In vivo evaluation of the protective capacity of sunscreen by monitoring urocanic acid isomer in the stratum corneum using Raman spectroscopy

Background/purpose: Urocanic acid (UCA) is a major ultraviolet (UV) ray‐absorbing component in the epidermis, and it isomerizes from the trans to the cis form upon exposure to UV radiation. Continuous measurement of UCA isomers in the skin at the same area is not available using conventional methods. This study aimed to evaluate the protective capacity of sunscreen by non‐invasively monitoring the trans‐UCA (t‐UCA) amount in the stratum corneum (SC) by confocal Raman spectroscopy. Methods: In vivo Raman spectra of the skin at the cheek or volar forearm were obtained from 27 healthy Japanese volunteers of different ages (age range, 22–53 years) throughout a whole year using confocal Raman spectroscopy. Eighteen healthy male Japanese volunteers (age range, 25–52 years) were enrolled for the evaluation of the protective capacity of sunscreen. The concentration depth profile of t‐UCA relative to keratin was calculated from the Raman spectra in the 400–2200 cm⁻¹ region. Then, the integrated amount within the depth of 0–12 μm was calculated, which represented the total amount of t‐UCA in the SC. Results: The integrated amount of t‐UCA in the cheek skin was significantly lower than that in the volar forearm skin throughout a year. The amount of it in the volar forearm skin was significantly the lowest in summer, but not in the cheek skin. The amount of t‐UCA decreased immediately after UV exposure even below 1 minimal erythema dose, remained low for 1 week, and gradually increased up to the initial level about 2 weeks after UV exposure. The decrease in the t‐UCA amount was hindered by the application of sunscreen on the skin surface. There were no statistical differences in response to UV exposure between the erythema‐positive and erythema‐negative groups. Conclusions: The monitoring of the amount of t‐UCA in the SC by confocal Raman spectroscopy is a good method to assess the efficacy of sun protective substances.     

Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration

BACKGROUND: Dermatologists and cosmetic scientists are becoming increasingly interested in stratum corneum (SC) hydration because the SC plays an important role in keeping the skin surface soft and smooth. However, conventional in vivo noninvasive methods do not provide direct information about the depth profiles of water content or SC components that hold water. OBJECTIVES: To study the depth profiles of water and SC components in vivo by Raman spectroscopy, and to analyse the changes due to age, anatomical location, season and water application. METHODS: In vivo Raman spectra of the skin of the cheek and the volar forearm were obtained from 50 healthy Japanese volunteers of different ages (age range 22-76 years) with a confocal Raman spectrometer. The depth-dependent profiles of water and of SC water-binding components were calculated from the respective Raman spectra. RESULTS: The depth profile of the SC water content was observed in a pattern ranging from around 30% (water/wet tissue) at the outermost layer of the SC to about 70% at the deeper living layer. Although the water content at a depth of 10-30 microm in the forearm skin tended to be lower in older subjects than in younger subjects, no such difference was found in the much thinner SC of the cheek. Moreover, there was no seasonal difference in depth profile of water content from the mid part of the SC to an 80-microm depth from the skin surface both in the cheek and in the forearm. The water content of all the evaluated SC components showed a gradual decrease from the surface to deeper portions. The mean amounts of lactate in the forearm skin and cholesterol in the cheek skin were significantly higher in younger subjects than in older subjects. In contrast, the levels of free amino acids and trans-urocanic acid were higher in the forearm skin of older subjects than in younger subjects. The relative amounts of urea and lactate were the highest in summer, when that of trans-urocanic acid was the lowest. Prolonged water application on the forearm skin even for 90 min resulted in a remarkable increase in water content throughout the SC, even reaching the granular layer, which was only gradually released from the upper part of the SC after discontinuation of the hydration procedure. CONCLUSIONS: Our present findings suggest that changes in the concentration depth profiles of water, free amino acids and lipids in the skin depend on age, anatomical site and season. These findings indicate the important roles played by various water-holding substances in the SC in the regulation of SC water content.     

