Connective tissue alterations in the skin of ultraviolet irradiated hairless mice

Connective tissue alterations were induced in hairless mouse skin by ultraviolet (UV) irradiation. Hairless mice were irradiated three times a week for 10 weeks with sunlamps (UVA and UVB) and the skin was examined using immunochemical and biochemical techniques. Indirect immunofluorescence was performed with antibodies directed against elastin, microfibrillar proteins, and fibronectin. Increased fluorescence was observed in the actinically damaged skin for elastin, microfibrillar proteins, and fibronectin. The elastic fiber components, elastin and microfibrillar proteins, were then isolated and quantified. Control skin contained approximately 0.1% by dry weight of elastic fiber components, whereas actinically damaged skin contained 0.2% by dry weight. These data are consistent with previous observations of elastic fiber hyperplasia in UV irradiated mice. In addition, irradiated mouse skin contained 1.12 mg of extracted fibronectin per gram wet weight as compared with 0.59 mg in control skin. Irradiated mouse skin contained increased quantities of hyaluronic acid and chondroitin sulfate (uronic acid content). These studies further support the validity of the UV irradiated hairless mouse as a model of human dermal photoaging.     

Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice

BACKGROUND: It is recognized that UV radiation produced apoptotic cells (sun burn cells) in the epidermis of mice. However, the relationship between apoptosis and cell proliferation after UV exposure in the skin of hairless mice are still unclear. OBJECTIVE: To investigate the effects of ultraviolet (UV) radiation on molecular events associated with apoptosis and proliferation in SKH1-hr mouse skin. METHODS: Mice were irradiated with daily UVB exposure of 0.1 or 0.25 J/cm(2) for 14 days. The skin tissues were analyzed at 2 and 24 h after the end irradiation for the presence of apoptotic cells and Bromodeoxyuridine (BrdU)-positive cells. We measured the expression of p53, p21, bcl-2, bax and E2F-1. RESULTS: The results indicated that UVB irradiation caused to increase apoptotic cells in the epidermis of mice. The expression of p53 and p21 was increased at 2 and 24 h after irradiation compared with the control. UV radiation induced high levels of bax at 2 and 24 h after irradiation with a concomitant decrease in bcl-2 expression. The expression of E2F-1 in the skin was also increased at 2 and 24 h after irradiation. Coinciding with these changes, BrdU positive cells increased at 2 and 24 h after UVB exposure at the epidermis of hairless mice, which observed the apoptotic expression. CONCLUSION: These results suggest that UVB irradiation of mouse skin induces apoptosis and is mediated by the p53/p21/E2F-1/bax pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia.     

Sunscreens with low sun protection factor inhibit ultraviolet B and A photoaging in the skin of the hairless albino mouse.

We examined the chronic effect of long daily suberythemal, fluorescent solar-stimulated radiation (FSSR; ultraviolet B (UVB)+A(UVA)) and UVA alone on female Skh-1 hairless albino mouse skin. Mice were dorsally irradiated 8 h every weekday for 16 weeks with FSSR or UVA, or 32 weeks with UVA alone. Various topical, low concentration, UVB and/or UVA sunscreens were applied before irradiation. Damage was assessed by skin-fold thickness, histology and biochemically by changes in the proportion of type III collagen. All FSSR-exposed mice showed increased skin thickening, elastic fibre hyperplasia, collagen damage and an increased proportion of type III collagen. Application of the UVB sunscreen (2.00%) resulted in marked protection for all nonbiochemical endpoints. There was no obvious advantage of adding 0.75% UVA sunscreen to the UVB sunscreen, but adding 2.00% UVA sunscreen reduced biochemical changes and connective tissue damage. Sixteen weeks of UVA irradiation caused skin thickening and laxity but the histology and biochemistry were indistinguishable from the controls. The mice irradiated with UVA for 32 weeks showed slight elastic fibre hyperplasia and collagen damage histologically, and increased skin thickening and laxity; these changes were unmodified by the 0.75% UVA sunscreen. These mice showed a significant increase in the proportion of type III collagen against which the UVA sunscreen offered protection. Our data suggest that UVA may be important in photoaging and that the use of low sun protection factor UVB+ UVA sunscreens on a day-to-day basis may offer some protection from solar photoaging.     

