Biological changes of human cutaneous nerves caused by ultraviolet irradiation: an ultrastructural study

Three white male volunteers were irradiated by long wave ultraviolet (UV-A) and by solar simulating radiation (SSR). An acute change of cutaneous nerves was found following a single exposure of UV-A irradiation. Non-myelinated Schwann cells and perineural cells in the papillary and reticular dermis were degenerated. Axoplasms appeared to be electron-dense, but were less affected than Schwann cells. After long-term repeated exposure, many free nerve endings were found in the dermo-epidermal junction above the basal lamina and some of them made a terminal enlargement. In one instance an axon made a swelling. Dermal free nerve endings also seemed to be increased in number and some of them were situated immediately beneath the melanocytes which were active in melano-genesis. Multiplication of basal lamina of the Schwann cells and perineural cells was observed. Amorphous material was precipitated around the non-myelinated Schwann cells. After a single exposure of SSR irradiation the degeneration of Schwann cells and axons in the dermo-epidermal junction was less severe than after UV-A and changes were minor in the reticular dermis. After repeated exposure, intra-epidermal proliferation of free nerve endings was detected. Dermal nerves were slightly affected. No intra-epidermal free nerve endings were observed in controls. The intra-epidermal proliferation of free nerve endings was confirmed following repeated UV exposures. Melanocyte-nerve association is suggested to be the cause of stimulating melanocyte activity.     

Long-lasting molecular changes in human skin after repetitive in situ UV irradiation

It is known that UV modulates the expression of paracrine factors that regulate melanocyte function in the skin. We investigated the consequences of repetitive UV exposure of human skin in biopsies of 10 subjects with phototypes 2-3.5 taken 1-4 years later. The expression of melanogenic factors (TYR, MART1, MITF), growth factors/receptors (SCF/KIT, bFGF/FGFR1, ET1/EDNRB, HGF, GM-CSF), adhesion molecules (beta-catenin, E-cadherin, N-cadherin), cell cycle proteins (PCNA, cyclins D1, E2) as well as Bcl-2, DKK1, and DKK3, were analyzed by immunohistochemistry. Most of those markers showed no detectable changes at > or = 1 year after the repetitive UV irradiation. Although increased expression of EDNRB protein was detected in 3 of 10 UV-irradiated subjects, there was no detectable change in the expression of ET1 protein or in EDNRB mRNA levels. In summary, only the expression of TYR, MART1, and/or EDNRB, and only in some subjects, was elevated at > or = 1 year after UV irradiation. Thus the long-term effects of repetitive UV irradiation on human skin did not lead to significant changes in skin morphology and there is considerable subject-to-subject variation in responses. The possibility that changes in the expression and function of EDNRB triggers downstream activation of abnormal melanocyte proliferation and differentiation deserves further investigation.     

UV irradiation and topical vitamin A modulate retinol esterification in hairless mouse epidermis

The formation of fatty acyl esters of retinol, the major form of vitamin A in epidermis, is catalysed by microsomal enzymes. To study the regulation of retinol esterification, we exposed hairless mice to ultraviolet (UV) irradiation which destroys vitamin A and to topical retinol treatment. Vitamin A (retinol and retinyl esters) in serum and epidermis was analysed by high-performance liquid chromatography at 0-12 days after a single irradiation with UVB (280-320 nm; 0.34 J/cm2) or UVA (320-400 nm; 1.0 J/cm2). The immediate vitamin A reducing effects of UVB and UVA were similar, but UVB elicited a more rapid replenishment of epidermal vitamin A with a corresponding transient depletion of serum retinol after 2-3 days. The activity of retinyl ester synthetase, measured by an in vitro radiochemical assay, was unaffected by the irradiations. By contrast, the acyl-CoA:retinol acyltransferase (ARAT; EC 2.3.1.76) activity increased to 167% on the 2nd day after UVB-irradiation and to 124% after topical retinol, but was otherwise quite constant. The UVB- and retinol-induced ARAT activity was less dependent on exogenous palmitoyl-CoA than that of control microsomes and experiments indicated that this might be due to an increased endogenous concentration of long-chain acyl-CoA in the microsomes. We conclude that extreme variations in the vitamin A supply to epidermis, such as a rapid influx of unesterified retinol, may modulate the epidermal ARAT activity.     

