298 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS plays an important role in the formation of permanent wrinkles, there have been few studies of the factors associated with wrinkle formation using hairless mice. We previously evaluated the effects of temporary skin fixation on wrinkle formation using the back skin of hairless mice (13). In the group exposed for 20 weeks to UV-B irradiation immediately after production of the artificial groove parallel to the midline, a wrinkle formed at right angles to the groove, which is an unusual direction for wrinkle formation. Therefore, in this study, we examined the first ten-week period quantita- tively, using image analysis to determine how early wrinkle formation can occur, and we also studied the effects of production of a temporary groove in the skin. MATERIALS AND METHODS ANIMALS HR/ICR albino hairless mice were used in this study. This strain was derived by crossing hairless mice (HR/HR), originally obtained from Nisseiken Corporation, Japan, and the albino strain HaM/ICR. The HR/ICR strain represents a line maintained under con- ventional conditions in our laboratory for several generations of hairless brother/ phenotypically normal haired sister mating. All experiments were performed with hair- less female mice only, which had free access to food and water throughout the experi- mental period. Animals were housed in rooms where the lighting without UV emission was automatically regulated on a 12-hour light/dark cycle. RADIATION SOURCE AND PRODUCTION OF ARTIFICIAL TEMPORARY GROOVE The hairless mice were divided into five groups from the same stock at six weeks: group 1, UV-B irradiation immediately after production of the artificial temporary groove group 2, production of the artificial temporary groove immediately after UV-B irradia- tion group 3, production of the artificial temporary groove only (no irradiation) group 4, UV-B irradiation only group 5, no treatment. Group 1 and 2 UV-B mice were placed in cages in groups of nine animals each and were irradiated by a bank of six Toshiba SE lamps with no filtering for UV-B. The distance from the lamps to the animals' backs was 35 cm. The animals were exposed to UV-B at a dose of 65 mJ/cm 2 (1 MED = 70 mJ/cm 2) five times weekly for ten weeks, yielding a total dose of 32.5 J/cm 2. The energy output of the lamps (at 35 cm) was measured with a Topcon Co. Ltd. UV-radiometer 305/365DII. The spectral irradiance of these lamps was measured with an Optical Science Co. Ltd. MSR7000 radiospectrometer, the spectral output of which is shown in Figure 1. The mice in the test groups were anesthetized with ether ten minutes before irradiation, and the back skin was fixed with cyanoacrylate resin to produce an artificial groove parallel to the midline (Figure 2). The control groups were not treated or were treated similarly but without UV irradiation. During the period of exposure, the animals could move around freely in their cages. The template (cyanoacrylate resin)-induced groove seen after completion of the UV irradiation protocol was confirmed to be restored to the previous state after one to two hours. GRADING OF VISIBLE CHANGES Skin wrinkling in hairless mice was assessed weekly as described by Bisett et al. (8), as

SKIN MORPHOLOGY AND WRINKLE FORMATION 299 o 200 300 400 500 Wavelength (nm) Figure 1. Spectral irradiance of the unfiltered SE lamps as measured with the radiospectrometer. follows: grade 0, no coarse wrinkles grade 1, a few shallow coarse wrinkles grade 2, some coarse wrinkles grade 3, several deep coarse wrinkles. The scale ranged from 0 for normal animals to 3 for the heavily wrinkled skin. For convenience in grading, mice were held by the tail with their feet resting against a solid surface to diminish move- ment. WRINKLE MEASUREMENT After ten weeks, impressions were made of the back skin of nine unrestrained mice, using EXAFINE hydrophilic vinyl polysiloxane impression material (GC Corp. Tokyo, Japan). We set the impression of wrinkles on the sample stand so that the measurement surface was horizontal, and produced wrinkle shadows by illumination with light of a fixed intensity at 30 ø, using a fiber optic light source (Nikkon). The shadow images were photographed with a still video camera (MS-C1100) and a digital image recorder (MS- R1100, Minolta) with a macro50 lens system and were input into an image analyzer (LA555 personal image analysis system, PIASS Co Ltd., Japan). Figure 3 shows examples of a shadow image and binary image obtained by extracting shaded areas of the image at a constant gray level. We measured the shadow area for all shadows in one image, using the image analyzer, and calculated the ratio of wrinkle area (%), defined as the ratio of the sum of the shadow area to the measured area. STATISTICS The wrinkle grading score and wrinkle area (%) were expressed as mean + standard deviation. Differences between means were checked for significance using Student's t-test. RESULTS From grading of visible changes, original wrinkling on the back skin of hairless mice