cm2 of 96% UVA/4% UVB using a solar simulator (Sol3A Class AAA Solar Simulator, Newport Corporation, CA). The 20J/cm2 and 100J/cm2 UV doses falls into an average range of a 3MED and 13MED respectively based on skin phototypes II-III.23 The 20J/cm2 condition simulated the effects of a 1-week exposure to the maximal level of daily UV condition, while the 100J/cm2 demonstrated an elevated and severe non-physiological level of UV exposure and serves as a contrast group.24 Following irradiation, skin explants were cultured in a 12-well transwell at an air-liquid interface in Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum and 1% Penicillin- Streptomycin at 37°C and 5% CO2. The group receiving 20J/ cm2/5x over 1 week were subjected to daily UV-exposure conditions and returned to the incubator. Following the 8-day culture period, all biopsies were processed for histological and transmission electron microscopy analysis.
H&E and Immunofluorescence Staining
Skin explants were processed for hematoxylin and eosin staining (Tejas Pathology, Trumbull, TX) and frozen sectioning. Frozen section samples were fixed in methanol/acetone and blocked with 10% normal goat serum for 1 hour at room temperature. Tissue sections were then incubated with TUNEL (Reveal Biosciences, San Diego, CA), Rabbit Polyclonal to Anti- Transglutaminase 1 (Novus Biologicals, Centennial, CO), Rabbit Polyclonal to Anti-Involucrin (Abcam, Cambridge, MA), Mouse Monoclonal to Anti-Desmoglein 1 (Abcam, Cambridge, MA), Rabbit Polyclonal to Anti-Claudin 4 (Abcam, Cambridge, MA), or Anti-Laminin 5 y2 chain (Millipore Sigma, Temecula, CA) primary antibody. Following the primary staining, tissue sections were then incubated with secondary antibody Goat Anti-Rabbit IgG H&L Alexa Fluor 594 or Goat Anti-Mouse IgG H&L Alexa Fluor 594 (Invitrogen, Carlsbad, CA) and counterstained with DAPI. Frozen sections were stained with Rabbit Polyclonal to Anti- Filaggrin (Abcam, Cambridge, MA) according to the Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam, Cambridge, MA). All sections were then imaged with a fluorescent microscope (Leica DM500, Wetzlar, Germany).
Transmission Electron Microscopy (TEM)
Tissue explants were fixed in 5% glutaraldehyde solution and prepared for TEM as previously described by Van den Bergh et al.25 Sections were imaged on Jeol 1200 EX Transmission Electron Microscope (Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ) at 80kV.
H&E and Immunofluorescence Staining
Skin explants were processed for hematoxylin and eosin staining (Tejas Pathology, Trumbull, TX) and frozen sectioning. Frozen section samples were fixed in methanol/acetone and blocked with 10% normal goat serum for 1 hour at room temperature. Tissue sections were then incubated with TUNEL (Reveal Biosciences, San Diego, CA), Rabbit Polyclonal to Anti- Transglutaminase 1 (Novus Biologicals, Centennial, CO), Rabbit Polyclonal to Anti-Involucrin (Abcam, Cambridge, MA), Mouse Monoclonal to Anti-Desmoglein 1 (Abcam, Cambridge, MA), Rabbit Polyclonal to Anti-Claudin 4 (Abcam, Cambridge, MA), or Anti-Laminin 5 y2 chain (Millipore Sigma, Temecula, CA) primary antibody. Following the primary staining, tissue sections were then incubated with secondary antibody Goat Anti-Rabbit IgG H&L Alexa Fluor 594 or Goat Anti-Mouse IgG H&L Alexa Fluor 594 (Invitrogen, Carlsbad, CA) and counterstained with DAPI. Frozen sections were stained with Rabbit Polyclonal to Anti- Filaggrin (Abcam, Cambridge, MA) according to the Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam, Cambridge, MA). All sections were then imaged with a fluorescent microscope (Leica DM500, Wetzlar, Germany).
Transmission Electron Microscopy (TEM)
Tissue explants were fixed in 5% glutaraldehyde solution and prepared for TEM as previously described by Van den Bergh et al.25 Sections were imaged on Jeol 1200 EX Transmission Electron Microscope (Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ) at 80kV.
RESULTS/DISCUSSION
The health risks associated with prolonged UV exposure on unprotected skin are well-documented. While the shorter wavelength of UVB light is less able to penetrate the skin, excessive exposure to solar UVB irradiation induces DNA damage and inflammation.26 The longer wavelength of UVA light penetrates deeper into the skin and is one of the key exogenous factors promoting premature skin aging and inducing oxidative stress. To date, there are limited studies that investigated the relationship of prolonged sun exposure with skin barrier function.11,14,22 The objective of this study was to elucidate the effects of exposure to high doses of UV irradiations on structural and molecular properties of skin barrier using ex vivo human skin, a model that is able to closely represent physiologically relevant changes. To accomplish this, a solar simulator was utilized with a filter that allowed a balanced ratio of 4% UVB/96% UVA in order to provide physiologically relevant UV energies. The doses elected for this study are 1) 5x exposure to 20J/cm2 for 1 week and 2) 1x exposure to 100J/cm2. The 20J/cm2 group is designed to simulate the effects of 1-week exposure to the maximal level of daily UV condition, while the 100J/cm2 group serves as a contrast group when an extreme, above-physiological level of UV irradiation is applied. Multiple skin lots from different donors have provided consistent results.
Structural Changes in UV-Irradiated Tissue
The relationship of UV-exposure with tissue structure was evaluated by hematoxylin and eosin (H&E), Laminin 5 staining, and TUNEL staining to assess epidermal apoptosis. One-time exposure of 20J/cm2 UV dose displayed no structural changes while a daily exposure of 20J/cm2/5x demonstrated localized apoptosis in the stratum granulosum (Figure 1A). A one-time
Structural Changes in UV-Irradiated Tissue
The relationship of UV-exposure with tissue structure was evaluated by hematoxylin and eosin (H&E), Laminin 5 staining, and TUNEL staining to assess epidermal apoptosis. One-time exposure of 20J/cm2 UV dose displayed no structural changes while a daily exposure of 20J/cm2/5x demonstrated localized apoptosis in the stratum granulosum (Figure 1A). A one-time