A study of Q-switched Nd:YAG laser irradiation and paracrine function in human skin cells

BACKGROUND AND OBJECTIVES: This preliminary laboratory-based study looks at the paracrine release from human skin cells subject to sublethal Q-switched Nd:YAG 532 nm laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: Human dermal fibroblast and keratinocyte cultures were exposed to sublethal energy using the Nd:YAG 532 nm laser. Altered gene expression was then screened using RT-PCR for a range of paracrine factors known to affect melanogenesis, basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), stem cell factor (SCF), melanocyte stimulating hormone (MSH), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and protease-activated receptor-2 (PAR-2). Enzyme-linked immunosorbent assay (ELISA) was used to confirm protein production. Conditioned medium was used to assess altered melanogenesis in a melanoma cell line. Results: Fibroblasts exposed to sublethal radiation showed upregulation of b-FGF, HGF and SCF. This contrasts with keratinocytes which showed upregulation of IL-6. Elevated protein levels of b-FGF and SCF were confirmed by ELISA assay. Conditioned fibroblast medium was shown to stimulate melanogenesis in a melanoma cell line. CONCLUSIONS: This preliminary laboratory study reports, for the first time, specific gene upregulation using the Q-switched Nd:YAG 532 nm laser.     

Cyclobutane pyrimidine dimer formation and p53 production in human skin after repeated UV irradiation

Substantial differences in DNA damage caused by a single UV irradiation were found in our previous study on skin with different levels of constitutive pigmentation. In this study, we assessed whether facultative pigmentation induced by repeated UV irradiation is photoprotective. Three sites on the backs of 21 healthy subjects with type II-III skin were irradiated at 100-600 J/m(2) every 2-7 days over a 4- to 5-week period. The three sites received different cumulative doses of UV (1900, 2900 or 4200 J/m(2)) and were biopsied 1 day after the last irradiation. Biomarkers examined included pigment content assessed by Fontana-Masson staining, melanocyte function by expression of melanocyte-specific markers, DNA damage as cyclobutane pyrimidine dimers (CPD), nuclear accumulation of p53, apoptosis determined by TUNEL assay, and levels of p21 and Ser46-phosphorylated p53. Increases in melanocyte function and density, and in levels of apoptosis were similar among the 3 study sites irradiated with different cumulative UV doses. Levels of CPD decreased while the number of p53-positive cells increased as the cumulative dose of UV increased. These results suggest that pigmentation induced in skin by repeated UV irradiation protects against subsequent UV-induced DNA damage but not as effectively as constitutive pigmentation.     

脂质体影响荧光素钠透皮和皮肤贮留量的实验研究

目的：观察脂质体包裹荧光素钠对人皮肤透皮性能和在皮肤分布的影响。方法：采用Franz扩散池、荧光显微镜和荧光分光光度计研究真、表皮中荧光素钠的分布和含量。结果：荧光素钠脂质体同其溶液和乳剂比较,在表皮内荧光分布深,在真皮荧光强度小;脂质体荧光素钠真皮透过量少,各时间点表皮内荧光含量较溶液剂型高,而真皮内荧光含量较乳剂低,不同剂型各时间点的差异均具有统计学意义（P〈O．05）。结论：皮肤外用制剂中脂质体包裹水溶性药物可在人皮肤表皮层保持较高浓度,真皮贮留量和累积透皮量小,故适用于病变表浅的皮肤病,以及经透皮吸收后有不良反应的药物。     

Effects of UV irradiation with one minimal erythema dose on human afferent skin lymph in vivo

Ultraviolet (UV) irradiation of the skin induces complex local and systemic immunomodulatory reactions. The biological effects of UV irradiation on human skin derived afferent lymph however are unknown. The aim of this study was to examine the effects of a single combined UV-A and UV-B irradiation with 1 minimal erythema dose (MED) on human skin derived lymph in vivo. After cannulation of a superficial lymph vessel on the lower leg, lymph flow and cell output per hour were determined before and for 6 days after UV irradiation of the lymph draining skin area in 5 volunteers. Furthermore, expression of CDla, CD4, CD8, CD28, CD54, CD80, CD86 and HLA-DR on migrating lymph cells and cytokine levels (IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-13, TNF-α and IFN-γ) in the afferent lymph were analyzed by cytofluorometry and ELISA. After UV irradiation a small initial enhancement in the daily lymph flow per hour was noticed in correlation with the slight erythematous skin reaction. Following resolution of the skin reaction, a delayed increase in cell output in correlation with an additional peak in the lymph flow was found between the 4th and 6th day after UV irradiation. However, no changes in the expression of CDla, CD4, CD8, CD28, CD54, CD80, CD86 and HLA-DR on migrating lymph cells were detectable. Interestingly, in parallel to the increased lymph flow and cell output, only elevated IL-8 protein levels were reproducibly detected in the afferent lymph after UV irradiation. Furthermore, using immunohistochemistry positive staining for IL-8 was found on migrating mononuclear lymph cells. In conclusion, our data demonstrate that a single UV irradiation of the skin with 1 minimal erythema dose leads to a delayed enhancement of lymph flow, number of migrating lymph cells and cytokine levels of IL-8. Moreover, we provide evidence that migrating lymph cells, besides resident epidermal and dermal cells, may contribute to the detected levels of IL-8 in the afferent lymph.     

