| BackgroundSkin photoaging refers to skin aging or accelerated skin aging due to the long-term sun exposure. People usually call the ageing, which due to genetic and irresistible factors (such as gravity, the important organ physiological dysfunction and so on) are intrinsic aging or natural aging, environmental factors such as ultraviolet (UV), radiation, smoking, wind blowing and exposure to harmful chemicals are called exogenous aging. Because the solar UV radiation is the most important factor in environmental factors resulting in skin aging, it is usually said that exogenous skin aging is skin photoaging.Long-term, excessive sunlight impacts many cell components and structures of the skin, such as epidermal uneven thickening or atrophy, melanocytes irregular hyperplasia or reduced, dermal capillary disordered expansion, bending, dermal inflammatory cell infiltration and so on. The most characteristic change is dermal matrix components change due to the sunlight.Dermal matrix components including all the intercellular substance except water, the main ingredients are elastic fibers, collagen fibers, amino polysaccharide and Protein polysaccharide. These substances are synthesized by dermal fibroblasts. During skin natural senescence, elastic fibers degradation and fragmentation disappeared. UV irradiation can cause degeneration of elastic fibers, deformation, fiber thickening, torsion, and bifurcation.As time passed, the degeneration of elastic fibers arranges in mass accumulation, the flexibility and adaptability of the skin will loss, and skin laxity crack after excessive stretching.another matrix related to photoaging is collagen fiber. Adult skin is mainly formed by type I and type III collagen. Type I collagen accounts for about80%of skin collagen in the dermis, accumulates to the coarse fiber bundles, parallels with the surface, and interweaves a net. with high mechanical stability, it is important components to maintain the skin tension, bearing force, and keep the skin plump. Type III collagen, which is naive, slender collagen fibers, is the main components of reticular fibers, solar irradiation inhibits type I collagen formation, relatively increases type III collagen, eventually lead to the mature collagen bundles decreases, skin laxity and wrinkles. Photoaging is in relation to other ingredients in the matrix, such as amino polysaccharide and protein polysaccharide.The ultraviolet radiation of the sunlight is not destructing the dermal cells and matrix components directly. It can cause dermal inflammation, especially activated perivascular macrophages and mast cell infiltration. Inflammatory mediators and cytokines can lead to tissue lytic enzymes releasing, such as elastase, collagenase, and slow dissolute the matrix components. Reactive oxygen species (ROS) in photoaging plays an important role. UV can increase ROS concentration through a variety of ways. Skin components and high concentration ROS produce complex chemical reaction, generate multiple biological effects. There is growing evidence that ROS in UV induced skin photoaging plays an important role. Large amounts of natural UV base color in the skin, base color molecules absorption UV photon energy in the excited state, generating type I or type II photodynamic reaction with molecular oxygen in the skin immediately, and under the action of a variety of enzymes and transition metal ions generate ROS. In addition to the direct induction of ROS, UV irradiation can also increase ROS concentration through some indirect ways. ROS can also inactivate matrix metalloproteinase inhibition factor to inhibit precollagen synthesis and damage the normal collagen fibers directly. In skin, collagen damage of photoaging also play a role. Growth factor receptors and cytokine receptors trigger skin photoaging formation. Research shows that ligand effects of the action of UV radiation and receptor are similar. It can activate the cell surface receptors for growth factors and cytokine receptors, and receptor activation leads to downstream unique signaling path component activation. UVB is strongly activated agent for JNK, and EGF collaborate with IL-1or TNF also induced JNK activation, when these3ligands (EGF, IL-1and TNF) binding its receptor, activation of JNK is similar to UVB after radiation.UV irradiation leads to skin photoaging mainly associated with immune suppression and immune tolerance. Either the cell or molecular level, the inhibitory effect of UV often manifests as a complex, interconnected network system.The pathogenesis and prevention mechanism of skin photoaging inevitable to invasive sampling, due to occur in exposed areas and relative ethical requirements, to build a strong simulation, simple operation, clear evaluation index of photoaging animal model is needed. Mouse, rat, and guinea pig always use as photoaging animal model. Some people use Wistar rat as an experimental