Impacts Of Intense Pulse Light Irradiation On Balb/c Mouse Skin

Background and objective:Photo rejuvenation technique is a kind of non-denudation and non-virulence treatment using continuous wave-lengh (515～1200nm) intense pulse light (IPL) at low energy density. Photo rejuvenation technique is mainly applied in the treatment of certain skin diseases such as photo aging,telangiectasis,spider veins, pigmented lesions and wrinkles. Ultraviolet irradiation of sunlight including UVA and UVB is the most powerful wavelength which causes sunburn, photoaging and skin tumors. The main histologic characteristics of photoaging skin is the change of matrix constitute, including decrease in collagen and deposition of abnormal elastic fibers. It is well known the collagen in dermis is mainly composed of type I(80%)and III(10%) collagens, which is responsible for the elasticity and intensity of skin to produce a healthier skin texture. Fibroblasts can produce and secrete procollagen that consist of collagens. It was reported by Talwar that the procollagen type I and III levels decreased in intrinsic aging and photoaging skin.It has been proved that UV irradiation could promote the human fibroblasts to generate matrix metalloproteinases (MMPs), MMPs could specifically degrade almost all of the extracellular matrix component, and they play an important role in the skin aging and photoaging. Vascular endothelial growth factor (VEGF) is the only growth factor which has a specific effect on angiogenesis. The vascular endothelial cells could secrete considerate VEGF after various kinds of stimuli to be involved in the process of inflammation and carcinogenesis. The main mechanism of the biological effect of IPL is the photothermy and the photochemistry effects generated from the skin tissue irradiated by IPL. However, the study about effects of IPL on animals is rare. This study focused on investigating the consecutive effects of IPL radiation on mRNA expressions of procollagen types I and III, VEGF, MMPs genes as well as protein expressions of collagen types I and III in Balb/c mouse skin from 1day to 8 weeks after twice IPL treatments and confirm the possible mechanisms of IPL radiation with longer potency.Methods:1. Experimental animals: Forty-five female BALB/c mice were randomly divided into 9 subgroups with different experimental terminals, i.e. one non-IPL treated and 8 IPL treated groups. The dorsal hairs of Balb/c mouse skin were shaved with electric clippers. Before treatment and from 1 day to 8 weeks (1d, 3ds,1wk, 2 wks, 3 wks, 4 wks, 6 wks and 8 wks) after treatment, the irradiated skin specimens were taken at above mentioned specific time points, respectively.The biopsied specimens were then conserved in the frozen condition (-70℃).2. IPL irradiation: The shaved skin was first smeared with cooled ultrasonic gel and then irradiated by IPL(560～1200 nm wavelength , 8 x 35 mm spot size, pulse duration 4.0/4.0 milliseconds, pulse delay 30 milliseconds, fluence 15 J/cm²). Each mouse was given twice treatments separated by 2 week intervals.3. Histopathology examination and immunohistochemical analysis: the skin biopsies were placed in 10% phosphate-buffered formalin, then dehydrated in ascending concentrations of ethanol, cleared in xylene and embedded in paraffine. After conventional treatment, 4μm thickness of tissue sections were made for regular hematoxylin-eosin staining or immunohistochemical staining.4. Detection of mRNA expressions of procollagen types I and III, MMP-1, MMP-2 and VEGF genes: Total skin RNA was harvested by utilizing a total tri-Reagent. The mRNA levels of procollagen type I and type III, VEGF, MMP-1 and MMP-2 genes were detected by reverse transcription and polymerase chain reaction (RT-PCR) assay. 1. Histopathologic changes after IPL irradiation: Under microscope, the irradiated skin and non-irradiated control illustrated the pathology results of HE-stained specimens seen in different groups of BALB/c mice. The epidermis showed no change, even through while observing it for 8 weeks after IPL irradiation.In contrast , the dermis became thicker and collagens increased gradually from the 2^nd weeks, showing the largest amount till the end (the 8^th week) of experiment. At the same time, collagen fibers were arranged in a better order and in a paralleled way to the epidermal surface.2. Immunohistochemistry changes after IPL irradiation: In correspondence with HE examination,IPL exposed mouse skin appeared thicker and deeper brown staining for collagen I and collagen III proteins in whole dermis in IPL irradiated groups than non-IPL irradiated groups from 2～8 weeks, which mainly appeared in the upper papillary layer of dermis and around appendages, such as hair follicles and sweat glands. There was statistical difference pre and post IPL treatment from 2 ,3, 4 and 6 weeks to 8 weeks(p< 0.05).3. mRNA expressions of type I and type III procollagens after IPL irradiation: There was no statistical difference of the mRNA expression between the irradiated group and the control group during the first 2 weeks after IPL treatment, but the up-regulation of above genes was achieved starting from the 2nd week and persisted till the 8^th week. There was a significant statistical difference of mRNA expressions between IPL treated and non IPL treated groups(p< 0.05～0.001), which implied IPL irradiation could promote type I and type III mRNA expression in a time-dependent manner.4. mRNA expressions of MMPs and VEGF after IPL irradiation: In contrast to the above results, the mRNA expressions of MMP1 and MMP2 were decreased in time dependent pattern from 2 weeks up to 8 weeks after IPL irradiation(p< 0.05～0.001) even though there was no any modulation of MMPs during the first week. However, the band density of VEGF PCR product on agarose gel electrophoretogram seemed to have no noticeable change during the whole experimental period and the analysis of its fluorescence intensity also revealed no statistical difference (p>0.05).Conclusions:In summary, during such a continuous study from 1 day to 8 weeks, IPL irradiation can induce the mRNA transcription of the procollagen type I and III and promote the protein production of collagen type I and III in a time-dependent fashion as well as rearrangement of the collagen fibers in order. On the other hand, IPL can downregulate mRNA expressions of MMP1 and MMP2 genes in a time dependent way, The data confirm directly the part of mechanism of the photorejuvenation by IPL.