The effect of UV irradiation and photochemotherapy (PUVA) on histamine weal and flare in human skin

It is recognized that ultraviolet light has a beneficial effect in various inflammatory disorders of the skin. We have used histamine weal and flare as a model testing the effect of ultraviolet light on vascular response. No difference in weal and flare reaction following intradermal injection of 0·01-10·0 μg of hisiamine was found between covered and exposed skin following UV radiation or photochemotherapy (PUVA).     

Improvement of the experimental setup to assess cutaneous bioavailability on human skin models: dynamic protocol

Human skin models, such as EpiDerm and Episkin, are not easily mounted into static or dynamic diffusion cells that are commonly used to perform bioavailability studies with human skin ex vivo. For various reasons, such as fragility, small sample size, and other morphological constraints, skin absorption studies with human skin models are often carried out on the delimited skin surface obtained by gluing a ring onto the reconstituted epidermis and manually exchanging the receptor solution. However, such an experimental setup is prone to artifacts. Discontinuous removal of the receptor fluid leads to alternating sink conditions, and an area of application smaller than the area in contact with the receptor fluid, as well as imperfect seal of the glued ring, may result in inaccurate penetration rates. Human skin models were shown to be relatively easily mounted into In-Line cells (PermeGear Inc.), vertical diffusion cells which appear to be appropriately designed for such a purpose. In-Line cells allowed accurate determination of solute penetration as well as automated sampling of receptor fluid. Excised human skin can be mounted into these cells as well, making it possible to compare penetration rates through different types of skin samples under identical conditions. Using mannitol as a reference compound, penetration profiles and epidermal distribution similar to those obtained with human skin ex vivo were obtained both with EpiDerm and Episkin. Under the present conditions, human skin models were more permeable to mannitol than excised human skin, which was only slightly permeable to mannitol. Due to these experimental innovations and to the good agreement with the absorption characteristics through human skin ex vivo, EpiDerm and Episkin seem to be promising human skin models for testing the cutaneous bioavailability of topical products in vitro.     

Low concentrations of formaldehyde induce DNA damage and delay DNA repair after UV irradiation in human skin cells

Long-term occupational exposure to formaldehyde (FA) increases the risk for nasopharyngeal squamous cell carcinoma. As the skin is also in contact with FA by environmental exposure, we tested the genotoxic properties of appropriate low concentrations (<100 microM) of FA on cultured keratinocytes and fibroblasts of human skin. The initial DNA damage was assessed by comet assay. The induction of DNA protein crosslinks was measured by the ability of FA to reduce DNA migration induced by methyl-methane-sulfonate. Upon 4 h of exposure to FA, significant (P < 0.05) crosslink formations were observed in fibroblasts (50 microM FA) and in keratinocytes (25 microM FA). Upon 8 h of exposure to FA (25 microM FA), significant crosslink formations were observed in both the cell types. FA is known to inhibit different DNA repair pathways. Therefore, we studied the effect of FA on UV-induced repair. Human keratinocytes and fibroblasts exposed to 10 microM FA prior to UV irradiation showed disturbed repair kinetics after UVC and UVB, but not after UVA irradiation. Single-strand breaks (SSBs) derived from nucleotide excision repair disappeared 6 h after solely UVC (3 mJ/cm2) or 3 h solely UVB (30 mJ/cm2) exposure in both the cell types. In the presence of FA, SSBs were still present at these time points containing a reference to a delay in DNA resynthesis/ligation. FA at a concentration not inducing micronuclei (12.5 microM) caused significant increase of UVC-induced (4 mJ/cm2) chromosomal damage. Proliferation of keratinocytes and fibroblasts was in parallel to observed DNA damages. In conclusion, our data suggest that environmental exposure to FA may contribute to UV-induced skin carcinogenesis.     