Measuring the effects of topical moisturizers on changes in stratum corneum thickness, water gradients and hydration in vivo

Background Moisturizers are the most commonly used topically applied product for the treatment of dry skin conditions. They affect many properties and functions of the stratum corneum but some moisturizers have been reported to be detrimental to barrier function. Stratum corneum barrier function is a composite of its total structure and thickness but few studies have taken this into account. As a biosensor, the stratum corneum (SC) will change its structure in response to treatment and a swelling effect has been clearly demonstrated by skin hydration. Recently several moisturizing agents have been shown to have an effect on SC swelling behaviour with conflicting results. However, there is a paucity of data reported for measuring the effects of long‐term usage of moisturizers on SC thickness in vivo as, until recently, traditional techniques did not have the resolution to measure the effects of moisturizers on nonpalmoplantar body sites. The development of confocal Raman spectroscopy for use in human subjects provides noninvasive, real‐time, in vivo measurement of SC water concentration profiles and we have also used this state of the art equipment to measure the effect of the long‐term use of moisturizers on SC thickness for the first time. Objectives To validate the use of confocal Raman spectroscopy (CRS) to measure SC thickness and then use it to investigate the short‐ and long‐term effects of moisturizers (one of which is known to improve SC barrier function) on SC thickness, water gradients and hydration. Methods Two studies were conducted: (i) to validate the use of CRS for measuring SC thickness through comparison with optical coherence tomography (OCT); and (ii) once validated to use CRS to measure the long‐term effects of three commercially available moisturizers (A, B, C) on SC thickness and water gradients, together with total hydration, over a 3‐week period (2 weeks of treatment and 1 week regression) and compare the spectroscopy‐derived hydration value with instrumentally derived capacitance hydration values. Results (i) A strong, positive correlation in SC thickness was obtained between CRS and OCT (OCT‐derived thickness = 0·96 × CRS‐derived thickness, r² = 0·93; P <0·0001). OCT was shown, however, to have a lower resolution than CRS in distinguishing SC thickness on thinner nonpalmoplantar body sites. Using the CRS method, differences in SC thickness were readily apparent on different body sites (cheek 12·8 ± 0·9 μm, volar forearm 18·0 ± 3·9 μm, leg 22·0 ± 6·9 μm). (ii) Examining the effects of moisturizers in a blinded, randomized 3‐week study in human volunteers (n = 14) demonstrated that only one commercially available formulation (A) changed SC water gradients, thickness and hydration as measured by CRS. These hydration data did not directly correlate with capacitance hydration values. Conclusions (i) In vivo CRS was validated as a technique to measure SC thickness on both palmoplantar and, particularly, on nonpalmoplantar skin sites. (ii) Moisturizers improve skin moisturization but in this study only formulation A improved SC thickness, water gradients and hydration as measured by CRS. We hypothesize that this was due to compositional differences between the products. We believe that niacinamide (nicotinamide, vitamin B₃) is probably contributing significantly to this effect, as it has been proven to increase epidermal lipogenesis and SC barrier function in other studies. These results show that by using CRS, we were able for the first time to determine the effect of moisturizer on multiple SC barrier endpoints including SC thickness, and water content as a function of depth and total SC water content.     

Changes of human skin in subepidermal wound healing process

Background/purpose: The wound healing process involves unexplained mechanisms. An aberration in this process is known to cause dermal disorders such as keloid or hypertrophic scars, but the mechanism by which these scars are formed remains to be elucidated. Here we examined the usefulness of a non‐invasive optical imaging device to clarify mechanisms of wound healing and of scar formation. Methods: An 8 mm experimental wound was made in the forearms of six subjects by a suction blister method. To observe chronological changes associated with wound healing, horizontal cross‐sectional images were non‐invasively obtained of the wounded area from the skin surface down to 129 μm below at 21.5 μm intervals using in vivo laser confocal scanning microscopy (LCSM). Results: The wounds were covered with a new epidermis by week 2, at which time the dermal papilla count decreased while the thickness from the skin surface to the apex of the dermal papilla increased. The count and the thickness returned to the initial levels when the wound was healed. In two out of six subjects, fibrous tissues were observed in the upper dermis, whereas in one other subject, melanocyte‐like dendritic cells were observed in the epidermis–dermis border in later phases of wound healing. Conclusion: This non‐invasive method using in vivo LCSM revealed chronological changes in the dermis and epidermis during wound healing. In addition, although a scar was not formed in any of study subjects, this microscopy revealed aspects similar to the fibrous tissue overgrowth or to melanocyte migration, both of which may relate to wound healing. These results indicate the usefulness of this non‐invasive method in studies of wound healing and of scar formation.     