The contributions of UVA and UVB to connective tissue damage in hairless mice

UVA, in high-dose single exposures, can, like UVB, be deleterious to skin. Dermal damage resulting from chronic exposure to UVA has not been studied. To investigate the long-term effects, we irradiated albino hairless mice for 30-34 weeks with UVA radiation, alone, from two sources with differing spectral qualities, and in combination with UVB as solar-simulating radiation. The results were compared to UVB alone. Like UVB, the UVA waveband, especially that with a spectral distribution similar to solar UVA, caused elastic fiber damage, increased glycosaminoglycan levels, and produced hypertrophy of deep dermal tissues. There were, however, striking differences between UVB- and UVA-irradiated skin. A combination of UVA and UVB summated the effects of both wavebands. Substantial protection against these effects was afforded by a broad-spectrum sunscreen.     

Role for tumour necrosis factor-alpha receptors in ultraviolet-induced skin tumours

The biological effects of tumour necrosis factor (TNF)-alpha are mediated through either the TNFR1 or the TNFR2 receptor. In the present study, the effects of ultraviolet (UV) irradiation on skin pathology and tumour promotion were studied in hairless mice deficient in either the TNFR1 or the TNFR2 receptor. SKH-1 hairless mice were crossed with either TNFR1 knockout (KO) mice or TNFR2 KO mice to develop a strain of hairless mice deficient in either of these receptors. Elastosis and other pathological indications of UVB irradiation were not affected by the loss of either receptor. The absence of either receptor, however, resulted in a highly significant reduction in skin tumours in response to UVB irradiation. Inflammatory cell influx following chronic UV irradiation was virtually eliminated in the TNFR1 KO mice, while the TNFR2 KO mice responded to UV irradiation with the normal increase in inflammatory cells throughout the lower and upper dermis. Contact hypersensitivity responses were eliminated in the TNFR2 KO mice, whereas the TNFR1 KO mice retained normal contact hypersensitivity reactions. These studies suggest that TNF-alpha plays no part in the accumulation of excessive elastin in the skin during chronic UVB exposure. However, there appears to be an important role for TNF-alpha in mediating tumorigenesis, distinct from its role in initiating cutaneous immune responses.     

IL-3 production as an in vitro marker of the genetically determined traits of UVB susceptibility and UVB resistance.

The capacity of low-dose ultraviolet B (UVB) radiation to disrupt epidermal Langerhans cell function and to prevent the induction of contact hypersensitivity (CH) is genetically determined in mice and men. In mice, Tnf alpha and Lps are the genetic loci at which reside alleles that dictate susceptibility and resistance to the deleterious effect of UVB radiation. Detection of the UVB-susceptibility (UVB-S) and UVB-resistance (UVB-R) traits relies upon the in vivo end point of contact hypersensitivity, and is cumbersome, labor intensive, and time consuming. It has recently been reported that hapten-immune murine T cells can secrete interleukin-3 (IL-3) in vitro when exposed to hapten-derivatized syngeneic stimulator cells. To determine whether this assay might be useful in distinguishing UVB-R from UVB-S mice, panels of UVB-susceptible (C57BL/10, C3H/HeN) and UVB-resistant (A/J, BALB/c, C3H/HeJ) mice were sensitized epicutaneously with dinitrofluorobenzene (DNFB). When challenged in vitro 6 d later with dinitrophenyl-derivatized stimulator cells, T cells from all strains proliferated and secreted IL-3. Moreover, T cells from UVB-R mice that were sensitized through UVB-treated skin also made copious amounts of IL-3. However, T cells from UVB-S mice whose abdominal skin had been UVB irradiated prior to epicutaneous application of DNFB failed to secrete IL-3 in vitro, although the cells did proliferate. We conclude that following application of a sensitizing dose of hapten to UVB-exposed skin of UVB-S mice a) hapten-specific T cells are selectively unable to secrete IL-3 in vitro in response to hapten stimulation, and b) this inability is a reliable marker of the UVB-S trait. The IL-3 assay may prove useful in elucidating the mechanism by which UVB-exposed Langerhans cells activate regulatory T cells, and in detecting the UVB-S and UVB-R traits in humans.     

