The Effect Of IPL On Collagen In Rat Skin And On Its Modulating Factors

Skin aging includes chronological aging and photoaging. Their morphological phenotypes are different, but both of them display prominent alterations in components of ECM at the histological level. Chronological aging happens in sun-protected skin that shows an atrophy of the skin with fine wrinkling and reduced elasticity. Photoaging of the skin is caused by long affecting of environmental factors at the basis of chronological aging. Clinically, photoaging is characterized by deep wrinkling and furrowing, laxity, a leathery appearance, reduced resilience, increased fragility and impaired wound healing. The main environmental factor that damages the skin is UV radiation from the sun. The treatment of photoaging skin has experienced invasive and non-invasive stages. Because of its more side-effects, clinical application became less and less. With non-invasive specialty and less side-effects, non-invasive therapy got more and more commonly used in clinic and has gotten good results. IPL technique is a new developed non-ablative method for treating photoaging skin. It has been proved that IPL can improve many symptoms of photoaging skin in clinical application. Although there were some reports that IPL can enhance the content of collagens inskin in clinical studies, the quantitative analysis of the alteration of collagens has not yet been made. So, in theory, objective basis that supports the clinical application of IPL technique is still lacking. Overseas study reports about other molecular mechanisms of IPL action are still few, otherwise domestic study on this aspect is still vacant. Our experiment aimed at the quantitative analysis of the alteration of collagens after radiation of IPL and providing objectively theoretic basis for application of IPL technique in clinic. At the same time, we also observed the changes of some cytokines and growth factors that regulate metabolism of collagens on the chance of partially elucidating molecular mechanisms of IPL action.15 cats were served as study objectives in our experiment. Three dorsal sites of each cat were radiated respectively with IPL system at the same parameters. Skin specimens were taken at both non-treated and treated locations at day 1, 3, 5, 7, 15, 30 after IPL irradiation. We studied as follows in four parts:Part 1: Three stains methods were used on each specimen, including HE stains, V.G stains ( staining collagens) and type I/III collagens immunohistochemical stains. The area ratios of type I/III collagens stains were mensurated respectively by pathological image quantitative analysis system, observing the alteration of collagens content after IPL treatment. Our results revealed that HE stains of dermis at treated sites at day 15, 30 became stronger than that at non-treated areas and fibres arrayed more compactly and the amount of fibroblasts increased, especially obviously at superficial areas of dermis. V.G stains showed more and compactly arraying collagen fibres after treatment. Immunohistochemical stains of type I/III collagens at day 15, 30 after irradiation were stronger than that atnon-treated sites. The results of area ratios of type I/III collagens mensurating and statistical analysis showed that there was a extremely significance difference(P<0.001) comparing post-treatment verse pre-treatment. The increasing of type I collagen was seen in whole dermis, but more obviously at superficial areas of dermis. The increasing of type III collagen was seen mainly at superficial areas of dermis and surrounding areas of cutaneous appendages. Part 2: To each specimen, TGF-Pi mRNA in situ hybridization and bFGF immunohistochemical stains were employed to observe the changes of TGF-β1 mRNA and bFGF after irradiation in order to elucidate the part that growth factors operate in IPL action mechanism. Our results revealed that TGF-β1 mRNA and bFGF have no expression in normal skin. TGF-β1 mRNA started to express at day 1 after treating. Its expressions were seen in both epidermis and dermis layers. The expression in epidermis located the whole layer. Some expressions of endothelial cells and inflammatory cells were seen in dermis. To day 3, 5 after treatment, the expressions became stronger and at day 7 reached the highest. At day 15 after treatment the TGF-pi mRNA expressions got weaker mainly in epidermis in which only a part of basal cells expressed TGF-pi mRNA. But the expressions of dermis did not weaken at this time. The expressions of epidermis bated further and that the expressions of dermis cells still presented stronger at day 30 after treating. The expressions of bFGF were different from that of TGF-Pi mRNA. The expressions of bFGF showed negative at day 1 after treatment and just presented weak positive articles in superficial epidermis at day 3, but the expressions in dermis were still negative at that time. Not only can we observe the stronger expressions of bFGF in whole epidermis, but also canobserve the expressions of endothelial and inflammatory cells in dermis as well as the positive articles around some cells at day 5 post-treatment. With the time going, the expressions of bFGF gradually