Exploitation of long-lasting ultraweak photon emission to estimate skin photodamage after ultraviolet exposure.

BACKGROUND: Establishing a noninvasive method to estimate skin damage immediately after ultraviolet (UV) exposure is required to minimize the anticipated severe symptoms triggered by early phase UV-induced reactions in the skin. To develop a suitable method, we focused on ultraweak photon emission (UPE) immediately after UV exposure to characterize the relationship of UPE to skin photodamage caused by the UV exposure. MATERIALS AND METHODS: Analysis of the correlation between UV-induced UPE and erythema formation characterized by skin redness was conducted in a clinical study. To clarify the source of UPE, time-dependent lipid oxidation was analyzed in human epidermal keratinocytes in vitro using a fluorescence indicator as well as the lipid hydroperoxide (LPO) assay. RESULTS: The average amount of UV-induced long-lasting UPE per second, especially from 1 to 3 minutes compared to other time periods after the UV radiation, increased in a dose-dependent manner and was highly correlated with the intensity of cutaneous redness 24 hours after UV exposure. In addition, cellular examinations elucidated that both the long-lasting UPE signals and the increased amounts of LPO 2 minutes after UV radiation were significantly suppressed by Trolox (a vitamin E derivative), which has been shown to inhibit UV-induced erythema formation in human skin. CONCLUSION: Long-lasting UPE generated between 1 and 3 minutes immediately after UV exposure, which is associated with LPO production, is a valuable indicator to estimate and/or avoid severe cutaneous photodamage.

Evaluation of unique elastic aggregates (elastic globes) in normal facial skin by multiphoton laser scanning tomography.

BACKGROUND: There is no reliable marker to estimate the degree of skin aging in vivo. It now has become possible to quantitatively determine the dermal characteristics of the extracellular matrix (ECM) in vivo using multiphoton laser tomography (MLT). METHODS: Fifty-seven healthy Japanese female volunteers, aged from 20 to 60 years old, were examined using multiphoton depth-resolved measurements of autofluorescence (AF) and second harmonic generation (SHG) at three sites on their right cheek. Paraffin-embedded skin specimens obtained from the faces of 12 normal individuals aged 38-68 years old were stained with Elastica van Gieson (EVG). RESULTS: We found unique elastic aggregates at a 20 µm depth from the dermo-epidermal junction (DEJ) in vivo which increased in size with aging of subjects from 20 to 60 years old. SHG fibers seemed to surround those elastic aggregates. Histological examination of specimens from normal individuals stained with EVG confirmed the occurrence of elastic aggregates with varied sizes just beneath the epidermis or hair follicles. CONCLUSIONS: The elastic aggregates are morphologically similar to previously described 'elastic globes' and can serve as a marker of the early stage of photoaging. MLT will contribute to determine age-related dermal changes using a non-invasive technique.

Seasonal and annual variation in the intensity of facial wrinkles.

BACKGROUND/AIMS: We studied annual, seasonal and gender-dependent changes in the intensity of facial wrinkles. METHODS: The study included 32 Japanese subjects (16 men, 16 women, aged 35-47). Wrinkles on the forehead, corner of the eye, lower eyelid and nasolabial groove of each subject were captured in photos and in replicas at 3-month intervals from March, 2009 to March, 2010. At each occasion, skin conductance in the stratum corneum and skin color were measured on the cheek. The photos obtained were used for visual scoring of wrinkle intensity, and the quantitative 3-D data of replicas were obtained. RESULTS: An apparent annual variation in skin conductance, which is supposed to be caused by skin dryness, and in the level of skin brightness, which is supposed to be caused by sun tan, was found for both genders. In contrast, no seasonal changes were determined in wrinkle intensity. Three-D analysis revealed no annual variation in women but revealed annual variation in the depths of eye wrinkles in men. CONCLUSION: There was a gender-dependent difference in the annual variation of wrinkle depth in subjects around 40 years of age. Seasonal environmental changes may affect on the eye wrinkles depth in men within a year.

Gender-dependent differences in degree of facial wrinkles.