Early changes in dermal collagen of mice exposed to chronic UVB irradiation and the effects of a UVB sunscreen

Our previous studies have shown that hairless albino mice receiving chronic UVB irradiation show an increase in type III collagen, in the irradiated skin, after 12-36 weeks treatment. In this study we wished to find the earliest time at which such collagen changes were detectable and also to ascertain whether a topically applied UV-B sunscreen could prevent such changes. Groups of 10 hairless albino mice were irradiated, dorsally, with a daily dose of 62 mJcm-2 UVB for 3, 6, 9, and 12 weeks. Three parallel groups were used. They received a) no topical treatment, b) sunscreen vehicle alone, and c) sunscreen (containing 2-ethylhexyl 4'-methoxy-cinnamate) applied dorsally at 2 microliter cm-2. Dorsal and ventral (non-irradiated) skin samples were taken and the types I and III collagen quantified densitometrically after cyanogen bromide digestion and polyacrylamide gel electrophoresis. The ratio of dorsal/ventral type III collagen (D/V III) was determined for each mouse. The topical treatments caused no change in D/V III in the non-irradiated animals. In the non-topically treated group the irradiated animals showed a considerable increase in D/V III, compared with controls, at 12 weeks (P less than 0.001). The group treated with sunscreen vehicle alone also showed a significant increase in D/V III at week 12 (P less than 0.01). Both these groups showed a slight elevation in D/V III at week 6. The group treated with sunscreen showed no significant difference in D/V III between irradiated and control animals at any time.     

Numbers of murine dermal mast cells remain unchanged during chronic ultraviolet B irradiation

Dermal mast cell numbers reportedly increase in response to chronic ultraviolet irradiation in both humans and in the HRS/Skh-1 mouse model of human photoaging. It has been hypothesized that these increased numbers of mast cells are responsible, at least in part, for the damage in this chronically irradiated or photoaged skin. However, few actual quantitative data have been reported to support this claim of increased dermal mast cell numbers caused by chronic ultraviolet irradiation. We sought to quantify the numbers of dermal mast cells in the skin of chronic ultraviolet-irradiated and control HRS/Skh-1 hairless mice. Dermal mast cells from irradiated and age-matched control mice were quantified by digital image analysis during a 20-week period of exposure to ultraviolet B (UVB) radiation. During the entire course of irradiation, there was no difference in the numbers of dermal mast cells between the irradiated and nonirradiated age-matched control mice. Visible physical evidence of the effects of chronic UVB irradiation, i.e., skin wrinkling, was evident after 6 weeks of treatment. The numbers of dermal mast cells in unirradiated age-matched NSA (CF-1) haired mice were three- to four-fold lower than those in either ultraviolet-exposed or unexposed HRS/Skh-1 mice. These findings indicate that dermal mast cell numbers in HRS/Skh-1 mice are not increased by chronic exposure to UVB radiation.     

