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S.Y. Lee et al. / Journal of Photochemistry and Photobiology B: Biology 88 (2007) 51–67 53 fit the contour of the patient’s face optimally. The red light head (Omnilux reviveTM, Photo Therapeutics Ltd.) con- sisted of four panels containing 420 LEDs each arranged in the same way. The treatment heads delivered symmetri- cal peak wavelengths; 830 ± 5 nm for the infrared light and 633 ± 6 nm for the red light. The irradiance was 55 mW/ cm2 for the infrared light and 105 mW/cm2 for the red light at a distance of 1 to 10 centimeters from the light source. The radiant fluences, or doses, during a single treatment for twenty minutes were 66 J/cm2 and 126 J/cm2 for the infrared and red treatment heads, respectively. 2.3. Study design All patients were randomly divided using computer-gen- erated random numbers into four groups of 28 patients each. Group 1 was treated with the 830 nm head alone, group 2 with the 633 nm head alone, group 3 with a com- bination of the 830 and 633 nm heads by alternating them in that order, and group 4 with a sham treatment light as the control group. We used the standby mode of the 633 nm LED head as the sham treatment. In all groups, the patients were treated only on the right half of the face with the left half being occluded. The split- face model within the control group was set to detect any possible effects of the sham treatment. This study design allowed us two ways to compare the clinical efficacy; a within-patient comparison between the treated right side and the covered left side, and a group-to-group comparison between the treated sides of three treatment groups and those of the control group, which offered a tool of dou- ble-checking the differences. The use of objective methodol- ogy which offered numerical values representing the clinical efficacy technically enabled this ‘double comparison’. Objective instrumental measurements of the melanin level and the skin elasticity were carried out before treat- ment; at every treatment session for the former, at every other treatment session for the latter. After the measure- ments, each patient washed his/her face and was treated for twenty minutes in the supine position with the wave- length of light as set by the protocol of his/her group. The distance between the irradiating head and the patient’s nose was about 3-5 cm. In group 3, we alternated sessions of the 830 nm and 633 nm LED treatment in succession, with the 830 nm treatment head being used first. Goggles were worn to protect the retinae from direct illumination. When the treatment was over, the instrumental measure- ments for the melanin level were performed in the same way as before treatment. In this manner, the therapy was performed twice a week for four weeks at a three to four- day interval between each session. 2.4. Clinical assessment Digital clinical photography (Canon 300 D) of both peri- orbital areas of each patient was taken at baseline, at week 3 during the treatment period, and at 2, 4, 8, and 12 weeks after the treatment completion. Profilometric evaluation using silicon imprints (Visiometer SV600TM, Cour- age+Khazaka, Ko ̈ln, Germany) was performed on the outer canthus of both periorbital areas (so-called crow’s feet zone) at baseline and at 2, 4, 8, and 12 weeks after the final treatment session. Objective instrumental mea- surements of the melanin level were carried out with a MexameterTM (Courage+Khazaka) before and after each treatment session, and measurements of the skin elasticity were performed with a CutometerTM (Courage+Khazaka) at baseline, at weeks 2, 3 and 4 during the treatment period, and at 2, 4, 8, and 12 weeks after the final treatment ses- sion. The sites of evaluation were identical for a given mea- surement through all assessment points. All measurements were performed by the same physician. 2.5. The subject and the investigator global assessments The subjects were kept unaware as to which group they had been randomly assigned throughout the study period. The subject global assessment, or satisfaction level, was evaluated at week 3 during the treatment period and at 2, 4, 8, and 12 weeks after the treatment completion by rating on a six-point scale (worse, no change, fair, good, and excellent) by the patients themselves. The investigator’s assessment was performed by two blinded dermatologists through evaluation of the serial clinical photos with random codes. Their assessment was rated on a five-point scale and scored as follows; 1 for worse, 0 for no change, 1 for mild improvement, 2 for mod- erate improvement, and 3 for marked improvement. The results of the assessment were gathered and analyzed retaining the random coding. The codes were broken only after the assessments were finished. 2.6. Tissue assay evaluation 2.6.1. Preparation of the specimens A total of 19 patients volunteered for punch biopsies; 5 ingroup1,6ingroup2,5ingroup3,and3ingroup4. From among them, two patients randomly chosen for each group had 3 mm punch biopsies on the extensor surface of their left forearms before the first treatment and 20 minutes after the last treatment. In these patients, their left forearm received real or sham treatment identical to that delivered to their face. The specimens from these patients were pro- cessed for real time reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the messenger ribonucleic acid (mRNA) levels of interleukin-1ß (IL-1ß), tumor necrosis factor-a (TNF- a), interleukin-6 (IL-6), intercellu- lar adhesion molecule-1 (ICAM-1), and connexin-43 (Cx43). The remaining volunteers from each group had 2 mm punch biopsies on the lateral aspect of their right cheeks before the first treatment and 2 weeks after the last treat- ment. The specimens taken before the first treatment from these patients were embedded in paraffin and processed forPDF Image | LED phototherapy for skin rejuvenation
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