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography

Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography. Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth‐resolved intensity curves after correlation with multiphoton images. Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.     

Integrated real‐time Raman system for clinical in vivo skin analysis

Background: Raman spectroscopy is a non‐invasive optical technique that can probe the molecular structure and conformation of biochemical constituents. The probability of Raman scattering is exceedingly low (∼10⁻¹⁰), and consequently up to now the practical application of Raman spectroscopy to clinical medicine has been limited by either the weak spectral signal or by the long data acquisition times. Recent advances in Raman hardware and probe design have reduced spectral acquisition times, paving the way for clinical applications. Methods: We present an integrated real‐time Raman spectroscopy system for skin evaluation and characterization, which combines customized hardware features and software implementation. Real‐time data acquisition and processing includes CCD dark‐noise subtraction, wavelength calibration, spectral response calibration, intensity calibration, signal saturation detection, cosmic ray rejection, fluorescence background removal, and composition modeling. Real‐time in vivo Raman measurement of volar forearm skin is presented to illustrate the methods and modeling. Results: The system design implemented full‐chip vertical hardware binning to improve the signal‐to‐noise ratio by 16‐fold. The total time for a single in vivo measurement with analysis can be reduced to 100 ms with this implementation. Human skin was well modeled using the base Raman spectra. Conclusion: In vivo real‐time Raman can be a very promising research and practical technique for skin evaluation.     

Morphological skin ageing criteria by multiphoton laser scanning tomography: non-invasive in vivo scoring of the dermal fibre network

Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. Methods: In vivo MLT was used to image two‐photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age‐dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. Results: The resulting MLT‐based Dermis Morphology Score is correlated with age (R² = ?0.90) and with the previously published SHG to AF Ageing Index of Dermis (R² = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001). Conclusions: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi‐quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.     

Non‐invasive imaging of mid‐dermal elastolysis

Mid‐dermal elastolysis (MDE) is a rare disease that is characterized histopathologically by selective loss of elastic tissue in the mid dermis. We aimed to assess MDE using noninvasive skin imaging techniques in vivo. We examined both the lesional and adjacent healthy skin of a woman with the reticular variant of MDE, using confocal scanning laser microscopy, optical coherence tomography (OCT) and high‐frequency ultrasound (HFUS). The median diameter of blood vessels at the top of dermal papillae was significantly increased in erythematous lesional skin compared with healthy skin. The mid‐dermal signal intensity detected by OCT was higher in healthy skin than in lesional skin. With HFUS, mid‐dermal density values were significantly higher in healthy skin than in lesional skin. Our preliminary findings indicate that noninvasive skin imaging methods such as OCT and HFUS might be suitable techniques for the evaluation and monitoring of elastolytic skin disorders such as MDE.     

The impact of carbonylated proteins on the skin and potential agents to block their effects

Carbonylated proteins (CPs) are synthesized by reactions between amino groups in proteins and reactive aldehyde compounds (RAC) yielded from lipid peroxidation initiated by reactive oxygen species (ROS). In the skin, CPs are detected in a higher frequency at sun‐exposed sites of the skin in elderly subjects. Since CPs in the stratum corneum (SC) have been reported to correlate with skin water content and transepidermal water loss, it is considered that the accumulation of CPs in the SC involves the loss of skin moisture functions. However, the roles of CPs in the dermis on skin physiology are still unclear. The purpose of this study was to investigate the roles of CPs in the dermis during the progression of photoaged skin and to propose a method to prevent or reduce the synthesis of CPs. The exposure of human normal dermal fibroblasts to CPs increased intracellular ROS levels and the synthesis of intracellular CPs. In addition, CPs caused morphological changes of fibroblasts. Furthermore, CPs caused alterations of mRNA expression levels of dermal matrix‐related proteins, such as upregulating MMP‐1 and IL‐8. These results indicated that CPs disrupt construction of the dermal matrix. On the other hand, α‐tocopherol and β‐carotene suppressed the synthesis of RAC during lipid peroxidation which resulted in the reduction of UVA‐induced CPs in the SC. From these results, we propose that extracellular CPs increase intracellular ROS levels and contribute to alterations of the dermal matrix. To prevent the synthesis of CPs, the application of α‐tocopherol or β‐carotene could be effective.     

