fig. 7 is significant enough to invalidate permeation time-series data if not accounted for.

 

a)

b)

c)

d)

Figure 7: Radial contraction of porcine skin due to laser-induced drying over a 60-minute period.  From a) to d), 0, 20, 40, 60 minutes respectively post-mounting.  Scale bar represents 20 µm.

We found a novel solution to the problem, utilising the intrinsic XZ scan capability of our LabVIEW-based imaging software.  By capturing a single 13 second XZ image between each XYZ stack, we were able to keep track of the changing surface position, and adjust the long XYZ time-series parameters to ensure it was always incorporated.

5.3 Discussion

The scope of this chapter was to provide a side-by-side comparison of two skin types that could serve as a replacement for human skin in in-vitro penetration studies. It is concluded from the data taken with three different CRS modalities, that pig skin was the most suitable model of those available in the absence of human tissue.  Therefore, overestimation of drug penetration into human skin by extrapolation from experiments with porcine skin appears unlikely. This is in accordance with published reports of an adequate correlation of skin penetration into human and porcine skin [147-148].

A comparison of the skin of white mice, which are frequently used in toxicological investigations, with the skin of domestic pigs revealed significant differences under the three CRS microscopy techniques of this study. This is in line with the report that the skin of unmodified white mice yields permeation coefficients substantially dissimilar to human skin [149].

Human reconstructed skin models could, in principle, offer an alternative, if the penetration barrier of these skin equivalents were similar to that of human skin. In fact, such skin equivalents have been suggested for penetration studies [150].  Continuous efforts to reconstruct human skin in vitro are reflected by numerous reports on the development of different culture systems and their assessment as penetration models [151-163].  Thus far, conclusions from these studies suggest the barrier properties of the model systems are weak when compared to fresh or frozen human abdominal skin; the quality of the barrier was, in most cases, equivalent, or even inferior, to that of mouse, rat or guinea-pig skin.

Because of striking histological similarities between human and pig skin, pig is recognized as the most suitable model to study the cutaneous delivery of medicine. Therefore, improving the knowledge on swine skin dendritic cell (DC) subsets would be highly valuable to the skin vaccine field. A study by Marquet, Bonneau, et al. [148] showed that pig skin DC comprise the classical epidermal Langerhans cells and dermal DC (DDC) that could be divided in 3 subsets according to their phenotypes: (1) the CD163neg/CD172aneg, (2) the CD163highCD172apos and (3) the CD163lowCD172apos DDC. These subtypes have the capacity to migrate from skin to lymph node since we detected them in pseudo-afferent lymph. Extensive phenotyping with a set of markers suggested that the CD163high DDC resemble the antibody response-inducing human skin DC/macrophages whereas the CD163negCD172low DDC share properties with the CD8+ T-cell response-inducing murine skin CD103pos DC. This work, by showing similarities between human, mouse and swine skin DC, establishes pig as a model of choice for the development of transcutaneous immunisation strategies targeting DC.  Finally, the skin structure similarities between man and pig, now comforted by immunological homologies, suggest the use of pig as a model of choice for the expanding field of transcutaneous vaccinations [148].

5.4 References 

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8. Carruthers, C. and V. Suntzeff, SOME CHEMICAL CHANGES INDUCED BY METHYLCHOLANTHRENE IN THE TRANSFORMATION OF MOUSE EPIDERMIS TO SOUAMOUS CELL CARCINOMA. Cancer Research, 1947. 7(1): p. 46-47.

9. Costello, C.J., C. Carruthers, and M. Kamen, UPTAKE OF P-32 IN THE PHOSPHOLIPID FRACTION OF MOUSE EPIDERMIS UNDERGOING CARCINOGENESIS BY METHYLCHOLANTHRENE. Cancer Research, 1946. 6(9): p. 486-486.

10. Costello, C.J., et al., THE UPTAKE OF RADIOPHOSPHORUS IN THE PHOSPHOLIPID FRACTION OF MOUSE EPIDERMIS IN METHYLCHOLANTHRENE CARCINOGENESIS. Cancer Research, 1947. 7(10): p. 642-646.

