Tetsuji Hirao

In vivo estimation of stratum corneum thickness from water concentration profiles obtained with Raman spectroscopy.

Stratum corneum thickness was estimated from water concentration profiles of the skin measured by a confocal Raman spectrometer. Stratum corneum apparent thickness (SCAT) was defined as the depth where the water content reached an almost constant value. Site variations were determined using 15 healthy Japanese subjects (6 males, 9 females), and age variations at the cheek and forearm were examined using 27 female Japanese subjects. There were marked site variations in mean SCAT; 16.8 microm for cheek, 22.6 microm for volar forearm, 29.3 microm for back of the hand, and 173.0 microm for palm. These variations were similar to reported stratum corneum thickness values obtained in biopsy tissues. The SCAT tended to become age-dependently thicker at the forearm, but not at the cheek. In addition, SCAT was increased up to two-fold by hydration for 90 min, while lesser increases were seen with shorter hydration periods.

Identification of immature cornified envelopes in the barrier-impaired epidermis by characterization of their hydrophobicity and antigenicities of the components.

Abstract: Cornified envelopes (CEs), rigid and insoluble structures in the stratum corneum, which are assembled by crosslinking of several precursor proteins by transglutaminases, provide a hydrophobic foundation for barrier function; omega‐hydroxyceramides are covalently attached to the outer surface of CE components, and onto this hydrophobic assembly, lamellar layers of intercellular lipids are organized. Morphologically irregular, fragile CEs are found in the deep layer of the stratum corneum or in certain disorders, such as psoriasis, whereas most CEs from healthy subjects are rigid and polygonal. We have established a staining method to characterize such fragile CEs as immature and less hydrophobic CEs, and employed it to examine regional differences in the properties of CEs, especially in relation to the barrier function of the skin. CEs from the outermost stratum corneum of the trunk and extremities of healthy subjects were relatively uniform in morphology with larger shape, and were homogeneous in hydrophobicity as judged from the use of an environment‐sensitive fluorescent dye, Nile red. However, CEs from the face were strikingly heterogeneous, and consisted of both rigid and fragile CEs. Rigid CEs were Nile red‐positive and little stained by anti‐involucrin. In contrast, fragile CEs were Nile red‐negative but strongly stained with anti‐involucrin, as detected by indirect immunofluorescence. Thus, CEs from the face were stained with Nile red or involucrin in a mutually exclusive manner. Fragile CEs were stained with antibodies against other CE components, including loricrin, envoplakin, filaggrin, and isopeptides. Such fragile, involucrin‐positive CEs were detected not only in the face, but also in the deep layer of the stratum corneum of the arm. In addition, experimental barrier disruption resulted in the appearance of involucrin‐positive CEs in the outermost stratum corneum. These results suggest that involucrin‐positive, fragile CEs are immature and less hydrophobic, and that their occurrence is closely related to impairment of the barrier function of the skin

Improvement of mild inflammatory changes of the facial skin induced by winter environment with daily applications of a moisturizing cream. A half-side test of biophysical skin parameters, cytokine expression pattern and the formation of cornified envelope.

Objective: Based on our previous findings that, reflecting mild inflammation, the exposed facial skin shows much poorer functional properties of the stratum corneum (SC) in the dry and cold winter than those evaluated in the same individuals in the warm and humid summer time, we conducted a half-side test on the face to determine how the facial skin changes induced by a winter environment are improved by daily applications of a moisturizing cream as assessed by non-invasive biophysical and cytological methods. Methods: One side of the face of 16 young females was treated with a moisturizing cream twice daily for 6 weeks, with the other side serving as the non-treated control. Before treatment, 3 and 6 weeks after the start of the treatment, high-frequency conductance as a parameter of the skin surface hydration, transepidermal water loss (TEWL), a parameter of the water barrier function of the skin, and the skin surface lipid level were measured on the cheeks. Obtaining the SC from the skin surface by adhesive tape, interleukin (IL) 1α and IL-1 receptor antagonist (IL-1ra) in the SC and cornified envelope (CE) maturation were determined. Results: At first, baseline measurements conducted before treatment showed rather high TEWL values suggestive of an impaired skin barrier. During the treatment with the moisturizing cream, significantly higher conductance values and lower TEWL values were found on the moisturizer-treated side, accompanied by a decreasing IL-1ra/IL-1α ratio and immature CEs. Conclusion: These results suggested that the daily application of a moisturizing cream is effective in improving mild subclinical inflammation that is induced on the facial skin by the winter environment.