体外重建皮肤与正常人皮肤基本特征的对比研究

目的 用正常人的皮肤角质形成细胞和黑素细胞在体外重建皮肤,为将来用于临床、生物及药理学研究奠定基础.方法 从正常儿童的包皮分离、培养角质形成细胞、黑素细胞和成纤维细胞.用成纤维细胞和I型鼠尾胶原制备真皮类似物.将角质形成细胞和黑素细胞接种到真皮类似物表面进行培养,制备表皮类似物.将重建的皮肤类似物和正常人皮肤进行对比研究.结果 皮肤类似物的组织形态包括基底层、基底上层、角质层与正常人皮肤相似.与基底膜形成有关的标志,在皮肤类似物和正常人皮肤中的表达是相似的.角蛋白10、泛角蛋白在皮肤类似物和正常人皮肤中的表达也是相似的.结论 组织形态和所检测的各种标志的表达显示,该皮肤类似物与正常人皮肤相似.     

Age-related mechanical properties of human skin: an in vivo study

We have investigated in vivo how various viscoelastic parameters that describe the mechanical properties of the human skin may vary with age. Accordingly, we have used a mechanical device that records the torsional extensibility of the skin. When submitted to a low torque, the time-response curve of the skin affords the determination of the immediate extensibility (UE), the immediate recovery (UR), the viscoelastic part of the deformation (UV), the elastic recovery (UR/UE), and the creep relaxation time (tau). Because the skin thickness varies with age and primarily governs the mechanical properties, it was measured through an ultrasound technique at the same sites (forearm) where the torque was applied. The results show that the skin maintains its thickness and extensibility up to the seventh decade as opposed to its elasticity or recovery capacities, which decrease from an early age. The viscous part of the deformation is constant through life, whereas the creep relaxation time decreases linearily with age. Except for skin thickness, no differences in these parameters between men and women were detected. The significance of these results are discussed in terms of structure alterations. The determination of the elastic recovery (UR/UE) appears to be a parameter of choice for illustrating skin aging.     

Effect of UV irradiation on cutaneous cicatrices: a randomized, controlled trial with clinical, skin reflectance, histological, immunohistochemical and biochemical evaluations

The aim of this study was to examine the effect of ultraviolet (UV) irradiation on human cutaneous cicatrices. In this randomized, controlled study, dermal punch biopsy wounds served as a wound healing model. Wounds healed by primary or second intention and were randomized to postoperative solar UV irradiation or to no UV exposure. Evaluations after 5 and 12 weeks included blinded clinical assessments, skin reflectance measurements, histology, immunohistochemistry, and biochemical analyses of the N-terminal propeptide from procollagen-1, hydroxyproline, hydroxylysine, and proline. Twelve weeks postoperatively, UV-irradiated cicatrices healing by second intention: (i) were significantly pointed out as the most disfiguring; (ii) obtained significantly higher scores of colour, infiltration and cicatrix area; and (iii) showed significantly higher increase in skin-reflectance measurements of skin-pigmentation vs. non-irradiated cicatrices. No histological, immunohistochemical or biochemical differences were found. In conclusion, postoperative UV exposure aggravates the clinical appearance of cicatrices in humans.     

Effect of chemical peeling on the skin in relation to UV irradiation

Chemical peeling is one of the dermatological treatments available for certain cutaneous diseases and conditions or improvement of cosmetic appearance of photoaged skin. However, it needs to be clarified whether the repetitive procedure of chemical peeling on photodamaged skin is safe and whether the different chemicals used for peeling results in similar outcomes or not. In this article, we reviewed the effect of peeling or peeling agents on the skin in relation to ultraviolet (UV) radiation. The pretreatment of peeling agents usually enhance UV sensitivity by inducing increased sunburn cell formation, lowering minimum erythematous dose and increasing cyclobutane pyrimidine dimers. However, this sensitivity is reversible and recovers to normal after 1-week discontinuation. Using animals, the chronic effect of peeling and peeling agents was shown to prevent photocarcinogenesis. There is also an in vitro study using culture cells to know the detailed mechanisms of peeling agents, especially on cell proliferation and apoptotic changes via activating signalling cascades and oxidative stress. It is important to understand the effect of peeling agents on photoaged skin and to know how to deal with UV irradiation during the application of peeling agents and treatment of chemical peeling in daily life.     