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21. Krasilnikova, N.V., [24-HOUR RHYTHM IN MITOTIC ACTIVITY IN REPARATIVE REGENERATION OF THE SALIVARY GLAND, LIVER AND EPIDERMIS OF WHITE MICE AND RATS]. Biulleten’ eksperimental’noi biologii i meditsiny, 1963. 56: p. 96-9.

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25. Rusch, H.P., D. Bosch, and R.K. Boutwell, THE INFLUENCE OF IRRITANTS ON MITOTIC ACTIVITY AND TUMOR FORMATION IN MOUSE EPIDERMIS. Acta Unio Internationalis Contra Cancrum, 1955. 11(6): p. 699-703.

26. Setaelae, K., E.E. Niskanen, and M. Nyholm, UNMASKING OF DORMANT CARCINOGENETIC TRAITS IN EPIDERMIS OF HIGHLY TUMOR-SUSCEPTIBLE SWISS MICE. Naturwissenschaften, 1961. 48(7): p. 226-&.

27. Setala, K., [The opposing action of carcinogen and tumor producers on keratinizing tissue (mouse epidermis)]. Verhandlungen der Deutschen Gesellschaft fur Pathologie, 1961. 45: p. 223-6.

28. Setala, K., et al., MECHANISM OF EXPERIMENTAL TUMORIGENESIS .6. ULTRASTRUCTURAL ALTERATIONS IN MOUSE EPIDERMIS CAUSED BY LOCALLY APPLIED CARCINOGEN AND DIPOLE-TYPE TUMOR PROMOTER. Journal of the National Cancer Institute, 1960. 25(5): p. 1155-&.

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34. Tatum, E.L., et al., VITAMIN CONTENT OF MOUSE EPIDERMIS DURING METHYLCHOLANTHRENE CARCINOGENESIS .1. BIOTIN, CHOLINE, INOSITOL, P-AMINOBENZOIC ACID, AND PYRIDOXINE. Journal of Biological Chemistry, 1946. 163(3): p. 675-682.

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36. Vinogradov, G.A., [Effect of hepatic reparative regeneration on the mitotic activity of the corneal epithelium and epidermis in mice]. Biulleten’ eksperimental’noi biologii i meditsiny, 1960. 50: p. 105-8.

37. Bullough, W.S. and M. Johnson, THE ENERGY RELATIONS OF MITOTIC ACTIVITY IN ADULT MOUSE EPIDERMIS. Proceedings of the Royal Society Series B-Biological Sciences, 1951. 138(893): p. 562-575.

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39. Cooper, Z.K. and H.C. Franklin, Mitotic rhythm in the epidermis of the mouse. Anatomical Record, 1940. 78(1): p. 1-8.

40. Elgjo, K. and K.L. Reichelt, PURIFICATION OF AN ENDOGENEOUS MITOTIC INHIBITOR (CHALONE) IN MOUSE EPIDERMIS. Cell and Tissue Kinetics, 1984. 17(3): p. 275-275.

41. Evensen, A., Changes in the synthesis of deoxyribonucleic acid (DNA) and in mitotic count in epidermis of hairless mice after a single application of one per cent 3:methyl-cholanthrene in benzene. A preliminary report. Acta pathologica et microbiologica Scandinavica. Supplement, 1961. Suppl 148: p. 43-52.

42. Green, H.N. and F.N. Ghadially, RELATION OF SHOCK, CARBOHYDRATE UTILIZATION, AND CORTISONE TO MITOTIC ACTIVITY IN THE EPIDERMIS OF THE ADULT MALE MOUSE. British Medical Journal, 1951. 1(4705): p. 496-498.

43. Hopsu, V.K. and M. Harkonen, Mitotic activity, succinic dehydrogenase and SH-groups in the epidermis and hair follicles in mouse skin throughout the hair growth cycle. Acta Pathologica Et Microbiologica Scandinavica, 1960. 48: p. 89-93.

44. Iversen, O.H., G. Harket, and R. Rabe, EFFECTS OF HYDROXYUREA ON THE MITOTIC-ACTIVITY AND THE G2 PHASE IN HAIRLESS MOUSE EPIDERMIS. Cell and Tissue Kinetics, 1986. 19(3): p. 257-265.

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