| BackgroundDepression is a complex cause,multiple symptoms,high incidence of affective disorder type disease.In order to cope with the increasing pressure of depression,countries around the world will invest more in its research.At the same time,the development of more rapid,effective,economical antidepressant drugs requires a lot of experimental research,which will be at the expense of consuming a large number of experimental animals.However,our country is trying to carry out the standardization of the ethics review of experimental animal welfare,and has formulated and introduced the "new national standard",emphasizing the principle of substitution,reduction and optimization(3R).Therefore,on the basis of Science and Technology Planning Project of Guangdong Province," Research on alternative methods for animal experiments on depression using somatic animals and hippocampal nerve cells ",this paper carries out part of the research on the correlation between corticosterone induced cell injury and CUMS depression rat model and the protective effect of β-asarone were studied,and the partial replacement methods of depression animal experiment were further found in order to reduce the experimental cost,improve the efficiency and implement the new standard.ObjectiveThe depression model of SD rats and the model of nerve cell injury induced by corticosterone were established by chronic unpredictable mild stimulation of(CUMS).Based on the experimental results and the correlation analysis,the possibility of partly replacing or reducing the use of experimental animals was discussed.MethodsSPF SD rats were divided into normal group,model group and fluoxetine group.CUMS depression model was established by swimming,electric shock,tail clamping,water ban,fasting,day and night reversal,crowding and so on.The whole process lasted for 6 weeks.According to the appearance and morphology of the rats,the depression score was carried out,and the changes of body weight were observed at the same time.At the 4th week,fluoxetine group began to intervene.6 weeks later,the model was successfully established according to the depression score and weight change of rats.At the end of the experiment,the rats were taken from the brain and the serum containing drugs was prepared.the contents of corticosterone(CORT)and neurotransmitters(5-HT,DA,NE),the contents of neurotransmitters and IL-10,TNF-α,BDNF,pERK and VGF in brain tissue were measured.The hippocampal tissue of the rat was cultured in a group for cell culture,and the neurotransmitters and the cell viability were detected after the drug-containing serum was used.At the same time,the correlation analysis of the data results of the above-mentioned experiments was carried out to find out the factors which are closely related to depression in the model.The cell model was established by using cort damaged cells to detect the survival rate of hippocampal neurons in neonatal rats and pc12 cells,and the survival rate of hippocampal neurons in CUMS depressed rats was compared with that in cell model,so as to explore the feasibility of using this cell model to carry out the experiment.The content of IL-10,TNF-α,BDNF,pERK and VGF in the cell model was detected by the intervention of the cell model by using the β-asarone ether,and the cell model and the detection index of the rat depression rats were analyzed,and the method of replacing or reducing the amount of the experimental animal was explored.ResultsThe depression score of the model group was significantly higher than that of the normal group and fluoxetine group.The depression score of the model group was significantly different from that of the normal group and fluoxetine group.The depression score of fluoxetine group was lower than that of model group,but higher than that of normal group.There was no significant difference in the behavior of the rats before the model.At the beginning of the second week,the weight of the model group and the fluoxetine group decreased,the level of the open field experiment,the difference of the vertical movement and the spontaneous activity were significantly reduced,the preference degree of the sugar water decreased,the forced swimming time was shortened,and there was a significant difference with the normal group.At the 4th week,after the drug intervention,the difference between fluoxetine group and normal group decreased gradually,and the difference between fluoxetine group and model group increased gradually and began to show significant difference.The content of corticosterone in serum of the model group was significantly increased,which was significantly different from that of the normal group.The content of serum corticosterone in fluoxetine group decreased.The content of NE,DA,5-HT in the serum of the rat model group was significantly reduced,and there was a significant difference with the normal group.The content of NE,DA,5-HT in the serum of fluoxetine group was higher than that of the model group,but there was no significant difference with the normal group.The contents of NE,DA,5-HT in hippocampus,cortex,midbrain and striatum of cums depressed rat model group were significantly lower than those of normal group.The contents of NE,DA,5-HT in hippocampus,cortex,midbrain and striatum of fluoxetine group were higher than those of model group.Except DA,5-HT of midbrain,the contents of NE,DA,5-HT in other parts of brain tissue were higher