Enlarged rete pegs with excessive accumulation of melanosomes leading to darker aging spots revealed by histomorphological measurements of internal structures of the epidermis.

OBJECTIVE: Various histological studies of facial pigmented spot sites such as solar lentigo have been reported, but few studies have used quantitative indices by histomorphometric analysis of the internal structure of pigmented spot sites using non-invasive methods. In the present study, to quantitatively elucidate morphological changes in the epidermis in male, darker-pigmented spots and female, light-pigmented spots, indices that characterize the internal structure of the epidermis in pigmented spot sites were measured using in vivo confocal laser scanning microscopy (CLSM). METHODS: The darkness of pigmented spots on the cheeks of 69 women and 43 men was analysed using image analysis software. The L* value was calculated from RGB values obtained from facial images. The internal structures of pigmented spots on the cheeks of 13 subjects were observed by CLSM. Various parameters were measured using CLSM images from the surface of the stratum corneum to the bottom of the dermal papillae, including the thickness of the epidermis, melanosome content, and shape of the dermal papillae. RESULTS: Mean ΔL* values between pigmented spots and non-pigmented areas of male subjects were significantly increased in the 40s and 50s compared with those of female subjects. Conspicuous pigmented spots increased in the 40s in male subjects and the 50s in female subjects. In CLSM observations, significant increases in the thickness of the epidermis and melanosome content were confirmed in pigmented spots compared with surrounding non-pigmented areas. In particular, melanosome content in the male subject group with dark-coloured pigmented spots increased significantly to about eight times that of non-pigmented areas, and more than double that of the male subject group with light-coloured pigmented spots. CONCLUSION: From the measurements of quantitative parameters, morphological changes in the epidermis were clearly related to the surface colour tone of pigmented spots. Darker pigmented spot sites tended to show longer rete pegs in the epidermis. Accumulation of melanosomes in epidermal basal cells could be considered to increase with the degree of elongation of rete pegs at pigmented spot sites and, thus, induce darker pigmented spots.

OBJECTIF: Même si diverses études histologiques des taches pigmentées du visage, tels que les lentigos solaires, ont été publiées, il n'existe que peu d'études ayant utilisé des indices quantitatifs par analyse histomorphométrique de la structure interne des taches pigmentées via des méthodes non invasives. Dans la présente étude, afin d'expliquer quantitativement les changements morphologiques dans l'épiderme des taches pigmentées plus foncées chez l'homme et des taches pigmentées légères chez la femme, les indices qui caractérisent la structure interne de l'épiderme dans les taches pigmentées ont été mesurés par microscopie confocale à balayage laser (MCBL) in vivo. MÉTHODES: L'aspect foncé des taches pigmentées sur les joues de 69 femmes et 43 hommes a été analysé à l'aide d'un logiciel d'analyse d'images. La valeur L* a été calculée à partir des valeurs RVB obtenues des images du visage. Sur les joues de 13 sujets, les structures internes des taches pigmentées ont été observées par MCBL. Divers paramètres ont été mesurés à l'aide des images provenant de la MCBL, de la surface de la couche cornée jusqu'au bas des papilles dermiques, y compris l'épaisseur de l'épiderme, la teneur en mélanosome et la forme des papilles dermiques. RÉSULTATS: Les valeurs moyennes de ΔL* entre les zones de taches pigmentées et non pigmentées des hommes ont augmenté de manière significative chez les sujets dans la quarantaine et la cinquantaine par rapport aux valeurs des femmes. Chez les hommes, les taches pigmentées visibles ont augmenté dans la quarantaine, tandis qu'elles ont augmenté dans la cinquantaine chez les femmes. Dans les observations par MCBL, des augmentations significatives de l'épaisseur de l'épiderme et de la teneur en mélanosome ont été confirmées dans les zones de taches pigmentées par rapport aux zones de taches non pigmentées environnantes. Dans le groupe d'hommes présentant des taches pigmentées de couleur foncée en particulier, la teneur en mélanosomes a augmenté de façon significative jusqu'à environ 8 fois celle des zones non pigmentées, et jusqu'à plus du double de celle du groupe d'hommes présentant des taches pigmentées de couleur claire. CONCLUSION: D'après les mesures des paramètres quantitatifs, les changements morphologiques dans l'épiderme étaient clairement liés à la couleur à la surface des taches pigmentées. Les sites de taches pigmentées plus foncées montraient généralement des extensions des crêtes épidermiques dans l'épiderme. On pourrait envisager que l'accumulation de mélanosomes dans les cellules basales épidermiques augmente selon le degré d'allongement des crêtes épidermiques au niveau des sites de taches pigmentées, et entraîne ainsi des taches pigmentées plus foncées.

Evaluation of intercellular lipid lamellae in the stratum corneum by polarized microscopy.

BACKGROUND: Intercellular lipids contain a lamellar structure that glows in polarized images. It could be expected that the intercellular lipid content be estimated from the luminance values calculated from polarized images of stratum corneum strips. Therefore, we attempted to develop a method for simple and rapid evaluation of the intercellular lipid content through a procedure. Herein, we demonstrated a relationship between the luminance value and the amount of ceramides, one of the main components of intercellular lipids. MATERIALS AND METHODS: The stratum corneum was collected from the forearm using slides with a pure rubber-based adhesive, which did not produce unnecessary luminescence under polarizing conditions. Images were analyzed using luminance indices. The positive secondary ion peak images were obtained using the time of flight-secondary ion mass spectrometry; the polarized and brightfield images were obtained using a polarized microscope. The ceramide and protein amount was measured by high-performance liquid chromatography and bicinchoninic acid protein assay after microscope imaging. Images and quantitative values were used to construct evaluation models based on a convolutional neural network (CNN). RESULTS: There was a correlation between the highlighted areas of the polarized image to overlap with the area where ceramide-derived peak was detected. Evaluation of the CNN-based model of the polarized images predicted the amount of ceramides per unit of stratum corneum. CONCLUSION: The method proposed in the study enabled a large number of specimens to provide a simple, rapid, and efficient evaluation of the intercellular lipid content.

Antimelanogenic activity of hydrocoumarins in cultured normal human melanocytes by stimulating intracellular glutathione synthesis.

The antimelanogenic activity of six hydrocoumarins and alpha-tocopherol (alpha-Toc) in normal human melanocytes was evaluated in both cell culture systems and cell homogenates. The inhibitory effects of hydrocoumarins depended upon their substituent groups. alpha-Toc and some of the hydrocoumarins inhibited melanogenesis in cultured normal human melanocytes, although they did not influence melanin synthesis in enzyme solution prepared as cell homogenates. In addition, alpha-Toc and the hydrocoumarins stimulated intracellular glutathione (GSH) synthesis. In particular, 7-allyl-6-hydroxy-4,4,5,8-tetramethylhydrocoumarin strongly inhibited melanogenesis and intracellular GSH synthesis in normal human melanocytes, more so than alpha-Toc. Furthermore, hydrocoumarins exhibited higher scavenging and quenching activities against with tert-butyl peroxyl radicals and singlet oxygen species. These results suggest that 7-allyl-6-hydroxy-4,4,5,8-tetramethyl hydrocoumarin would be useful as an antimelanogenic agent for the prevention or improvement of skin pigmentation induced by reactive oxygen compounds and free radicals, and may inhibit melanogenesis, including tyrosinase transfer and melanosome differentiation, by interrupting melanization by increasing the intracellular GSH content.