| Objective: Morphine dependence is a chronic and relapsing brain diseasecharacterized by absence of control and compulsive continuous drugadministration. It not only harms the physical and mental condition of addictsbut also society, so it has been known as a focus issue by people. Recentresearch into morphine dependence has almost been confined to function andmetabolism, while pathological morphology has been under investigated. Amass of data indicate that the mesencephalic dopamine system is believedinvolved in almost the whole reward effects of drug dependence, and isregarded as the last path of reward effect. So the mesencephalic dopaminesystem is regarded as the most critical areas of the brain in drug addiction.Previous studies have indicated that opioids could damage manyimportant organs to varying degrees. Acute and chronic cerebral ischemia andhypoxia induced by morphine, resulting from pathological damage of the heartand lung, as well as inhibition of the central nervous system and respiratorycenter, can lead to pathological changes in nerve cells and fibers. However, thepathological morphology research has not been published, so in order tosystematically survey the effect of morphine on dopaminergic neurons of thesubstantia nigra (SN) and VTA in morphine dependent rats, we usedimmunohistochemical staining to label tyrosine hydroxylase (TH), a marker ofdopaminergic nerve cells. The results showed a significant decrease indopaminergic neurons in the SN and VTA in long-term morphine dependentrats.In order to explore the problem that whether this decrease was owing toapoptosis or neurodegeneration, we studied from two aspects of the overalllevel and cell level. First, through establishment of different duration ofmorphine dependent rat model, we systematically survey the effect of morphine on dopaminergic neurons of SN and VTA in morphine dependentrats. Second, using dopaminergic MN9D cells, observe effects of differentmorphine stimulation on dopaminergic neurons from the cell level. Thus laythe foundation for the explore of function change that associated withmorphological change.Methods:1Adult female Wistar rats, weigh300±20g. The rats were randomlydivided into the following groups: control,2week,4week and6weekmorphine dependent groups (n=8).The three groups of morphine dependentanimals were injected subcutaneously in the back with morphinehydrochloride, twice daily (8:00,20:00) for5days. The initial doseadministered was10mg/kg and was increased by10mg/kg every other dayuntil the5th day of treatment. Control rats received an equal volume of saline.Animals were thoroughly disinfected with alcohol (75%) prior to everyinjection with a disposable needle/syringe. Rats were then confirmed to bedependent on morphine after5days administration. Following this assessment,30mg/kg morphine was administered twice daily (8:00,20:00) until2,4or6weeks post-establishment of dependence, while control groups were injectedthe same measurement of physiological saline. After models were established,all brain tissues of groups were got in reference to Brain stereotaxic map.After dehydrated, paraffined and sliced, we used immunohistochemistry andimmunofluorescence with TH-antibody in order to observe the changes ofdopaminergic neurons of SN and VTA. Besides, Fluoro-Jade B staining wasused to detect the degeneration and necrosis, and terminal deoxynucleotidyltransferase-mediated dUTP nick-end-labeling (TUNEL) to detect apoptosis ofmesencephalic dopaminergic nerve cells during different stages.2This experiment adopts the dopaminergic MN9D cells, usingimmunofluorescence to detect three opioid receptors of mu, kappa, sigma.After morphine exposure of48h,24h and12h, using Fluoro-Jade B staining toobserve degeneration and necrosis of dopaminergic MN9D cells.Statistical analysis was performed using an independent-samples t test. Results are presented as mean±SEM. The threshold for statisticalsignificance was defined as p <0.05.Results:1. The results of immunohistochemistry indicated that TH-positive cellswere shrinking, dyeing density increased and the number of TH-positive cellsdecreased significantly in the long time morphine dependent rats.2. The results of Fluoro-Jade B were showed that there were scattereddegeneration and necrosis cells in SN and VTA of long-duration morphinedependent rats.3. TUNEL staining showed that apoptotic cells were not be observed.4. The results of Fluoro-Jade B were indicated that there were scattereddegeneration and necrosis cells in MN9D cells after the long time morphinesituationConclusion:The number of SN and VTA dopaminergic nerve cells decreased withincreasing periods of morphine dependence, which was most likely due todegeneration and necrosis of nerve cells induced by morphine toxicity. |
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