Changes in the depth profile of water in the stratum corneum treated with water

Background/purpose: Water in the stratum corneum (SC) plays an important role in keeping the skin surface soft and smooth. Information regarding the depth profile of the molecular composition of the skin, such as water and free amino acids, can be obtained non‐invasively using the recently developed confocal Raman spectrometer. In this study, we investigated changes in the depth profile of water in the SC treated with water. We also unveil a relationship between the depth profile patterns of water content in the SC and cutaneous sensation. Methods: Depth profiles of Raman spectra in the region 2600–4000 cm^?1 were obtained using a 671‐nm laser at 2 μm intervals from the skin surface toward the interior with a confocal Raman spectrometer. Water content (mass%), expressed in grams of water per 100 g wet tissue, was calculated from the water‐to‐protein ratio of the Raman band. Skin surface temperature was measured by an infrared irradiation thermometer. Changes in the depth profile of water and skin temperature were measured at 1, 5, and 10 min after the application of water to the skin surface. In addition, questionnaires regarding cutaneous sensation were administered. Results: Water content in the middle to lower part of the SC increased with increased water‐application time. Warming of the skin during water application increased the water‐penetration amount, depth, and holding time in the SC. Steamer application increased water content particularly in the upper part of the SC. Increasing water content in the upper part of the SC was associated with cutaneous sensations, such as hydrate and water‐rich feelings. On the other hand, the increasing water content in the middle and lower part of the SC was associated with water penetration and tension feelings. Conclusion: The water content in the upper part of the SC changed easily. When water was applied externally, the water content in both the upper and middle/lower part of the SC increased with increasing water‐application time. In addition, warming of the skin during water application increased the water‐penetration depth, amount, and holding time of water in the SC. Thus, we were able to control the depth profile of water in the SC by externally applying water. The location of water in the SC also affected cutaneous sensations.     

In vivo measurement of human dermis by 1064 nm-excited fiber Raman spectroscopy.

BACKGROUND/AIMS: Although chemical information on the dermis in vivo is highly important in skin research, an efficient method for gathering this information is yet to be developed. Here, we demonstrate that newly developed near-infrared (1064 nm) excited Raman spectroscopy is a powerful method for chemical analysis of human skin in vivo. METHODS: We used a laboratory-constructed Raman spectrometer equipped with a highly sensitive near-infrared detector (Hamamatsu Photonics), an optical fiber probe and a 1064 nm Nd:YAG laser. Raman spectra of porcine skin (in vitro) and human skin (in vivo) were measured with this spectrometer. RESULTS: The Raman spectrum of porcine skin measured from the outer side resembles that of the dermis more than that of the epidermis. The Raman spectra of human skin (cheek, forehead, inner forearm, outer forearm, palm) depend on the portion measured with the probe. The spectra of the forehead and inner forearm show larger lipid signals than that of the palm. CONCLUSIONS: The Raman spectrum of skin measured with the 1064 nm Raman system primarily reflects the chemical composition of the dermis. The 1064 nm excited Raman spectroscopy is useful for research of the dermis and skin appendages.     

The effect of mechanical forces (vibration or external compression) on the dermal water content of the upper dermis and epidermis, assessed by high frequency ultrasound

An ultrasound scanner, is used to detect changes in water content of the upper dermis. This has previously been found to vary with age and to show diurnal variation. Furthermore, oedema due to venous disease can be shown, using this technique, to respond to elevation. In this study, the water content of the upper dermis and epidermis of the leg in 16 subjects is increased following vibration for 10 minutes using a passive exercise system. A study of pressure applied to the skin of the heel for 10 minutes in 14 volunteers also showed an increase in water content of epidermis and dermis in young persons, but less so in the elderly. It is postulated that the anatomical structure of the vascular bed of the upper dermis predisposes to transsudation when pressure to the skin is applied, thereby maintaining the resilience of the skin in the young, but less so in the elderly.     