BACKGROUND/PURPOSE: This study aimed to reveal gender-dependent differences in the degree of facial wrinkles. METHODS: Subjects comprised 173 Japanese men and women, divided into four groups according to age. Photographs were taken from nine facial regions and used to classify the intensity of wrinkles into five grades. In addition, replicas were taken from five facial sites and used to measure surface roughness. Data were compared between men and women within each age group. RESULTS: In all age groups, men showed increased forehead wrinkles compared with women. In contrast, no gender-dependent differences were found in upper eyelid wrinkles. Other facial wrinkles were greater in men than in women in all except the oldest group (age, 65-75 years), in which wrinkles in women were greater than or equal to those in men. CONCLUSION: Our results showed that gender-dependent differences exist in the degree of facial wrinkles. In general, men tend to have more severe wrinkles than women. This tendency disappeared or was reversed in some regions of the face and in individuals more than 60 years old.

A new method for the visualization and quantification of internal skin elasticity by ultrasound imaging.

BACKGROUND/PURPOSE: Skin elasticity has been assessed previously only in the surface layer. We developed a new method that uses tissue strain imaging (TSI) technology, and the aim of this study was to test this new method to assess internal skin elasticity. METHODS: Using a pressure device with a 12 MHz ultrasound transducer, constant and linear compressions were applied to the cheek skin of 35 volunteers (aged: 20-60 years). The elasticity of each layer (dermis, subcutaneous and muscle) was measured and analyzed using the TSI application software incorporated into the Toshiba Aplio(™) XV ultrasound system. A skin tissue-equivalent phantom, which is a block of material with the acoustic velocity (1530 m/s) of human skin, was collaboratively developed by OST Inc. This phantom was placed between the skin and the transducer as a reference material. RESULTS: Skin elasticity was clearly visualized and quantified in each layer of the skin. Age-dependent decreases in elasticity were determined in all layers of the skin. Among the three internal skin layers, the highest elasticity was determined in the subcutaneous layer followed by the muscle layer. CONCLUSION: These findings support the validity and sensitivity of the TSI method to assess the elasticity of various layers of skin.

Relationship between the echogenicity of subcutaneous tissue and the depth of forehead wrinkles.

BACKGROUND/PURPOSE: Subcutaneous tissue is rarely studied in research on wrinkles. We used diagnostic ultrasonography to produce images of subcutaneous tissue of the forehead, one of the areas where age-related wrinkles form. We quantified the resulting echogenicity and investigated its relationship with wrinkle depth. METHODS: One hundred and seventy-three Japanese subjects were divided into four age groups (21-28 years, 35-41 years, 47-59 years, 65-75 years). Maximum wrinkle depth (Rmax) was measured in the forehead. On an ultrasound image, which was captured by scanning perpendicular to the deepest wrinkle, we set a 12.5-mm-wide region of interest by aligning the deepest point of the wrinkle at its center and then obtained the ratio of the area exceeding this threshold to the total area as the echogenicity. RESULTS: R max increased and the echogenicity decreased with age. A negative correlation was found in the oldest group but was not found in other groups, presumably due to individual differences in the echogenicity among younger subjects. CONCLUSION: This study suggests that the internal structure of subcutaneous tissue, as observed by ultrasonography, is related to wrinkle depth. The relationship between wrinkle depth and echogenicity of subcutaneous tissue is particularly pronounced in the elderly.

Relationship between the echogenicity of subcutaneous tissue and the depth of eye corner wrinkles.

BACKGROUND/PURPOSE: A close relationship was found between the internal structure of subcutaneous tissue and wrinkle depth in our previous study of human forehead. The present study examined the relationship in the eye corner. METHODS: One hundred and seventy-three Japanese subjects were divided into four age groups (21-28 years, 35-41 years, 47-59 years and 65-75 years). The maximum wrinkle depth (Rmax) of an eye corner wrinkle was obtained. On an ultrasound image, which was captured by scanning perpendicular to the wrinkle, we set a 12.5-mm-wide region of interest (ROI) by aligning the deepest point of the wrinkle at its center and then obtained the ratio of the area exceeding an echogenicity threshold to the total area as the echogenicity. RESULTS: No correlation was found between the Rmax and echogenicity. The relationship was further analyzed by dividing the ROI into two: one above and the other below the deepest point of the wrinkle. A negative correlation was detected in the lower ROI of the oldest group. The difference in echogenicity between the upper and the lower ROIs of the oldest group showed a positive correlation with the Rmax. CONCLUSION: These results suggest that the subcutaneous tissue structure is related to wrinkle depth.

