| Objective: Stress,a non-specific systemic reaction to body,which is induced by various internal or external environmental factors and social or psychological factors.Endoplasmic reticulum,as an important part of the homeostasis of cells,undergoes accumulation of misfolded or unfolded proteins when stimulated by external stimuli,thus affecting normal physiological functions of cells and forming ER stress,while GRP78?ATF6?CHOP is the mark of ERS.The hypothalamus is a major component of the hypothalamus-pituitary-adrenal axis and is also an important regulatory center of stress response,arcuate nucleus and periventricular nucleus are the two major dopaminergic neuron nuclei in hypothalamus.The dopamine nervous system plays an important role in stress,therefore,investigating the pathological changes of the hypothalamic dopamine neurons in the stress response is greatly significant.The study was to investigate the effect on neurons of arcuate nucleus and periventricular nucleus in hypothalamus as well as the changes of tyrosine hydroxylase positive cells and ERS protein in dopaminergic neurons after different durations of stress exposure,which could finally provide morphological evidence for studying the mechanism of nerve injuries induced by stress.Methods:1 SD male rats of 6 weeks and weighing(200 ± 20)g were randomly divided into six groups: 1 day,3 days,7 days,14 days,21 days and corresponding control group,6 rats in each group.Models of stress rats were established by being restrained for 6 hours(8:00-14:00)with fasting water and then given ice-water swimming for 5 minutes every day.The rats in the control group were given fasting water in the feeding cage when stress rats were restrained and ice-water swimming,all rats were freely fed at other times.After the model was established,killing the experimental and control group the next day,serum specimens were made for ELISA,and brain tissues were fixed,dehydrated,transparent,embedded and serial sections for various staining.1)Recording the weight changes of rats.2)Observing the concentration changes of glucocorticoid in serum by ELISA.3)Observing the morphological changes and Nissl body changes of AN and Pe by thionine staining.4)Observing the changes in the expression of TH protein by immunohistochemistry staining and panoramic tissue quantitative analysis system.5)Observing the changes of ERS-relative proteins in AN and Pe dopamine neurons.2 Statistical analysis: The number of TH positive cells was counted by Histo Quest software of panoramic tissue cell quantitative analysis system(TissueGnostics,Vienna,Austria).Data were analyzed by using SPSS 21.0 software and expressed as Mean ± SD.One-way analysis of variance(ANOVA)was used to compare the data with the least significant difference.The threshold for statistical significance was defined as P < 0.05.Results:1.The changes of weightWith the extension of experimental duration,the body weight of rats in the control groups showed an increasing trend.Compared with the control group,the weight of rats in 1 day experimental group decreased slightly,with the time prolonged,the body weight of the experimental group also showed an increasing trend,but the rate was significantly less than it's control group.2.Serum glucocorticoid test by ELISACompared with the control group,the level of serum glucocorticoid was significantly increased from the 3 days experimental group,peaked at 7 days,maintained a high level at 14 days group,but clearly decreased at 21 days,that is no significant differences with the 21 days' control group.3.Results of thionine stainingThe results showed no significant differences in the control group at each time point.Observing the hypothalamic AN and Pe under the microscope with 200 times magnified in each group respectively,in the control group,Nissl body was located in the cytoplasm,evenly distributed and more abundant.No significant pathological changes were observed in stress groups at 1 and 3 days.Neuron edema was found in stress group at 7 days and pyknosis of Nissl body was observed at 14 days.Disappearing of Nissl body and pyknosis of neurons at 21 days were much more than 14 days.4.Results of immunohistochemical staining(1)TH-positive cells observing and counting in ANThe results showed no significant differences in the control group at each time point.Histo Quest software(TissueGnostics,Vienna,Austria)was used to count the number of TH-positive cells in each group of AN under the microscope with 200 times magnified.Compared with the control group,there was no significant difference of positive cells at 1 day and 3 days after stress;the number of TH-positive cells were distinctly decreased at 7 days?14 days and 21 days,and with the extension of the stress duration decreased gradually.(2)TH-positive cells observing and counting in PeThe results showed no significant differences in the control group at each time point.HistoQuest software(TissueGnostics,Vienna,Austria)was used to count the number of TH-positive cells in each group of Pe under the microscope with 100 times magnified.Compared with the control group,there was no significant difference of positive cells at 1 day and 3 days after stress;the number of TH-positive cells were distinctly decreased at 7 days?14 days and 21 days,and with the extension of the stress duration decreased gradually.5.Results of immunofluorescent multiple staining(1)Results of immunofluorescent staining in AN1)Results of TH-GRP78 positive cells in ANCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,3 days group increased sharply,the number of positive cells in 7 days group decreased,with the time prolonged,the number of positive cells gradually reduced,no significant difference can be observed at 21 days compared with it's control group.2)Results of TH-ATF6 positive cells in ANCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,the number of positive cells began to rise at 3 days,peaked at 14 days,and decreased at 21 days,but no distinct differences in comparision with the control group.3)Results of TH-CHOP positive cells in ANCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,the number of positive cells increased sharply at 3 days group,peaked at 14 days,and decreased slightly at 21 days,but still much more than the control group.(2)Results of immunofluorescent staining in Pe1)Results of TH-GRP78 positive cells in PeCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,3 days group increased sharply,the number of positive cells in 7 days group decreased,with the time prolonged,the number of positive cells gradually reduced,no significant difference can be observed at 14 days and 21 days compared with it's control group.2)Results of TH-ATF6 positive cells in PeCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,the number of positive cells began to rise at 3 days,peaked at 14 days,and decreased at 21 days,but still much more than the control group.3)Results of TH-CHOP positive cells in PeCompared with the control group,there was no significant difference in co-expression of positive cells in 1 day stress group,the number of positive cells increased at 3 days and 7 days group,peaked at 14 days,and decreased slightly at 21 days,but still much more than the control group.Conclusions: The restraint and ice-water swimming model was successfully established,according to observing the thionine staining,immunohistochemistry staining,immunofluorescence staining and statistical analysis of the data,we could summarize that: long-term repeated stress could result in neuronal injury and the loss of dopaminergic neurons in AN and Pe of hypothalamus,the changes of ERS-related proteins GRP78,ATF6,CHOP in dopaminergic neurons suggested that ERS may be involved in the injury of hypothalamic dopaminergic neurons. |
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