Dermal echogenicity: a biological indicator of individual cumulative UVR exposure?

Dermal alterations due to chronic UVR exposure may influence dermal ultrasound echogenicity, and a subepidermal low-echogenic band has been proposed as a marker of photoaging. The aim of this study was to determine whether dermal echogenicity could be used as a biological UVR dosimeter. We included 201 subjects (138 healthy volunteers, 31 patients with basal cell carcinoma, and 32 patients with cutaneous malignant melanoma). The number of low-echogenic pixels in the upper dermis relative to the lower dermis (LEP_u/l) was determined in sun-exposed and sun-protected skin. Individual UVR exposure data were collected retrospectively and prospectively using a questionnaire and electronic personal UVR dosimeters. Age, but not sex, skin type, constitutive pigmentation or smoking, correlated significantly with LEP_u/l at all body sites. Different measures of individual UVR exposure were significantly positively correlated with LEP_u/l (together r²=0.39, dorsal forearm), but separately the correlations were poor (r²=0.04–0.19). LEP_u/l was higher in the dorsal forearm in a group with high UVR exposure compared to a low-exposure group (P=0.007). Skin cancer patients in general had a lower LEP_u/l than healthy subjects. The results indicate that the age-related increase in LEP_u/l might be attributed mainly to UVR exposure, and that the methods used to obtain the UVR exposure data might not be sufficiently sensitive or specific. Genetic factors might also influence LEP_u/l. We consider LEP_u/l to be a sensitive and specific marker for UVR exposure at the dorsal aspect of the forearm in healthy subjects.Abbreviations BCC basal cell carcinoma - LEP_u/l number of low echogenic pixels in the upper dermis relative to the lower dermis - MED minimal erythema dose - MM cutaneous malignant melanoma - PPF pigment protection factor - SED standard erythema dose - UVR ultraviolet radiation     

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.     

In vivo reflectance confocal microscopy characterization of silver deposits in localized cutaneous argyria

Localized cutaneous argyria is a rare condition secondary to skin deposition of silver following exposure to substances containing this metal. The clinical appearance and dermoscopy findings require deep melanocytic lesions and particularly melanoma metastasis to be ruled out. Silver deposits are usually confirmed by scanning electron microscopy and/or energy‐dispersive X‐ray spectroscopy. Herein we describe the in vivo reflectance confocal microscopy (RCM) features observed in one case of localized cutaneous argyria. These features include the presence of a hyperrefractile network in the papillary dermis and a periadnexal dotted bright pattern. In vivo RCM might be a useful tool for an early diagnosis of this uncommon entity.     

In vivo quantitative analysis of the effect of hydration (immersion and Vaseline treatment) in skin layers using high‐resolution MRI and magnetisation transfer contrast*

Background/aims: Many claims are made as to the efficacy of topical preparations in moisturising the skin, yet most of these claims cannot be substantiated by scientific study for the skin layers beneath the stratum corneum, and yield no information on the remainder of the epidermis and dermis. This argues for an in vivo quantitative method for measuring the effect of water loading extended to various layers of the skin. Methods: Detailed high‐resolution in vivo MRI studies of hydration and dehydration of finger pad skin layers were conducted on one normal subject using two moisturisation methods (topical white soft paraffin (Vaseline) and water immersion). The dehydration study was carried out immediately following removal from prolonged skin moisturisation. Inter‐individual variability for skin hydration (group study) was studied in seven healthy volunteers at 0 and 7 h hydration with Vaseline. Location dependence in skin hydration was investigated on the same subject by looking into the hydration of forearm and finger pad skin. System stability and measurement reproducibility was verified through a detailed phantom study. Results: Images of normal and hydrated human skin were obtained in vivo at voxel dimensions of 50 μm×150 μm×1000 μm. The effect of hydration and dehydration as a function of exposure to moisturiser (i.e. water and Vaseline) on the image signal intensity, observed T1, and interaction of free and bound water in specific tissues were identified and correlated with existing physiological knowledge. Swelling of stratum corneum due to hydration was expressed as an in vivo model of tissue hydration. Conclusion: Results of the dehydration study showed that the changes due to the previous hydration of the skin are reversible for all skin layers. For both moisturisation methods (i.e. Vaseline and skin bathing), the effects of hydration and dehydration on the skin were similar. The trends of the MRI parameters for finger pad and arm skin were similar. The group study showed low inter‐subject variability of hydration on stratum corneum and epidermis.     