animal, everyday UVA+UVB irradiation light source,8weeks later the typical skin photoaging performed. In the molding process, ultraviolet irradiation dose is the most important, and the most difficult to identify. Molding time should be more than a month in general. Photosensitizer is also used to accelerated molding process, Tsunemichi et al show ethoxypsoralen (8-MOP), which is often used in clinically photochemotherapy, be absorbed by skin can obviously promote shorter the work wavelength of UVA, thereby facilitate photoaging function, therefore, the use of photosensitizers can make photoaging model onset of action rapidly.There are a variety of ways to prevent photoaging, fractional laser has been used to the improvement of skin photoaging.The core of skin photoaging treatment is the stimulation of matrix components in dermal tissue, such as regeneration and rearrangement of collagen fibers, elastic fibers. The reaction of heat stimulation is considered to be very important therapeutic mechanisms. When laser beam diameter is regulated to smaller than hundreds of micrometers irradiated on skin, in certain power density, with either thermal denaturation or real aperture formation, this injury would start the process of wound healing and initiate the skin reconstruction repair procedure, eventually leading to full-thickness skin remodeling and reconstruction including the epidermis and dermis, to achieve the goal of treatment.From the point of view of repair and reconstruction to study fractional laser, it inevitably involves Toll receptors changes. Toll-like receptors(TLRs) belong to a class of natural immunity, are super family of interleukin1receptor/Toll like receptor, and distribute mainly in the immune cells as well as in the surface of epithelial cells which communicated with the outside world. TLRs plays an important role in the start of the defensive anti-pathogenic microbes immune response. This series of changes overlap with tissue repair processes, and more and more studies show that TLRs plays a key role in tissue regeneration, including the repair process of photoaging. For example, TLRs identify some endogenous ligands, such as some degradation of macromolecules, protein hydrolysate, intracellular components of rupture cells and inflammatory products. Through TLRs signal pathway, TLRs start and accelerate wound healing. After body injuried, necrotic cells can be recognized at least by the TLR2, recruited by neutrophils, initiate tissue repair process. The extracellular matrix hyaluronic acid can rapidly degraded in the tissue damage, soluble low molecular degradation products of hyaluronic acid are involved in a variety of inflammatory and repair processes, they are recognized by the TLR4within dendritic cells and endothelial cells activate signal transduction pathways.In clinic, ablative fractional laser can be treatment for photoaging has been confirmed. But present research mainly focus on the clinical experience of the fractional laser treatment, there is no much basic research to study the therapeutic mechanism from microcosmic organization structure, cellular, molecular level.ObjectiveTo discuss the application and significance of Wistar rat as animal model for experimental of skin photoaging. On this basis, fractional CO2laser was used to treat photoaging skin. To detect mechanism of fractional CO2laser in the treatment of photoaging, effect was observed before and post treatment.MethodsSelecting48Wistar rats,16were distributed to experimental group Al and A2respectively,11were in control group B,5were distributed to blank group C.0.5hours, before the ultraviolet lighting topic applications of8-MOP solution on bare skin were used in the experimental group A1. Group A2were received8weeks of continuous irradiation10mins once a day, and the irradiation strength were UVB0.12mW/cm2, UVA1.05mW/cm2respectively. With the same intensity of irradiation, Group Al were exposed to irradiation with UVA10mins and UVB120mins per day, while the control group B without irradiation treatment. Observe the histological appearance, peroxide and antioxidant indexes of MDA/SOD, hydroxyproline contents every two weeks to evaluate the effects of photoaging model. Group A2was randomly divided into group D (embrace10rats), group E (embrace5rats) after photoaging model made, selected as self-control, the bare back of animals in group D and control group B received different doses of fractional CO2laser treatment. Group D1and group B1were exposed with high energy fractional CO2laser(Active FXTM treatment probe, wavelength10600nm, spot size9mm x9mm, point0.125mm, a pulse energy of150mJ/cm2with10%coverage.) and another procedure using low energy CO2laser (with the same energy parameters, but with energy of50mJ/cm2) was performed in the region of D2and B2. While group E and blank group C were untreated. To observe the change of skin histology, peroxide and