The evaluation of skin friction using africtional feel analyzer

Background/aims: Sensory evaluation is an important factor for cosmetic products. Several devices for the measurement of sensory properties have been developed in recent years. The objective here is to measure skin surface friction using these devices and examine the correlation with other physiological parameters in order to evaluate the potential of physical measurement of tactile sensation.

A simple and non‐invasive visualization for assessment of carbonylated protein in the stratum corneum

Background/purpose: Stratum corneum (SC) is the interface of body and environment and is continuously exposed to oxidative stress, resulting in oxidative modification of proteins. Consequent carbonylated proteins (CPs) have so far been labeled with 2,4‐dinitrophenyl (DNP) hydrazine and subsequently detected with anti‐DNP antibody. We developed a simpler, non‐invasive method to assess CP level in the SC and applied it to following research.

Stratum corneum lipid profile and maturation pattern of corneocytes in the outermost layer of fresh scars: the presence of immature corneocytes plays a much more important role in the barrier dysfunction than do changes in intercellular lipids

Background  The functional characteristics of the stratum corneum (SC) of fresh scars as well as keloids and hypertrophic scars are characterized by elevated transepidermal water loss (TEWL) and increased SC hydration.

Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy

Optical probe methods for in vivo assessments of cutaneous photoaging are necessary in fields such as anti-aging dermatology and skin cosmetic development. We investigated the relation between wrinkle direction and collagen orientation in ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved second-harmonic-generation (SHG) microscopy. A polarization anisotropic image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation of dermal collagen fibers. Furthermore, collagen orientation in post-UVB-exposed skin with few wrinkles changed from that of UVB-exposed wrinkled skin to that of no-UVB-exposed skin. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous photoaging.

Ratio of immature cornified envelopes does not correlate with parakeratosis in inflammatory skin disorders.

Abstract:  We have previously established a non‐invasive method to evaluate the maturity of cornified envelopes (CEs), and have reported the appearance of immature CEs in the stratum corneum (SC) with poor barrier function, such as the SC of the face. The purpose of the present study was to evaluate CEs in inflammatory skin disorders, and to clarify the relationship between the appearance of the immature CEs and parakeratosis, which is often used as a marker for defective keratinization in inflammatory skin disorders.

In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser

Abstract. In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.

Regional Difference of Water Content in Human Skin Studied by Diffuse-Reflectance Near-Infrared Spectroscopy: Consideration of Measurement Depth

Diffuse reflectance (DF) spectra in the 1250–2500 nm region were measured in vivo for the skin of the forehead, cheek, jaw, elbow, volar forearm, palm, knee, and heel of seven healthy volunteers, using a Fourier transform near-infrared (FT-NIR) spectrophotometer with a fiber-optic probe. Apparent regional differences of water content in the skin, as estimated from the diffuse reflectance NIR spectra, are discussed in relation to the influence of measurement depth. The NIR spectra were collected with or without a 300 μm gap between the fiber-optic probe and the skin surface. For comparison, in vitro NIR spectra of stratum corneum sheets equilibrated at 41, 50, 63, and 81% relative humidity, at 25 °C, were also obtained. There was a difference in the ratio of the two water bands centered near 1450 nm and 1900 nm between the contact and non-contact measurements. In addition, regional differences of water content calculated from the peak height of the 1900 nm water band, which was normalized to the peak height of the 2175 nm amide band, were compared. The results of Monte Carlo simulation indicated that the apparent regional differences arise at least in part from differences in the measurement depth due to differences in specular reflection at the skin surface and in the thickness of the stratum corneum.