Evaluation of photoaging in facial skin by multiphoton laser scanning microscopy.

BACKGROUND/PURPOSE: It has been reported that autofluorescence (AF) and second harmonic generation (SHG) generated in the upper dermis are related with skin photoaging. In this study, we assessed the photoaging of facial skin exposed to daily sunlight using in vivo multiphoton laser microscopy to measure AF and SHG. METHODS: The intensities of AF and SHG in the upper dermis of cheek skin of 56 healthy volunteers aged 20-69 years were measured using a commercially available multiphoton laser microscope (DermaInspect(®) ). Correlations between the photo-signals and volunteer age were calculated. RESULTS: The intensity of SHG and the SHG-to-AF aging index of dermis (SAAID) correlated significantly with age (r=-0.48, -0.67, respectively). CONCLUSION: Our results suggest that SHG and the SAAID index are useful indicators of facial skin aging in vivo.

Imaging of melanin distribution using multiphoton autofluorescence decay curves.

BACKGROUND/PURPOSE: Multiphoton fluorescence lifetime imaging (FLIM) is a technique that produces an image based on differences in the decay rate of fluorescence from a sample. Based on this method, the DermaInspect was developed to observe human skin components non-invasively. In this study, we used the DermaInspect to study melanin in skin. METHODS: A human three-dimensional skin model containing melanocytes was embedded in an OCT compound, frozen and sectioned at 10 microm. The melanin distribution in each section was visualized by the DermaInspect using time-resolved single-photon counting and near-infrared femtosecond laser pulse excitation. The melanin distribution of the same sections was then visualized using the Fontana-Masson staining method. RESULTS: High-resolution images were generated from the ratio of a(1)/a(2) (a(1)e(-) (t/120)+a(2)e(-) (t/1100) was chosen to express the exponential fluorescent decay curve) obtained using the DermaInspect. Granules with a high a(1)/a(2) ratio, approximately 1 mum in diameter, were observed. Fontana-Masson staining identified these granules as melanin. This new technique was then applied for in vivo observation of melanin in human skin. 'Melanin caps' were visualized in the basal cell layer around the nuclei in images derived from the a(1)/a(2) ratio. CONCLUSION: Our study confirms that FLIM can non-invasively provide data of the melanin distribution with almost the same quality as the conventional Fontana-Masson staining method, and demonstrates that FLIM is useful for in vivo observation of melanin granules in human skin.

The effect of eye opening and closing on the result of facial wrinkle assessment.

BACKGROUND/PURPOSE: Since there is no standard protocol that specifies the opening or closing of eyelids in wrinkle assessment, the eyelid position of subjects at the time of assessment varies from study to study. This study aimed to reveal the effect of eye opening and closing on the result of wrinkle assessment. The study also attempted to analyze the age-, site-, and ethnicity-dependent variations in the effect. METHODS: Open- and closed-eyed photographs were obtained from 87 Japanese women of ages ranging from 21 to 73 years, and also from 80 American women comprising of Asians, Caucasians, Africans and Hispanics in their 30s. Their wrinkle intensities were scored separately at nine facial sites using a five-point photo scale. The obtained scores were compared between open- and closed-eyed photographs. The differences were then compared across age- and ethnic-groups. RESULTS: In Japanese subjects, a significant difference was found at the forehead wrinkle in all age groups, and also at the glabella, nasal root and eye corner in the middle and old age groups. In American subjects, significant differences were found at the forehead, nasal root, eye-corner, and upper and lower eyelids. The differences were more prominent in the Hispanic and Caucasian Americans than in the Asian and African Americans. CONCLUSION: These results suggest that the eyelid position should be considered as a variable in wrinkle assessment.

Ethnic differences in the structural properties of facial skin.

