Dopaminergic Regulatory Factors And Endoplasmic Reticulum Stress Proteins Are Associated With Neuronal Injury Of VTA And NAc Induced By Morphine Dependence

Objective:Morphine dependence is a chronic recurrent brain disease characterized by loss of control and forced continuous medication,which causes to severe damage of the central nervous system.The mesolimbic dopamine system,including two key neural nucleus of the ventral tegmental area(VTA)and its nerve projection area Nucleus accumbens(NAc),plays a vital role in neural pathological changes and rewarding effect induced by morphine dependence.However,it is still unclear that the pathological changes of VTA and NAc and their correlation with related regulatory proteins after different duration of morphine exposure.Among transcription factors involved in development and survival of neuron,glial derived neurotrophic factor(GDNF),homeobox protein transcription factor 1(EN1),and?-Synuclein(?-Syn)play critical roles.Therefore,one of the purposes of the study was to investigate the effect of morphine dependence on these transcription factors.In addition,morphine dependence is also a stress stimulus.Previous studies indicated that endoplasmic reticulum stress related proteins may play critical roles in pathological changes caused by morphine dependence.Thereby,the other purpose of the study was to investigate whether nositol-requiring Enzyme 1(IRE1)and protein kinase RNA-like endoplasmic reticulum kinase(PERK)were involved in neuron injury caused by morphine dependence.Most of previous studies on morphine dependence have investigated functioning and metabolic function,while pathological studies are lacking.So the present study focus on pathological changes of VTA and NAc after different duration of morphine exposure,and investigated the roles of dopaminergic regulation proteins and endoplasmic reticulum stress-related proteins in this process,which aims to provide evidence for the mechanisms study of nerve injury induced by chronic morphine dependence.Methods:1. The rats were randomly divided into the following groups:control(Con),and 1-week,3-week,and 6-week morphine(Mor)dependence(n=9rats per group).The model of morphine exposure was established by increasing subcutaneous injections of morphine hydrochloride.Withdrawal symptoms were induced by subcutaneous injection of naloxone hydrochloride,and the scores were given.Models of different durations of chronic morphine dependent rats were established after morphine dependence.2. Record the weight changes of rats.3. Open field test was used to observe changes behavior in rats.4. Tar violet staining was used to investigate the pathological changes of the VTA.5.HE staining was used to investigate the pathological changes of the VTA.6.Double-labeled immunofluorescence of NEUN and TH was used to investigate the changes of neuron and dopaminergic nerve fibers in the NAc.7.Multi-labeled immunofluorescence was used to investigate the expression changes of dopaminergic regulatory protein EN1,GDNF and endoplasmic reticulum stress-related protein IRE1 and PERK of the VTA.8.Immunohistochemical staining was used to investigate the changes of dopaminergic regulatory protein EN1,GDNF and?-Synin the NAc.Result:1.Compared with the control group,the scores of withdrawal symptoms were significant different in morphine dependent group,suggesting that the models of morphine dependent rats were successfully established.2.Weight changes of ratsWith prolonged of time,the rats weight were gradually increased in control and morphine dependent group.However,compared with control group,the weight of rats in morphine dependent group was lower and had significant difference.3.Open field experiment resultsCompared with the control group,the total distance of movement,the distance of central zone movement,and the time of central zone movement in the morphine-dependent groups at 1 week,3 weeks,and 6 weeks decreased gradually.Themorphine-dependent rats were anxious.4. Pathological changes in the VTAWith prolonged duration of morphine exposure,tissue was edema and neuronophagia,Nissl bodies were disappeared,nerve cells were pycnosis and deep dyeing.5. Pathological changes in the NAcWith prolonged duration of morphine exposure,tissue was edema and neuronophagia,neurophagy and pyknotic neurons were visible.6. Expression of NEUN and TH in the NAcWith prolonged duration of morphine exposure,neurons and dopaminergic nerve fibers were significantly decreased in the NAc.7. Expression changes of dopaminergic regulatory protein and endoplasmic reticulum stress protein in the VTA.Compared with the control group,the percentage of TH~+-EN1~+positive cells was significantly lower after 3 weeks and 6 weeks of morphine dependence,although there was no difference at 1 week of dependence.Compared with the control group,the percentage of TH~+-GDNF~+positive cells was significant decreased after morphine exposure.However,compared with 3 weeks of morphine dependence,the percentage of TH~+-GDNF~+positive cells was significant increased.The percentage of TH~+-p-PERK~+positive cells significantly increased after 3 weeks and 6 weeks of morphine dependence compared with the control group,although there was no difference after 1 week of dependence.The number of TH positive cells in the morphine dependent at 3 and6week groups was significantly reduced compared with control group.Compared with the control group,the number of p-IRE1 positive cells in the morphine-dependent group at 1 week,3 weeks,and 6 weeks was significantly increased.8. Expression changes of dopamine regulatory protein in the NAc.There was no significant changes of EN1 in morphine dependent group at1 week compared with control group.However,with prolonged of chronic morphine exposure,EN1 was decreased significantly.?-Syn was no significant difference between control and morphine dependent at 1 week.However,compared with control group,the expression of?-Syn was significantly increased after morphine exposure of 3 weeks.Compared with control group,the expression of GDNF was decreased after morphine exposure.However,compared with 3 weeks of morphine dependence,the expression of GDNF was significant increased.Conclusion:Based on successfully established rat models of different durations of morphine dependence,HE staining,tar violet staining,immunohistochemical staining,and immunofluorescence multiple labeling methods were used to research.The results indicated that chronic morphine dependence could damage nerve cells of the VTA and the NAc,and induce significant dynamic changes of dopamine regulatory protein EN1,GDNF,?-Syn and endoplasmic reticulum stress-related protein IRE1 and PERK,which suggested that these regulatory proteins may be associated with pathological changes of the VTA and the NAc.