became strong. The expressions got further strong at day 7, 15, and reached the highest in epidermis at day 15. But the expressions in dermis did not changed obviously during this period of time. We noted that the expressions of bFGF in epidermis still were at highest level at day 30 and at the same time the expressions in dermis attained the highest level, too. Besides endothelial and inflammatory cells, we also observed more fibroblasts expressing bFGF and more positive articles around cells than before.Part 3: We actualized MMP-1 and TIMP-1 proteins immunohistochemical stains dissection on each specimen to observe their changes after IPL irradiation in order to know their action in dermis remodeling. The results showed that no expressions of MMP-1 happened in normal skin. The expressions of MMP-1 were detected in middle- and up-layers of epidermis at day 1, but negative in dermis. At day 3, stronger expressions were seen in whole epidermis, however, no obvious expressions being detected in dermis. We found that the expressions of MMP-1 grew further strong at day 5 than day 3 and some endothelial and inflammatory cells presented positive expressions of MMP-1 in dermis at this time. The highest expressions of MMP-1 happened at day 7, showing brown positive articles and more positive articles in the dermal interstitial space. The expressions of MMP-1 in epidermis began to weaken showing weaker positive reaction at day 15 without changing of MMP-1 expressions in dermis in which a few positive fibroblasts also been detected. The expressions of MMP-1 in whole skin were at low levelat day 30. TIMP-1 did not express in normal skin being the same as MMP-1. Being different from MMP-1, TIMP-1 did not express at day 1. The expressions of TIMP-1 just emerged in epidermis at day 3, and that a few endothelial cells presented positive expressions in dermis. The expressions of TIMP-1 did not have obvious changing in epidermis at day 5, 7, but expressive positive cells increased obviously than at day 3, especially at day 7 the expressions of fibroblasts enhanced prominently. The highest expressions of TIMP-1 emerged in epidermis at day 15 with no obvious changing of expressions in dermis. To day 30 after treatment, the expressions in epidermis were basically the same as that at day 3, at the same time, the expressions in dermis were still at higher level. At total level, the expressions of TIMP-1 were stronger than that of MMP-1 at day 30. Part 4: HSP70 protein immunohistochemical stains were carried out on each specimen to observe the dynamic changing of HSP70 after IPL treatment in order to understand its action in dermis remodeling under the heat stress caused by IPL irradiation. The results revealed that HSP70 did not express at non-treated sites. At day 1 after IPL treatment, the expressions of HSP70 can be seen in both the epidermis and dermis. The expressions became further strong at day 3, 5 and reached the peak at day 7. We noted that the positive reactions began to weak gradually at day 15 and were weaker than that at day 5, 7 but stronger than that at day 1,3. Observing at day 30, we found that the expressions of HSP70 basically disappeared. The cells that positively expressed HSP70 protein were mainly endothelium, sebaceous gland cells and some inflammatory cells. Conclusion According to experiments above, we can conclude that: 1. By means of quantitative analysis of changing of collagens content,it can be confirmed that the irradiation of IPL can enhance the content of collagens in dermis. Because collagens are the important components, increased content of collagens is meaningful to remodeling of dermis after IPL irradiation.2. The changing of the expressions of TGF-β1 mRNA and bFGF after IPL irradiation can be explained that TGF-Pi and bFGF are very important to modulating metabolism of collagens during the remodeling of skin. TGF-β1 not only plays an important modulating role during the early stage of dermal remodeling, but also plays a certain role during the middle and late stages of remodeling. Being different from TGF-β1, bFGF acts mainly during the late stage of remodeling. Although bFGF plays a certain role to some extent during early stage of remodeling, its role is next to the TGF-β1's Ys role.3. During repairing of skin heat damaging caused by IPL, not only does MMP-1 play an important modulating role in the early stage of breaking down denaturalization and necrosis tissues in dermis, but also does play an important role in balance of modulating metabolism of ECM in the late stage of repairing. TIMP-1 plays an important modulating role in breaking down denaturalization and necrosis matrix components by MMP-1 in the early repairing stage of skin heat damaging caused by IPL irradiation, namely preventing from excessive decomposing of ECM to hinder the process of ECM remodeling. The expressions of TIMP-1 in the late repairing stage exceeded that of MMP-1, this further explains that matrix deposit beyond matrix breaking up is beneficial to dermal remodeling.4. Mutual modulating relationships exist between TGF-β1 and bFGF, MMP-1 and TIMP-1, and among each factor.5. As an important factor that regulates heat stress reaction, HSP70...