Intrinsic aging- and photoaging-dependent level changes of glycosaminoglycans and their correlation with water content in human skin

Background

Glycosaminoglycans (GAGs) have various structural and physiological regulatory functions in skin, including tissue water maintenance, due to their high water-holding capacity.

Objective

To investigate changes of GAGs during intrinsic aging and photoaging of human skin and their correlations with water content.

Methods

Samples of sun-protected buttock and sun-exposed forearm skin were obtained from young male (21-30 years, n = 8) and female (20-33 years, n = 8) subjects, as well as old male (70-78 years, n = 8) and female (70-80 years, n = 8) subjects, and their epidermal and dermal contents of hyaluronic acid (HA), total sulfated GAG (tsGAG), total uronic acid (tUA), and tissue water were measured. HA content was determined by enzyme-linked immunosorbent assay using HA-binding protein, tsGAG by the sulfated GAG assay kit using 1,9-dimethylmethylene blue, tUA by carbazole reaction, and tissue water by subtraction of tissue dry weight from wet weight.

Results

In the buttock, HA was higher in dermis than in epidermis, while tsGAG and tUA were higher in epidermis. In intrinsically aged buttock, epidermal HA and dermal tsGAG and tUA decreased. However, when analyzed for each gender, epidermal tsGAG, tUA, and tissue water decreased only in females. Forearm/buttock ratios of each molecule were compared for determination of photoaging-dependent changes. Forearm/buttock ratios of HA, tsGAG, tUA, and tissue water increased in aged dermis, but showed no change in aged epidermis. When analyzed for each gender, ratios of epidermal HA and tissue water increased only in aged females, while ratios of epidermal tsGAG, tUA, and tissue water decreased only in aged males. Correlations of water content with HA, tsGAG, and tUA were found in epidermis, but not with tsGAG in dermis.

Conclusion

These intrinsic aging- and photoaging-dependent GAG changes and their correlations with water content provide new insights into the pathophysiology of dry skin in the elderly.     

In vivo reflectance confocal microscopy imaging of vitiligo,nevus depigmentosus and nevus anemicus

Background/purpose: Hypopigmentary skin disorders such as vitiligo, nevus depigmentosus and nevus anemicus are common diseases in clinic. The lesions of these diseases could be similar to some extent, although each of them has its own characteristic clinical appearance and histological features. Clinically, the atypical lesions are often difficult to be differentiated. In vivo reflectance confocal microscopy (RCM) is a non‐invasive, repetitive imaging tool that provides real‐time images at a nearly cellular histological resolution. Our aim was to investigate the RCM features of vitiligo, nevus depigmentosus and nevus anemicus. Subjects and Methods: A total of 135 patients with a clinical diagnosis of the aforementioned diseases were included in this study. The RCM images from depigmented skin, border of the white macules, adjacent normal‐appearing skin and distant normal skin for all patients at the dermo‐epidermal junction (DEJ) level were investigated. Results: In the active phase of vitiligo (AVP), the RCM demonstrated a complete loss of melanin in lesional skin in eight (53; 15.1%) patients. In 45 patients (53; 84.9%) of the AVP, part of the bright dermal papillary rings normally seen at the DEJ level disappeared or part of the rings lost their integrity and the content of melanin decreased obviously. In 20 patients (53; 37.7%) of the AVP, highly refractile inflammatory cells could be seen within the papillary dermis in the lesional and adjacent normal‐appearing skin, which may indicate the lesion progresses. In addition, part of the dermal papillary rings showed lack of integrity or their brightness decreased in adjacent normal‐appearing skin in all the patients of the AVP. It is important to know that the RCM demonstrated an ill‐defined border. In the stable phase of vitiligo (SPV), the RCM demonstrates a complete loss of melanin in lesional skin and a clear border in 31 (41; 75.6%) patients; the content of melanin and dermal papillary rings in adjacent normal‐appearing skin show no changes. In 10 (41; 24.4%) patients, the dendritic and highly refractile melanocytes arose in the recovery phase of vitiligo, which may indicate the repigmentation of vitiligo. There are three kinds of repigmentation patterns under RCM: marginal, perifollicular and diffuse. Distant normal skin showed no difference from controls in both the active and the SPV. In all the patients with nevus depigmentosus, the content of melanin decreases obviously but the dermal papillary rings are intact. The dermal papillary rings show no differences between lesional skin and adjacent normal‐appearing skin of nevus anemicus. Conclusion: Considering our results, RCM may be useful to non‐invasively discriminate vitiligo, nevus depigmentosus and nevus anemicus in vivo.     

