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S.Y. Lee et al. / Journal of Photochemistry and Photobiology B: Biology 88 (2007) 51–67 63 Table 5 Real time RT-PCR results showed a noticeable increase in the mRNA levels of IL-1ß, TNF-a, and Connexin 43 in the treatment groups. The changes in the mRNA levels of IL-6 and ICAM-1 did not show a consistent tendency but could not find any significant difference. We hypothe- size that the decrease in the melanin levels measured by MexameterTM were possibly as the result of reasons other than the direct decrease in the amount of melanin pigment, for example, changes in the reflection of light from the epi- dermis or alteration in the absorption and scattering char- acteristics of light in the dermis. Because the MexameterTM quantifies melanin pigmentation by comparing the amount of reflected light to that of a device-generated reference beam [49], morphological changes in the epidermis and even in the dermis may alter this reflected beam and cause a decrease in the measured levels without any actual decrease in the total melanin amount. Further studies with larger sample sizes and various biologic investigations will be necessary to find out the mechanism of action of improvement of the skin tone by red LED phototherapy. Of particular interest in our study was that the pro- inflammatory cytokines, IL-1ß and TNF-a, were induced by non-thermal LED treatment. It has been suggested that these primary cytokines are produced by inflammatory cells which are recruited to heal the intentionally formed photothermally-mediated wounds associated with laser treatments, and that this cascade of wound healing conse- quently contributes to new collagen synthesis [7,20, 22,42,50]. Our results suggest that LED therapy may be useful to induce this wound healing process, through the athermal and atraumatic induction of a subclinical ‘quasi-wound’, even without any actual wounding by ther- mal damage which can cause complications as shown in other thermal laser treatments. Some investigations have demonstrated that proteolytic clearance of photodamaged, fragmented collagen by MMPs might facilitate biosynthesis of new collagen [42]. Although we did not measure the amount of MMP-1 and MMP-2 early after LED therapy, we observed that there were not any significant changes in their status in the 2 weeks post-treatment specimens. It is not clear whether MMPs were not induced at all by LED therapy or if they were induced earlier after the therapy and decreased again to baseline during the subsequent 2 weeks. However, it is highly probable that MMPs might be induced in the early response, because IL-1ß and TNF-a, which were shown to be increased by LED therapy in our results, are cytokines that induce production of MMPs. Additionally, our results revealed a significant increase in the amount of TIMP-1 and TIMP-2 two weeks after LED therapy, which led us to hypothesize that the remark- able increase in the amount of collagen shown in the histo- logic evaluation might be related to induction of TIMPs because of their action of inhibiting MMP activities. Putting together these observations, we hypothesize that increased production of IL-1ß and TNF-a might have induced MMPs in the early response to LED therapy, which might clear the photodamaged collagen fragments to facilitate biosynthesis of new collagen fibers, and that subsequent events of normalization of MMPs status and increase in the amount of TIMPs might protect the newly Group 1 (830 nm alone) 13.3 1.6 0.14 1.39 1.31 Group 2 (633 nm alone) Group 3 (830 nm and 633 nm) Group 4 (Control sham light) 1.59 0.38 0.26 0.43 0.92 IL-1ß TNF-a IL-6 ICAM-1 Connexin-43 6.165 13.7 1.36 2.5 0.5 0.74 0.54 5.39 2.53 2.42 Values are fold change from baseline. that the dermal matrix remodeling is limited to the areas which are directly or indirectly affected by thermal damage by the nonablative rejuvenation devices [38,39], our study results revealed dermal changes even in an area deeper than the usually agreed optical penetration depth of 633 nm (550 lm) [45], although 830 nm is associated with much deeper penetration than 633 nm. It is unique that these changes were actually obtained without causing any ther- mal damage to the dermis, using a non-thermal LED light source, which we consider is a significant point of our results. The post-treatment TEM findings revealed highly activated fibroblasts containing increased numbers of dilated endoplasmic reticula and surrounded by abundant thickened collagen bundles and elastic fibers, which may probably be the source of the dermal matrix changes pro- ducing increased amounts of collagen and elastic fibers. In the present study, increases were noted in elastic fibers in addition to collagen fibers, after LED phototherapy. This finding also corresponds with other nonablative rejuvena- tion studies [19,20]. Although increased numbers of elastic fibers have also been found in photodamaged skin, such fibres demonstrate degenerative elastotic changes. However, in our study, Alcian blue staining ruled out this possibility, showing no evidence of deposition of acid mucopolysaccha- rides, which are known to exist in a large amount in elastotic material [46]. In addition, the elastic fibers shown in the TEM findings appeared normal-structured, containing amorphous, electrolucent elastin in the center with numer- ous fine microfibrils embedded largely in the periphery of the elastin, arranged parallel to the long-axis of the elastic fiber [47]. It is possible that this significant increase in elastic fibers may play a role in rejuvenating photoaged skin through improving the skin elasticity and resilience, as seen in the objective CutometerTM data, although this hypothesis is yet to be confirmed by further studies. The significant decrease of melanin levels after red LED irradiation has been already observed in our previous clin- ical study on acne treatment [48]. In addition, many sub- jects spontaneously reported their perception of brightening of the skin tone in the present study, as had been also the case in the previous study. We performed Schmorl’s staining to elucidate any changes in the amount of melanin pigment between before and after treatment,PDF Image | LED phototherapy for skin rejuvenation
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