Collagen metabolism in ultraviolet irradiated hairless mouse skin and its correlation to histochemical observations

Early biochemical studies of ultraviolet (UV) irradiated human skin reported a loss of insoluble collagen with a concomitant increase in the soluble fraction. Recent work has described an early increase in type III collagen during chronic irradiation of hairless mice as determined by cyanogen bromide digests of whole skin. In order to understand the correlation of these events and those seen with histochemistry, in the present study we irradiated hairless mice for up to 24 weeks with approximately 4 minimal erythema doses (MEDs) of UVB thrice weekly with Westinghouse FS-40 bulbs. Skin samples were taken at 4-week intervals from irradiated and age-matched control mice. Collagen was isolated from other skin proteins by acid extraction, pepsin digestion, and salt precipitation. Estimates of types I and III collagen were made by interrupted polyacrylamide gel electrophoresis and densitometric scanning. Compared with unirradiated controls, there was a small increase in the ratio of type III to total collagen after 8 weeks of UV. There were no significant increases at later time points until after 24 weeks of radiation. Total collagen in normal mouse skin, determined by hydroxyproline content, remained constant over the 24 weeks, while UV radiation produced significant increases at 4, 8, 12, and 16 weeks, returning to control levels at week 20. There was no change in the degree of hydroxylation at any time point in either group. Thus, chronic UV exposure resulted in increased collagen synthesis until late in the course of irradiation. Because there is a lack of consistent change in the ratio of type III to total collagen, the early increases in collagen content may represent both types I and III, synthesized in relatively unchanging proportions.     

Photoprotective effect of Pothomorphe umbellata root extract against ultraviolet radiation induced chronic skin damage in the hairless mouse

In this study we evaluated the activity of Pothomorphe umbellata root extract on hairless mice chronically exposed to UVB radiation (76.5 mJ/cm(2), 4 days per week for 22 weeks). Mouse dorsal surfaces were treated topically with 20 mg/cm(2) of a carbomer 940 gel (vehicle) with or without P. umbellata root extract to a final concentration of 0.1%, for 2 h before irradiation. Another irradiated group received no topical treatment. A fourth group received no treatment and was not irradiated. Visible skin wrinkling was evaluated using a scale ranging from 0 to 4, where 0 corresponds to no skin modification, and 4 to the maximum visual skin alteration observed in our experiments. Histological measurements were carried out on standard haematoxylin & eosin stained sections. The mean distances between the outermost surface of the epidermis (excluding the stratum corneum) and the dermal-epidermal junction were determined by morphometric analysis. These distances were statistically increased in the irradiated control groups when compared to the nonirradiated control group and to the irradiated group using P. umbellata root extract. These data demonstrate that P. umbellata may be successfully used as a topical skin-protecting agent against the deleterious effects of UV radiation.     

Skin-infiltrating monocytes/macrophages migrate to draining lymph nodes and produce IL-10 after contact sensitizer exposure to UV-irradiated skin

Low-dose UVB exposure induces antigen-specific unresponsiveness to antigen(s) introduced through UV-irradiated skin (tolerance). Analysis of cytokine expression in murine draining lymph nodes (DLNs) revealed that IL-12p40 mRNA and protein expression as well as IL-12p70 protein were upregulated after application of the contact sensitizer 2,4 dinitro-1-fluorobenzene (DNFB) to normal skin. The cellular source of IL-12p40 mRNA was CD11c+ cells. By contrast, following DNFB application to UV-irradiated skin (UV+DNFB), IL-12p40 mRNA was not upregulated, and DLN IL-12p40 and p70 proteins were reduced. UVB irradiation alone did not upregulate IL-10 mRNA, but UV+DNFB upregulated IL-10 mRNA as early as 3-6 hours after DNFB application, immediately preceding a decrease of IL-12p40 mRNA from the level induced by UVB. The infiltration of F4/80+ cells into UV-irradiated skin was followed by a rapid and remarkable increase of F4/80+CD11c(-) cells in DLN 3 hours following DNFB application. FITC/DNFB skin painting and subsequent enzyme-linked immunospot assay demonstrated that flow-sorted FITC+F4/80+CD11c(-) cells from the DLN produce IL-10. Thus, monocytes/macrophages that infiltrated into the skin following UVB exposure migrate to the DLN triggered by contact sensitizers. Production of IL-10 by migrating macrophages, in conjunction with IL-12 inhibition in the DLN, likely reflects a role as mobile suppressive mediators for locally induced UV tolerance.     