Interdigitation index – a parameter for differentiating between young and older skin specimens

Background/aims: Quantitative morphometry developed rapidly during the last decade due to advances is computers and software. We wish to establish a simple baseline for the morphometric differences due to intrinsic ageing between young and old cohorts: the interdigitation index. It is an expression of the shape of the border between the epidermis and dermis. Methods: We used volar forearm biopsies of women, since the volar forearm is usually not photodamaged. The biopsies were fixed in buffered formalin, embedded in paraffin and sectioned. Separate sections were stained by hematoxylin‐eosin, by orcein and by dimethyl‐methylene blue. We had seven female volunteers in each group; the young cohort had a mean age of 26.6 years, the older cohort 50.9 years. We chose a cohort that was just about postmenopausal, since in the future we wish to evaluate the effect of externally‐applied agents on postmenopausal female skin and the earlier it is applied the better its chance of being effective. Results: We found no difference between the young and older cohort with regard to epidermal thickness. We found a decrease of glycosaminoglycen (GAG) as measured by dimethyl‐methylene blue staining. The results of the elastic staining by orcein, although in line with the reports in the literature, are not useful for evaluating the intrinsic ageing process, at least not by the simple percentage of area stained procedure. We introduced a new parameter: the interdigitation index. It is a simple measurement of the interdigitation in the epidermal‐dermal junction, known to be diminished by age. This index was diminished by about 20% between the young and older cohort. Conclusions: Quantitative morphometry using simple epidermal and dermal measurements on biopsies of the volar forearm of women is suitable for following intrinsic ageing of the skin and offers a simple objective method for following the ageing process of the skin.     

In vivo study for the discrimination of cancerous and normal skin using fibre probe‐based Raman spectroscopy

Raman spectroscopy has proved its capability as an objective, non‐invasive tool for the detection of various melanoma and non‐melanoma skin cancers (NMSC) in a number of studies. Most publications are based on a Raman microspectroscopic ex vivo approach. In this in vivo clinical evaluation, we apply Raman spectroscopy using a fibre‐coupled probe that allows access to a multitude of affected body sites. The probe design is optimized for epithelial sensitivity, whereby a large part of the detected signal originates from within the epidermal layer's depth down to the basal membrane where early stages of skin cancer develop. Data analysis was performed on measurements of 104 subjects scheduled for excision of lesions suspected of being malignant melanoma (MM) (n = 36), basal cell carcinoma (BCC) (n = 39) and squamous cell carcinoma (SCC) (n = 29). NMSC were discriminated from normal skin with a balanced accuracy of 73% (BCC) and 85% (SCC) using partial least squares discriminant analysis (PLS‐DA). Discriminating MM and pigmented nevi (PN) resulted in a balanced accuracy of 91%. These results lie within the range of comparable in vivo studies and the accuracies achieved by trained dermatologists using dermoscopy. Discrimination proved to be unsuccessful between cancerous lesions and suspicious lesions that had been histopathologically verified as benign by dermoscopy.