BACKGROUND: Conspicuous facial pores are one type of serious aesthetic defects for many women. However, the mechanism(s) that underlie the conspicuousness of facial pores remains unclear. We previously characterized the epidermal architecture around facial pores that correlated with the appearance of those pores. OBJECTIVES: A survey was carried out to elucidate ethnic-dependent differences in facial pore size and in epidermal architecture. METHODS: The subjects included 80 healthy women (aged 30-39: Caucasians, Asians, Hispanics and African Americans) living in Dallas in the USA. First, surface replicas were collected to compare pore sizes of cheek skin. Second, horizontal cross-sectioned images from cheek skin were obtained non-invasively from the same subjects using in vivo confocal laser scanning microscopy (CLSM) and the severity of impairment of epidermal architecture around facial pores was determined. Finally, to compare racial differences in the architecture of the interfollicular epidermis of facial cheek skin, horizontal cross-sectioned images were obtained and the numbers of dermal papillae were counted. RESULTS: Asians had the smallest pore areas compared with other racial groups. Regarding the epidermal architecture around facial pores, all ethnic groups observed in this study had similar morphological features and African Americans showed substantially more severe impairment of architecture around facial pores than any other racial group. In addition, significant differences were observed in the architecture of the interfollicular epidermis between ethnic groups. CONCLUSIONS: These results suggest that facial pore size, the epidermal architecture around facial pores and the architecture of the interfollicular epidermis differ between ethnic groups. This might affect the appearance of facial pores.

Development of a novel method to measure the elastic properties of skin including subcutaneous tissue: new age-related parameters and scope of application.

BACKGROUND/PURPOSE: We have developed a new apparatus to measure the elastic properties of skin without contact using an air blown technique. METHODS: Real-time measurements of skin movement induced by air blown on the surface were measured at various times. We investigated age-related changes in the displacement of the skin surface caused by air using various parameters. Ninety-eight female Japanese volunteers, aged from 10 to 70 years, were used in this study. RESULTS: The maximum distance (the denting state) that cheek skin moved reached 2-5 mm within 10-15 ms. After that, the skin generally recovered to its original state within 40-50 ms. The average maximum speed of movement was 0.49+/-0.08 (average+/-SD) m/s and the average recovery speed was 0.25+/-0.06 m/s. Significant changes with age were not observed in the denting state, but significant correlations with age were observed in the recovery state. The maximum recovery speed decreased significantly with age and the time required for recovery increased significantly with age. Although similar results were obtained at the corner of the mouth or the inner upper arm, different results were obtained at the below eyes and the forehead. CONCLUSIONS: This apparatus differs from other conventional instruments in that it measures the elastic properties of skin including subcutaneous tissue without contact.

Comparison of age-related changes in facial wrinkles and sagging in the skin of Japanese, Chinese and Thai women.

BACKGROUND: Differences in skin aging features between Asians and Caucasians are commonly known, whereas little is known about such differences in various Asian populations. OBJECTIVE: A survey was carried out in Tokyo, Shanghai and Bangkok to identify specific features of skin aging in each population and to evaluate whether our conventional photo scale is an appropriate tool for this type of comparative study. METHODS: Eighty-seven women residing in Tokyo, 100 women residing in Shanghai, and 90 women residing in Bangkok were examined by a specialist. Facial wrinkles (forehead, glabella, upper eyelid, crow's feet, lower eyelid, cheek, nasolabial groove and mouth corner) and cheek sagging were evaluated using photo scales previously obtained from Japanese subjects. Comparisons were made according to 10-year age groups. RESULTS: Women in Bangkok showed the most severe level of wrinkles, followed by those in Shanghai in the three groups. Significant differences were observed between Thai and Japanese women in the intensity of wrinkles at many facial sites. Chinese women had significantly more severe wrinkles in the area around the eyes compared to Japanese women, while Thai women had significantly more severe wrinkles in the lower halves of their faces compared to Chinese women. In cheek sagging scores, significant differences were observed between Japanese and Thai women in their 30s and 50s, but not between Japanese and Chinese women or between Chinese and Thai women in all age groups. CONCLUSION: These results indicate variations in skin aging features among women from three Asian cities thereby suggesting the diversity of Asian skin. Our scaling method proved to be appropriate for facial wrinkles, but required modification to compare cheek sagging among Asian populations.