Metallothionein-null mice exhibit reduced tolerance to ultraviolet B injury in vivo

Events that induce expression of the metallothionein (MT) gene, such as injection of cadmium chloride, cold stress or topical application of 1,25-dihydroxyvitamin D3, can deplete the number of ultraviolet (UV) B-induced sunburn cells (SBC) in mouse skin in vivo. MT-null mouse skin explants exhibit reduced tolerance to UVB injury in vitro. However, the in vivo response of MT-null mice to UVB injury has not been investigated. In the present study, we investigated the role of the MT gene on UVB injury in vivo. MT-null mice that are deficient in MT-I and MT-II genes were studied and compared with homozygous wild-type mice. Mouse dorsal skin was irradiated with 0.05, 0.70 and 1.40 J/cm2 UVB. The thickness of the dorsal skin was measured with a spring micrometer before and 24 h after UVB irradiation. In addition, SBC were counted 24 h after UVB irradiation. No significant difference was found in the change of skin thickness between MT-null mice and control mice irradiated with low-dose UVB (0.05 J/cm2) (Student's t-test, t = 1.519, P = 0.167). At higher doses (0.70 and 1.40 J/cm2), the skin of MT-null mice became much thicker than that of control mice (Student's t-test, t = 6.576, P < 0.01 and t = 3.142, P = 0.007, respectively). More SBC were detected in MT-null mice skin irradiated with the highest dose of UVB (1.40 J/cm2) (Student's t-test, t = 4.258, P < 0.01). These results suggest that the MT gene in mice has a photoprotective role in vivo.     

Genetic factors in immunosuppression: precise genetic evidence that polymorphism of TNF-alpha dictates UVB-susceptibility in mice

Acute low-dose treatment of murine skin with ultraviolet B (UVB) light impairs induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) in certain inbred strains of mice (termed UVB-susceptible), but not in others (termed UVB-resistant). These deleterious effects of ultraviolet radiation (UVR) are mediated in part by TNF-alpha, which is released from UVR-exposed epidermal and dermal cells. To test the hypothesis that polymorphism of TNF-alpha governs the phenotype of UVB-susceptibility in vivo, various strains of mice received UVB radiation followed by hapten application to induce contact hypersensitivity. Results suggest that the polymorphism at the Tnf-alpha locus dictates UVB susceptibility in vivo.     

Protective effect of topically applied conjugated hexadienes against ultraviolet radiation-induced chronic skin damage in the hairless mouse

Albino hairless mice (SkH:HR-1) exposed chronically to suberythemal doses of ultraviolet B (UVB) radiation display visible skin wrinkling and tumors. Topical treatment of mice with solutions of conjugated dienes (2,4-hexadien-1-ol and derivatives of it) prior to each UVB radiation exposure reduces significantly the severity of these visible alterations. Chronic suberythemal doses of ultraviolet A radiation induce skin sagging, a distinctly different visible skin alteration. The severity of skin sagging is not reduced by topical application of the conjugated dienes tested here.     

Characteristics of skin wrinkling and dermal changes induced by repeated application of squalene monohydroperoxide to hairless mouse skin

