Chronic Stress-induced Depression In Mice Chloasma Experimental Animal Models

IntroductionChloasma complex etiology,the main pathogenic factors have ultraviolet radiation, endocrine disorders,emotional changes.Long-term ultraviolet radiation will enable the proliferation of melanoma cells,and melanocytes are tyrosinase formation of a series of oxidation and in vivo oxidation and anti-oxidation process anomalies,such as lipid peroxidation(LPO) increased,SOD decreased and so on,so Melanin can be through the skin pathomorphology,LPO derivatives of MDA and SOD volume change research Chloasma experimental animal models.This issue using chronic stress stimulation-induced depression in mice with estrogen(progesterone) to attack the body at the same time local irradiation of mice to ultraviolet Chloasma of experimental animal models,as well as with other mice Malacosoma experimental animal model of plaque-control study.PurposeThrough to local ultraviolet radiation mice injected with progesterone injection and stimulation of chronic stress-induced depression General role,the establishment of chronic stress-induced depression Chloasma type mice experimental model,and set up methods of ultraviolet radiation model comparison.Materials and MethodsMaterials1.AnimalHealthy female BALB/C mice,cleaning class(Academy of Military Medical Sciences Laboratory Animal Center).2.Experimental drugs and reagentsProgesterone Injection 20mg/ml,superoxide dismutase(SOD) kit, malondialdehyde(MDA) kit(Nanjing Institute of Bio-engineering completed),HMB45 rat monoclonal antibody(abcam UK companie).3.Experimental equipment GermanyDuaLightTM UVA/UVB therapy instrument high-energy ultraviolet(UV120-2), CMIAS true color multi-functional pathological image analysis system 2000.Methods1.Animal Grouping:30 mice were randomly divided into 3 groups:normal group 6,ultraviolet Group 12,chronic stress-induced depression group 12.Model time,the first batch of experiments in mice for 21 days for the second group of mice 28 days.2.Experimental packetThe Normal Group:Weekly unhairing 1～2 times every day by intramuscular injection water for injection,5ml/kg body mass.Continuous observation of mice 21 days unhairing Change skin.Ultraviolet Group:First through the pre-experiment BALB/c mouse skin UVA (UVA 330nm～380nm) the smallest amount of phototoxic(MPD) for 7J/cm 2 ultraviolet B(UVB 304nm～311nm) the smallest amount of erythema(MED) for 480mJ/cm 2.According to pre-experimental exposure methods,as follows:1st day to day 0.7MPD +0.7 MED irradiation time,irradiation for 6 days,7 days a slight mouse skin scab desquamative,stop exposure to 2 days;No.9 days,12 days 0.SMPD +0.5 MED irradiation time,the first 16 days,19 days 0.25MPD +0.25 MED irradiation time; the first group:21 days exposure.The second group at one week after cessation of stimulation executed.Chronic Stress-induced depression Group:In accordance with the classical model of depression to improve treatment methods:ultraviolet radiation with UV Ways Group. Daily intramuscular injection of 0.4%progesterone,5ml/kg body mass,the first batch of mice for 21 days.The second group of mice at one week after the cessation of all stimulation executed.3.Determination of depression model(1) Open-field experiment(2) Beginning the end of the experiment in mouse body mass(weight) Change (3)Liquid consumption test4.MDA and SOD Activity assayAt the same time,the liver and skin hair removal,using pre-cooling with normal saline(4℃) rinse,divisible impurities,drying,cut the skin 0.2g,liver 0.5g,respectively, there is 9 times Add to Organize a block of pre-cooling with normal saline small test-tube will be cut and break to pieces,organizations are slurry poured into the glass tube,the upper and lower rotating grinding 20 to 30 times,and fully subject to pulverisation,so organizations slurry.Are 3500r/min centrifugal slurry after 8 minutes, take the supernatant using Detect Kit MDA,SOD activity.5.Skin melanoma cells Pathomorphological observationWhen the first batch of mouse skin unhairing terribly noticeable(21 days exposure),taking a hair of skin(1cm×1cm),10%formalin-fixed,conventional paraffin-embedded sections.Again by dewaxing to water,antigen retrieval,the application of immunohistochemical method using HMB45 as the first antibody,DAB chromogenic hematoxylin counter-stained.With PBS in place of anti-1 negative control to do.The second group of mice in the experiment were killed 28 days after the skin, more than the pathological steps Ibid.6.Melanin-positive target by computer image analysis,Each slice at 40X microscope objective,the randomly selected five vision for color image acquisition and storage,to calculate each morphological horizon positive goals(target goals) in the number density,optical density parameters of the sum of the average,each group standard deviation between the parameters((?)±S),standard error, coefficient of variation.7.Statistical analysisSPSS 12.0 statistical software,data((?)±S) said that those engaged in analysis of variance,T test.ResultsDepression modelProgesterone+UV+chronic stress-induced depression in mice of wear grid frequency,vertical frequency,the number of grooming than the normal group decreased,and there is significant difference.Weight gain at least,dessert consumption was significantly lower than group(P＜0.05) are prompted to chronic stress in mice 21 days after the emergence of signs of depression.MDA and SOD levelsIn Group 5,the UV group,chronic stress model of depression in two groups of mouse liver and skin of the MDA level higher than the normal group,SOD activity than the normal group declined.Among them,the chronic stress-induced depression group mice 21 days after skin SOD treatment has a very statistically significant difference(p＜0.01),chronic stress-induced depression group of mice 28 days of skin, liver MDA has a very significant difference(P＜0.01).Prompted to set up such a model law for the mouse skin,liver MDA,SOD activity of the impact of very large.Cutaneous melanoma cells pathomorphology results1.Mouse epidermal melanosis quantityModel group mouse skin melanocyte number than an increase in the normal group, one of 21 days of chronic stress-induced depression group has significant difference. Model group was significantly higher than the number density of the normal group, especially the chronic stress model of depression can be seen 21 days group and normal group were significantly different.2.The depth of mouse epidermal melanosisModel mice skin Melanin positive goal average optical density was significantly higher than the normal group,there is statistically significant difference.ConclusionComparison of the role of UV in this experiment the same time and one week after cessation of exposure to experimental animal models in mice Chloasma changes in skin pigmentation,the result would be stress-induced depression in chronic mouse model of type Chloasma melanosis area expanded,deepened pigment Obviously,mice activity in skin melanocytes,and melanocytes to maintain calm a long time,it is better able to explain the reliability of the experiment.