| Objective: Morphine dependence is a serious obsessive-compulsive mental disorder,not only causing serious harm to the health of the dependents,but also posing a serious social and public safety problem.Morphine dependence causes pathophysiological changes of all systems in the body,especially of central nervous system.Previous studies have indicated that almost all drugs abuse could lead to a disorder in the release of dopamine neurotransmitters,thereby,dopamine neurotransmitters changes induced by morphine dependence may be associated with addictive behavior in abuse.Substantia nigra(SN)and striatum are two important dopamine regulatory areas in the central nervous system.SN is a typical dopamine nucleus group,whose projection area is striatum.So,any disruption of dopamine neurons in SN could inevitably affect the neurons in striatum.Previous studies in our group have indicated that chronic morphine dependence could result in damage of dopaminergic neurons in SN.However,it hasn't referred to the projection area and related proteins associated with the damage.Among many dopamine regulatory factors,glial derived neurotrophic factor(GDNF),homeobox protein transcription factor 1(EN1),?-synuclein(?-Syn)play critical in dopamine neurons,but their expression as well as functions are still unclear in chronic morphine dependence.Meanwhile,morphine exposure could be seen a classic stress.Whether endoplasmic reticulum stress stress(ERS)was involved in the dopamine neurons damage in morphine dependence should be clear.Therefore,the study observed the pathological changes of neurons in SN and striatum,and investigated the changes of dopamine regulator factors and ERS related proteins using immunohistochemistry and Tissue FAXS quantitative imaging system.These findings were aimed to provide morphological evidence for studying the mechanism of nerve injuries induced by morphine dependence.Methods: 1 Male,healthy,Sperague-Dawley(SD)rats(n=90),weighing 250 ± 20 g.The rats were randomly assigned into the following groups: control group,morphine dependence group for 1 week,3 weeks and 6 weeks(n=6 rats per group).The three groups of morphine dependent animals were injected subcutaneously in the back with morphine hydrochloride twice daily(8:00,20:00)for 5 days.The initial dose administered was 10mg·kg-1 and was increased by 10mg·kg-1 every other day until 5th day of treatment.The control rats received equal volumes of saline.The animals were thoroughly disinfected with alcohol(75%)prior the every injection with a disposable needle/syringe.The rats were then confirmed to be dependent on morphine after 5 days of morphine administration.Following this,30mg·kg-1 morphine were administered twice daily(8:00,20:00)until 1,3 or 6 weeks post-establishment of dependence.The next day after successfully established models,rats were anesthetized and their brains were removed and fixed with 10% formalin.After dehydration,clearing and paraffin embedding,all interesting tissues were prepared for various histopathological staining.1)Recorded the withdrawal symptoms of rats.2)Recorded the weight changes of rats.3)Open field tests were performed to observe spontaneous activity of rats.4)Tar violet and toluidine blue staining were used to observe the changes of Nissl body in SN.5)HE staining was used to investigate the pathological changes of the striatum.6)Immunohistochemical staining and immunofluorescence multiple labeling were used to observe the expression of related proteins.2 Cell counting: Microscopy based multicolor tissue cytometry(MMTC) was used to count the neurons and nerve fibers(brown)in the striatum.This software has been widely recognized at home and abroad.To determine the number of TH+ cells that exhibited GDNF/EN1/PERK/IRE1 in SN,cell counting were counted at 100× magnification.The average number of positive cells in each rat was calculated by two independent observers who were blinded to the experimental conditions.3 Results processing and statistical analysis: Data were imported into SPSS 21.0 statistical software for statistical analysis.Data are expressed as the mean ± standard error of the mean(SEM).Data were analyzed using one-way analysis of variance(ANOVA)followed by a post-hoc LSD t-test to determine specific group differences.Significance was defined as P<0.05 for all statistical tests.Result: 1.The Withdrawal symptoms in rats Rats in morphine dependent groups were showed obviously withdrawal symptoms(P < 0.05).Compared with the control group,the scores of withdrawal symptoms were obviously different in morphine dependent group(P < 0.01),suggesting that the