antioxidant indexes of MDA/SOD, hydroxyproline, and TLR2, TLR4contents on the time points1day,1week,2weeks,3weeks,4weeks after treatment. All the data was analyzed by statistical software SPSS13.0and every group of measurement data was indicated with (X±S); count data used t-test with two independent sample; first of all, we did normality test and homogeneity of variance test to the data, and comparison between every two groups by the least significant difference method (LSD); the one which didn’t fit the normal distribution or have homogeneous variance was analyzed by non-parametric tests. Mean of samples among groups was compared by variance analysis with repeated measures and for those Mauchly spherical test didn’t satisfy spherical hypothesis, we used Greenhouse-Geisser to correct the degree of freedom with statistical significance at P<0.05.Results1Results of photoaging animal modeling:1.1The typical clinical symptoms of chronic photoaging and histological appearances are found in experimental group A1and group A2at the fourth week, eighth week respectively. But the mortality of group Al is56%(9/16).1.2In the early moldelling, with the UV illumination, either group A1or group A2, the MDA contents in rat skin elevate from the second week, and increase ceaselessly along with the aging process, while the MDA contents in the control group B remain stable. 1.3In the early moldelling, with the UV illumination, either in the experimental group A1or A2, the contents of SOD in rat skin reduce from the second week, and continued to declining along with the aging process, while the contents of SOD in the control group B are stable.1.4In the early moldelling, with the UV illumination, either group A1or A2, the contents of hydroxyproline in rat skin reduce from the second week, and continue to declining along with the aging process. While the contents of hydroxyproline in the control group B keep stable.2Results of fractional laser treatment with different energy, including photoaging group D (group D1received high energy laser in the treatment, group D2received low), control group B (with high energy laser in the treatment was group B1, low energy was group B2), none treatment group E, the blank group C.2.1With time passed, the improvement of photoaging skin is obvious either from visual or histological appearance in the laser treatment group group D. The control group group B tissue slices display newborn collagen fibers increasing, and no treatment group E photoaging remain the same.2.2In the experiment, the blank group(group C) and group E (ptotaging untreated), MDA contents remain almost unchanged. With time extending, MDA contents in the laser treatment photoaging group decrease gradually, group D1with high energy laser in the treatment decrease faster, they have been almost restored the normal level of group C at the third week. There have a decline with low energy laser in the treatment of photoaging group, but do not reach the normal level of blank group(group C) at the fourth week. Contents in control group B rise in the early stage, decrease to normal level after lweek.2.3After laser treatment, SOD contents of group D increase gradually, remain relatively stable at the third and forth week, returning to normal level. From the third week, SOD contents of group D are independent with the energy level. SOD contents in control group B reach a peak at the second week, then begin to decline. At the fourth week, they return to normal levels before the treatment. SOD contents in control group B are not influenced by the energy level.2.4Along with prolongation of the treatment, hydroxyproline contents of group D(photoaging treatment group) and group B (control treatment group) continue to increase, hydroxyproline contents of group D restore to normal level at the fourth week. Influenced by the factor of energy level, high energy laser or low energy laser can cause hydroxyproline contents variation in photoaging group D; but the normal control group B do not been affected by the factor of energy difference.2.5Contents of TLR2/TLR4in group D(photoaging treatment group) fluctuate with time and energy. The expression of TLR2/TLR4show the peak on the first day after treatment, decline to minimum at the second week, then recover gradually. Control treatment group B do not been affected by energy difference effect.Conclusion1Wistar rat can act as photoaging model for human skin, its histologic performance and molecular biology index can imitate the characteristic of skin photoaging.2Fractional CO2laser can meliorate visual appearance and histologic performance of photoaging rat.3Fractional CO2laser can improve peroxide and antioxidant indexes of photoaging rat.4Fractional CO2laser can rate an increase of hydroxyproline in skin.5TLR2/4may provide index of efficacy for photoaging by fractional CO2laser therapeutic. |
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