Evaluation of carotenoids and reactive oxygen species in human skin after UV irradiation: a critical comparison between in vivo and ex vivo investigations

UV irradiation is one of the most harmful exogenous factors for the human skin. In addition to the development of erythema, free radicals, that is reactive oxygen species (ROS), are induced under its influence and promote the development of oxidative stress in the skin. Several techniques are available for determining the effect of UV irradiation. Resonance Raman spectroscopy (RRS) measures the reduction of the carotenoid concentration, while electron paramagnetic resonance (EPR) spectroscopy enables the analysis of the production of free radicals. Depending on the method, the skin parameters are analysed in vivo or ex vivo. This study provides a critical comparison between in vivo and ex vivo investigations on the ROS formation and carotenoid depletion caused by UV irradiation in human skin. The oxygen content of tissue was also determined. It was shown that the antioxidant status measured in the skin samples in vivo and ex vivo was different. The depletion in the carotenoid concentration in vivo exceeded the value determined ex vivo by a factor of about 1.5, and the radical formation after UV irradiation was significantly greater in vivo by a factor of 3.5 than that measured in excised human skin, which can be explained by the lack of oxygen ex vivo.     

Enzyme-independent NO stores in human skin: quantification and influence of UV radiation

Nitric oxide (NO) has many functions in the skin, including the mediation of inflammation and antimicrobial defense, wound healing, regulation of keratinocyte homeostasis, and regulation of apoptosis following UV radiation. NO is synthesized by a family of NO synthase enzymes, but its rapid release following UV exposure suggests the existence of preformed stores. NO can be converted into nitrite or nitrosothiols that are stable until cleaved by UV to release NO. Using dermal microdialysis, suction blister epidermal samples, and sweat collection, we demonstrated cutaneous concentrations of total NO-related products of 12+/-5.97 microM, 0.03+/-0.03 micromol mg(-1) epidermal protein, and 22+/-9.34 microM, respectively. The predominant oxyanion was nitrate (60-75%) followed by nitrite. S-Nitrosothiols were barely detectable. Serum total NO-related products correlated directly with those of the upper dermis and sweat (R(2)=0.62 and 0.3, respectively). UVA irradiation (10 mW cm(-2)) increased the yield of NO-related products by microdialysis, peaking after 30 minutes. Dialysis with noradrenaline abrogated this rise. Both the skin and the dermal vasculature contain biologically significant stores of NO, particularly nitrite, which can be directly mobilized by UVA irradiation. The level of circulating NO-related products probably determines skin-bound stores.     

Engraftment of precursor lesions of human cutaneous neoplasms onto C.B-17 SCID mice: A useful in vivo experimental model of carcinogenesis in human skin

Using a full-thickness skin grafting technique, lesional skin from various human neoplastic and preneoplastic skin diseases was transplanted onto SCID (severe combined immunodeficiency) mice. Of 27 grafted lesions, 21 were successfully accepted by the mice and maintained in good condition. All these accepted grafts were finally excised 10–101 days after transplantation for histological examination. In most grafts, the characteristic histological configurations of each disease were well preserved. Immunohistochemical study using monoclonal antibodies to human blood group antigens ABH revealed that some elements of the grafts such as sweat glands were clearly positive, confirming that the tissue was from human skin. Neoplastic (atypical) cells were detected in 9 of 17 accepted grafts containing neoplastic cells from the beginning. The detection rates for neoplastic cells were very high (90%) in grafts from precursor lesions of squamous cell carcinomas such as Bowen's disease (5/5 specimens) and thermal keratosis (2/3). In contrast, no definite neoplastic cells were found in two grafts from extramammary Paget's disease and five grafts from the radial growth component of malignant melanoma. In most of the grafts from latter two diseases, characteristic histological configurations such as elongation of the rete ridges were maintained, suggesting that the neoplastic cells were selectively eliminated from the grafts. Split-thickness grafts of normal human skin were accepted and remained in a good condition for as long as 6 months. Engraftment of human lesional and non-lesional skin onto SCID mice therefore may well provide a useful in vivo experimental model of human skin diseases.