models of morphine dependent rats were successfully established.2.Weight changes of rats With 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(P < 0.05).3.Open field test results Compared with the control group,the number of rat feces was significantly increased after morphine exposure.Compared with the control group,the total distance of movement,the distance of central zone movement,and the time of central zone movement were gradually decreased in the morphine-dependent groups at 1 week,3 weeks,and 6 weeks.4.The changes of Nissl body in SN In the control group,the neurons were rich in cytoplasm,and Nissl bodies were clearly visible in the cytoplasm.Compared with the control group,the staining of Nissl body was light,some nerve cells were edema at 1 week of morphine dependence.With increasing duration of morphine exposure,some Nissl bodies disappeared and pyknotic neurons were visible.Cellular damage was more obvious at 6 weeks,more neurons were pyknotic and dying,and more Nissl bodies disappeared.5.The changes of neurons in striatum With prolonged duration of morphine exposure,tissue was edema and neuronophagia,and pyknotic neurons were visible.6.The immunohistochemical staining in SN 6.1 Coexpression of GDNF and TH in dopaminergic neurons of SN The number of GDNF +-TH+ positive cells was not insignificant different among the control groups.Compared with the control group,the percentage of GDNF +-TH + positive cells was significantly decreased after morphine dependence at 1 week,3 weeks and 6 weeks.6.2 Coexpression of EN1 and TH in dopaminergic neurons of SN The number of EN1+-TH+ positive cells was not significantly different among the control groups.Compared with the control group,there was no significant difference of EN1+-TH+ positive cells in the morphine dependent at 1 week.But with increasing morphine exposure,the percentage of EN1+-TH+ positive cells was significantly reduced.6.3 Coexpression of p-PERK and TH in dopaminergic neurons of SN The number of p-PERK +-TH+ positive cells wasn't difference among the control groups.Compared with the control group,there was no significant difference of p-PERK +-TH+ positive cells in the morphine dependent 1 week.But with increasing morphine exposure,the percentage of p-PERK +-TH+ positive cells was significantly increased.6.4 The changes of p-IRE1 and TH in SN The number of TH positive cells was not insignificant difference among the control groups.Compared with the control group,there was no significant difference in the number of TH positive cells after 1 week.However,with increasing morphine exposure,the number of TH positive cells was significantly decreased.The number of p-IRE1 positive cells was not insignificant difference among the control groups.With duration of morphine exposure,the number of p-IRE1 positive cells was significantly increased.7 The immunohistochemical staining in striatum 7.1 The expression of GDNF There was no significant difference of GDNF positive cells among the control groups.Compared with the control group,the number of GDNF positive cells in the morphine dependent 1 week was not significantly different.But the positive expression of GDNF in the morphine dependent 3-week and 6-week groups was significantly reduced.7.2 The expression of EN1 There was no significant difference of EN1 positive cells among the control groups.Compared with the control group,the number of EN1 positive cells in the morphine dependent 1 week was not significantly different.But the positive expression of EN1 positive cells in the morphine dependent 3-week and 6-week groups was significantly reduced.7.3 The expression of ? – Syn There was no significant difference of ? – Syn expression among the control groups.Compared with the control group,the number of ? – Syn positive cells at 1 week was not significantly different.But the number of ? – Syn positive cells after morphine dependent at 3 or 6 weeks was significantly increased.Conclusion: Based on successfully established rat models of different durations of morphine dependence,tar violet staining,toluidine blue staining,immunohistochemical staining,and immunofluorescence multiple labeling methods were used to research.The results indicated that chronic morphine dependence could change the ability of behavior and damage nerve cells of SN and striatum,as well as dynamic changes of dopamine regulatory proteinEN1,GDNF,?-Syn and endoplasmic reticulum stress-related protein PERK and IRE1,which suggested that these regulatory proteins may be associated with pathological changes of SN and striatum. |
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