The Effect Of Stress On Midbrain Dopaminergic Neurons In Rats And Its Possible Mechanism

Objective:Stress is a systemic non-specific adaptive response induced by various internal and external environmental factors as well as social and psychological factors.Current studies have suggested that excessive stress could lead to changes of the body in psychology,physiology and metabolic function.Thus,this study was designed in conjunction with important scientific issues that need to be addressed in forensic practice.In the forensic practice,there are cases that someone died rapidly who had been attacked by a major mental strikes or their bodies had been suffered from restriction.On the premise of excluding all other causes of death,some scholars believe that it belongs to stress death,but there is still insufficient evidence.As we all known,stress results in changes of the central nervous system.Substantia nigra(SN)and ventral tegmental area(VTA)are the two most important dopamine nuclear groups in the midbrain.Previous study indicated that dopaminergic neurons may play a role in stress response,but the exact mechanism is not clear.Thereby,the present study was aim to systematically observe the pathological changes of dopamine neurons in SN and VTA under different durations of stress and to investigate the changes of tyrosine hydroxylase(TH)and the endoplasmic reticulum related proteins in the process,which will provide reliable pathomorphological basis for the study on the mechanism of stress injury.Methods:1 Male Sperague-Dawley(SD)rats(n=60),weighing 220±20 g,were bred at a 12/12-h light/dark cycle,temperature and humidity controlled room.The rats were given access to food and water with ad libitum.The rats were randomly assigned into the following groups:control group;restraint stress combined with ice water swimming(stress)groups at 1,3,7,14 and 21 d(n=6rats in per group).Rats were placed in the restrainer with no food and water for 6 hours(from 8:00 AM to 14:00 PM)each day.Then the restricted stress were placed in ice water to swim for 5 minutes each day.This stress process lasted for 1d,3d,7d,14d and 21d,respectively.The control group rats were left in the cages for the same time with no food and water.All the rats were decapitated 24 hours after all the experiment.Brain were removed and immediately fixed in neutral formalin,and then subsequently dehydrated in a graded ethanol series and embedded in paraffin.Sections were prepared for further study.1)To record the weight changes of rats.2)To observe the pathological changes in SN and VTA by HE staining.3)To observe the changes of Nissl body of neurons in SN and VTA by Thionine staining.4)To observe the changes of the number of dopaminergic neurons in SN and VTA by immunohistochemistry.5)To observe the changes of endoplasmic reticulum stress proteins GRP78(glucose-regulated protein 78),CHOP(C/EBP homologous protein)in dopaminergic neurons of SN and VTA by multiple fluorescent labeling.2 The positive cell counting and statistical analysis:The number of positive immunostaining cells in SN and VTA was quantified using HistoQuest?(Tissue-Gnostics)software.Microscopy based multicolor tissue cytometry(MMTC)has been used by previous researchers.Statistical analysis was performed by one-way ANOVA.Post hoc LSD-t test was used when comparisons were restricted to two experimental groups.The threshold for statistical significance was defined as P<0.05.Result:1.The weight changes of the ratsWith the extension of experimental duration,the weight of the control group was obviously increased.Compared with the control group,the weight of stress rats was slightly decreased in 1 day.And with prolonged time,the weight also increased.However,the weight of stress group was lower than its control group.2.The observation of cytopathological changes by HE stainingIn the control group,the neuronal structures were clear,and the volume were normal.The neurons in the stress group at 1d,3d and 7d were insignificantly different to those in the control group.However,after 14d and21d of stress exposure,deep stained and pyknotic neurons were found obviously.3 The Nissl body changes of neurons by Thionine staining3.1 The Nissl body changes of neurons in SNIn the control group,the structure of neurons was clear,and Nissl bodies were evenly distributed in the cytoplasm.In the RS+IS group,there were no significant changes in Nissl bodies at 1d.However,edema was found in the neurons and Nissl bodies were not clear at 3d.After 7d and 14d of stress exposure,some Nissl bodies disappeared,and pyknotic neurons were visible.Cellular damage was more obvious at 21d.3.2 The Nissl body changes of neurons in VTAThere were large and rich in cytoplasm of neurons in the control group.However,In the RS+IS group at 1d and 3d,edema was visible and deep stained in cytoplasmic in the neurons.At 7d and14d of stress expression,edema was obvious in the neurons and Nissl bodies were not clear.At 21d,pyknotic neurons were obvious and the neurons missing Nissl bodies were more.4 The changes of the number of dopaminergic neurons4.1 The changes of the number of dopaminergic neurons in SNCompared with the control group,there was no significant difference in the RS+IS group at 1d.However,with prolonged of duration of stress,the number of dopaminergic neurons were significantly decreased.4.2 The changes of the number of dopaminergic neurons in VTAThe result was similar with that in SN.With increased duration of stress expression,the number of dopaminergic neurons were obviously reduced,though there was no difference in the RS+IS group at 1d.5 The changes of TH~+-GRP78~+and TH~+-CHOP~+positive cells in dopaminergic neurons in SN and VTA5.1 The results of TH~+-GRP78~+positive cells in SN and VTAWith prolonged of stress exposure,the number of GRP78 colocalized with TH in SN and VTA was slightly decreased after significantly increase,and the peak was at stress at 3d.5.2 The results of TH~+-CHOP~+positive cells in SN and VTAWith prolonged of stress exposure,the number of CHOP colocalized with TH in SN and VTA was significantly increase,and the peak was at stress at14d.6.Correlation analysis between the number of dopamine neurons and the number of TH~+-CHOP~+positive cells.There was a significant negative correlation between the number of dopamine neurons and the number of TH~+-CHOP~+positive cells.Conclusion:For the present study,we successfully established rat models different durations of restraining combined with ice water swimming.After observing by HE staining,thionine staining,immunohistochemistry and multiple fluorescent labeling,we get the conclusion as follows:long-term stress expression could result in attenuation of the number of dopaminergic neurons in SN and VTA and neuronal injury,as well as dynamic changes in endoplasmic reticulum stress related protein GRP78 and CHOP in this process,which suggested that endoplasmic reticulum stress may be involved in the pathological changes of midbrain dopaminergic neurons.