than those in model group.The content of TNF-α and pERK in the hippocampus of the model group of the rat model group was significantly higher than that in the normal group.The content of TNF-α and pERK in the hippocampus of the fluoxetine group was decreased,and there was a significant difference with the model group.The content of IL-10,BDNF and VGF in hippocampus of cums depressed rat model group was significantly lower than that of normal group.The content of IL-10,BDNF and VGF in hippocampus of fluoxetine group was significantly higher than that of model group.The survival rate of hippocampal neurons in CUMS depressed rat model group was significantly lower than that in normal group.The survival rate of hippocampal neurons in fluoxetine group was significantly higher than that in model group.The content of NE,DA,5-HT in the hippocampus of the model group of the rat model group was significantly higher than that in the normal group.The content of NE,DA,5-HT in the hippocampal neurons of the fluoxetine group decreased,and the content of NE,DA was significantly different from that of the model group.In the model of the rat hippocampal cells,the cell survival rate of the model group was lower than that of the normal group,and there was a significant difference with the normal group.The cell survival rate of asarone low dose group,asarone high dose group and fluoxetine group was improved.The cell survival rate of asarone low dose group was lower than that of high dose group and fluoxetine group.In the model of pc12 cells,the cell survival rate of the model group was lower than that of the normal group,and there was a significant difference with the normal group.In the low-dose group of asarone and the high-dose group of asarone,the cell survival rate of the fluoxetine group was improved,but it was not as normal as the normal group.The survival rate of the low-dose group of asarone was close to the high-dose group,but the two groups were significantly lower than that of the fluoxetine group.The contents of TNF-α and pERK in hippocampal nerve cell injury model group were significantly higher than those in normal group.The contents of TNF-α in β-asarone group were significantly different from those in model group.The content of TNF-α and pERK in the fluoxetine group was lower than that of the model group,and it was close to the normal group level.The content of BDNF and VGF in the model group was lower in the model group than in the normal group,and there was a significant difference with the normal group.The content of IL-10 and VGF in β-asarone group was significantly higher than that in model group The content of IL-10,BDNF and VGF in fluoxetine group was significantly higher than that in model group.In the PC12 cell injury model,the content of TNF-α and pERK in the model group was significantly higher in the model group than in the normal group.The contents of TNF-α and pERK in P-asarone group were significantly lower than those in model group.The content of TNF-α and pERK in the fluoxetine group was lower than that of the model group,and it was close to the normal group level.In the PC12 cell injury model,the content of IL-10,BDNF and VGF in the model group decreased significantly,which was significantly different from that in the normal group.The content of IL-10 and VGF in β-asarone group was higher than that in model group,and the content of IL-10,VGF in P-asarone group was significantly different from that in model group.The content of IL-10 and BDNF in fluoxetine group was significantly higher than that in model group.The hippocampal neurons of neonatal rats and pc12 cells damaged by corticosterone were closely related to the hippocampal neurons of rats,and the cell survival rate of the three cells was basically the same.The content of IL-10,TNF-α,BDNF,pERK and VGF in the hippocampus of the model of the rat model was also consistent with that of the cortical ones.At the same time,the correlation between PC12 cell model and hippocampal cell model was consistent with that of depression,and there was a significant correlation between them.PC12 cell model was consistent with hippocampal cell model and there was a close correlation between PC12 cell model and hippocampal cell model.ConclusionThe depression of CUMS model rats was stable,and the score of depression morphology and appearance was consistent with the observation score of behavioral indexes.Excessive cort can not only damage the function of monoamine transmitter system,but also damage hippocampal nerve cells and inhibit their regeneration.The changes of cell survival rate,cytokines and neurotrophic factors in the cell model of cort injury were basically consistent with those of CUMS depressed rats,and there was a close correlation between the changes of cell survival rate,cytokines and neurotrophic factors.The cell survival rate and responsiveness to fluoxetine and β-asarone in pc12 cell model were basically consistent with those in neonatal rat hippocampal cell model,and the correlation was significant.Cell model can partly replace or reduce animals in depression related experiments such as cytokines,neurotrophic factors and so on,and can be used to screen antidepressants or active components and to study some mechanisms.At the same time,pc12 cell model can replace neonatal rat hippocampal cell model for related experiments. |
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