We have studied the effect of squalene monohydroperoxides (Sq-OOH), initial products of UV-peroxidated squalene, on the skin of hairless mice. Repeated topical application of 10 mM Sq-OOH to hairless mice for 15 weeks induced definite skin wrinkling. When image analysis was used to compare wrinkle formation induced by ultraviolet B (UVB) irradiation and Sq-OOH treatment, the degree of wrinkling in exposed skin was seen to be similar. However, the characteristics of wrinkles induced by either method differed markedly with regard to direction and distribution. Biochemical analysis revealed a significant decrease in collagen content per unit area and mass in Sq-OOH-treated skin, whereas no changes per unit area and decrease in collagen per unit mass were observed in UVB-irradiated skin. As for glycosaminoglycan (GAG) content per unit area, significant increases were observed in both Sq-OOH-treated skin and UVB-irradiated skin. These changes were not induced by organic hydroperoxides such as TERT-butylhydroperoxide or cumene hydroperoxide treatment. Histological observation revealed epidermal hyperplasia and dermal alterations such as collagen degradation and GAG increases in Sq-OOH-treated skin. Histological changes induced by Sq-OOH were not as pronounced as those induced by UVB irradiation. These results clearly suggest that the wrinkling and changes in dermal collagen content induced by Sq-OOH are qualitatively different to those induced by UVB exposure. This may provide a useful model for the study of skin aging, particularly with regard to collagen content.     

金属硫蛋白基因对小鼠UVB照射的保护作用

目的:研究金属硫蛋白基因对UVB照射反应的影响.方法:敲除小鼠金属硫蛋白基因,背部皮肤剃毛后24 h照射不同剂量的UVB(0.05 J/cm2,0.70 J/cm2,1.40 J/cm2),照光前、后24 h测量小鼠背部皮肤厚度,皮肤组织病理观察照光后24 h日晒伤细胞形成情况.正常野鼠作对照.结果:小剂量UVB照射时两种小鼠水肿程度无显著性差异,大剂量UVB照射时金属硫蛋白基因敲除小鼠背部水肿显著高于野鼠.病理显示金属硫蛋白基因敲除小鼠较野鼠有更多的日晒伤细胞产生.结论:金属硫蛋白基因对急性UVB照射具有保护作用.     

紫外线所致皮肤鳞状细胞癌小鼠模型的建立

目的 应用日光模拟器模拟日光照射SKH-1无毛小鼠,建立皮肤鳞状细胞癌（鳞癌）动物模型,探讨其生物学特性。方法 将91只SKH-1无毛小鼠随机分成7个实验组（每组10只）和7个对照组（每组3只）。实验组每天给予红斑量日光紫外线照射,对照组不做任何处理;在第4、8、12、16、20、24、28周分别处死小鼠行病理检查。实验过程中观察小鼠一般状态和照射区域皮肤变化,分析各期小鼠皮损特点及组织学变化。结果 10周后实验组部分小鼠陆续出现直径≥1 mm的丘疹,紫外线照射20周开始实验组剩余小鼠中39.3%出现肿瘤（11/28）,照射28周后成瘤率达100%（10/10）,累计UVB剂量约为26.99 J/cm2,UVA剂量约为242.91 J/cm2。对照组未见肿瘤形成。照射各时期组病理显示,12周30%、16周33.3%、20周60%、24周87%、28周100%的小鼠具有鳞癌特征。结论 紫外线照射可诱导SKH-1无毛小鼠皮肤组织增生,并随照射时间延长产生皮肤鳞癌。     

Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin

Abstract Generally, many wrinkles form on the human face, and temporary wrinkles eventually become permanent. We evaluated the effects of temporary skin fixation on wrinkle formation after UVB irradiation using the back skin of hairless mice. In the group treated with UVB irradiation immediately after production using cyanoacrylate resin of an artificial groove parallel to the midline, wrinkles formed parallel to the midline, an uncommon direction for wrinkle formation in this mouse model. These wrinkles did not disappear even when the skin was stretched. No such changes were observed in the group in which only the temporary groove alone was produced without UVB irradiation. In 3-D surface parameter analysis, all roughness parameters in the group treated with UVB irradiation immediately after production of an artificial groove were significantly increased relative to the age-matched control group. In contrast, no differences were observed between the group in which only the temporary groove alone was produced without UVB irradiation and age-matched controls. The results of this study suggest that both a temporary groove in the skin and UVB irradiation are necessary for wrinkle formation in the back skin of hairless mice.