| Objective: Morphine has an obvious analgesic effect, but it is also highly addictive. The abuse of morphine has increased greatly in recent years, which has seriously harmed the physical and mental health and also led to a series of undesirable effects on social stability and harmony. Morphine is often used to the treatment of patients with malignant tumor in the clinic, and the dosage is much larger than the conventional, which non-standard drugs causing serious consequences. Chronic morphine exposure refers to loss of control, compulsive continuous administration characterized by chronic recurrent brain disease. Acute morphine poisoning, the main clinical manifestation of coma, respiratory depth inhibition, shock resulting from severe hypoxia, circulation failture and even death, is more common in clinical critical disease. Our previous results indicated that degeneration and necrosis of local nerve cells in the brain was found after chronic morphine exposure, but the mechanism should be further studied. Previous studies also have shown that endoplasmic reticulum stress(ERS) may participate in the nerve injury induced by morphine. The ERS related proteins: sugar regulatory proteins 78(GRP78), Phosphorylated 2 alpha eukaryotic cells and translation initiation(p-p-eIF2?), activation of transcription factor 6(ATF6), and CHOP are the critical regulatory proteins to start the ERS pathway. However, the changes of these proteins associated with chronic morphine exposure and acute morphine poisoning remain unclear. Therefore, one of the purposes of the study was to investigate the changes of these ERS related proteins and the mutual relations after chronic morphine exposure and acute morphine poisoning, which finally providing a new insight for studying the dysregulation of neuron as well as the mechanism of nerve injuries induced by morphine exposure.Previous studies have indicated that morphine contributes to acute or chronic ischemic brain and hypoxia, leading to pathological changes in the nerve cells and fibers, and finally induces to obvious nerve injury. The present study shows that proliferation, differentiation and migration to the injured area could be observed in the endogenous neural stem cells after brain injury, which is of great significance for the repair of nerve injury. However, it is not clear that the proliferation and differentiation of endogenous neural stem cells after nerve injury induced by morphine exposure. Thereby, the other purpose of the study was to investigate the proliferation of endogenous neural stem cells as well as the expression of Ki67(the proliferating cell nuclear antigen) and Nurr1(newborn neuron specific marker) in the brain after nerve injury induced by morphine exposure, which is meaningful to provide a scientific basis for the reduction of nerve injury associated with morphine toxicity.Method:1 Adult male Wistar rats, weighing(300±20)g, were used to study. The rats were randomly divided into the following groups: control, 1 week, 3weeks, 6 weekmorphine dependent groups and acute morphine poisoning group(n= 8). The model of morphine exposure was established by increasing subcutaneous injections of morphine hydrochloride. 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 and was increased by 10mg/kg every other day until the 5th day of treatment. Animals were thoroughly disinfected with alcohol(75%) prior to every injection with a disposable needle/syringe. Rats were then confirmed to be dependent on morphine after 5 days administration. Following this assessment, 30mg/kg morphine was administered twice daily(8:00, 20:00) until 1, 3 or 6 weeks post-establishment of dependence.Acute group adopts health rats that morphine exposure model has not been established,and they were injected with morphine hydrochloride(30mg· kg-1)one-time,and were killed after two hours.Control rats received an equal volume of saline. After established the model rats, tissue used for staining was harvested and fixed, then dehydrated in a graded ethanol series, and finally embedded in paraffin.1) HE staining was used to observe the conventional pathology changes.2) Thionine staining was used to observe the Nissl body changes of nerve cells.3) Immunohistochemical staining was used to detect the expression changes of related proteins.2 Statistical methodsWith SPSS13.0 statistical program, data were analyzed using one-way analysis of variance(ANOVA) followed by a post hoc Newman-Keuls test, to determine specific group differences. Dates are presented as mean ± SEM. The threshold for statistical significance was defined as P < 0.05.Results: 1 Results of HE staining Structure of cerebral cortex was clear and neurons were arranged neatly. There was no obvious pathological change in the control groups. Compared with the control group, there were insignificantly difference in the morphine exposure at 1week, excepting that small perivascular spaces were slightly increased. However, never cells were shrunken, gaps around the small blood vessels and neurons were increased, and microglia was proliferated in the morphine dependent at 3 weeks. Tissues obtained from the morphine dependent group at 6 weeks displayed loose organizational structure, edema and shrinkage of nerve cells as well as marked hyperplasia of microglia. Some pyknotic cells appeared at acute poisoning group.2 Results of thionine staining Nissl body in neurons was easily identified in the control rats. There was no significantly difference between morphine dependent rats at1 weeks and control rats. By contrast, the number of nissl body was decreased and structure was unclear in the morphine dependent rats at 3 weeks. Furthermore, nissl body was disappeared and neurons were shrunken after 6 weeks of morphine exposure.And nissl body was shrunken at acute poisoning group.3 Immunohistochemical staining 3.1 Results of GRP78 immunohistochemical stainingThe positive expression of GRP78 in cerebral cortex neurons were increased in the 1 week and 3 weeks rats of morphine dependent when compared with those in the control group. However, with the duration of morphine exposure further prolong, the level showed a trend of decrease.Positive expression of acute group slightly higher than control group.The positive expression of GRP78 in hippocampal CA1 area neurons were increased in each morphine dependent group and acute poisoning group rats compared with those in the control group. But we did not see obvious difference between each group of morphine.3.2 Results of p-eIF2? immunohistochemical stainingThe positive expression of p-eIF2? in cerebral cortex neurons were increased in the 1 week and 3 weeks rats of morphine dependent when compared with those in the control group. However, with the duration of morphine exposure further prolong, the level showed a trend of decrease.Positive expression of acute group slightly higher than control group. p-eIF2? proteins in hippocampal CA1 area neurons from every morphine dependent group were insignificantly different when compared with those in the control group.Positive expression of acute poisoning group slightly higher than control group.3.3 Results of ATF6 immunohistochemical stainingThe positive expression of ATF6 in cerebral cortex and hippocampal CA1 area neurons were increased in the 1 week and 3 weeks rats of morphine dependent when compared with those in the control group. However, with the duration of morphine exposure further prolong, the level showed a trend of decrease.Positive expression of acute poisoning group slightly higher than control group.3.4 Results of CHOP immunohistochemical stainingThe positive expression of CHOP in cerebral cortex and hippocampal CA1 area neurons were increased in the 1week and 3 weeks rats of morphine dependent when compared with those in the control group. However, with the duration of morphine exposure further prolong, the level showed a trend of decrease.Positive expression of acute group slightly higher than control group.3.5 Results of Ki67 and MAP2 immunohistochemical stainingmorphine exposure groups of peripheral nerve cells in the rat brain ventricle Ki67 positive expression was decline compared with normal control group is on the decline,as the extension of morphine exposure on time.Compared with the control group, there were insignificantly difference in the acute poisoning group.3.6 Results of Nurrl immunohistochemical stainingThe neurons of morphine exposure groups in cerebral cortex Ki67 positive expression was decline compared with normal control group is on the decline,as the extension of morphine exposure on time.Compared with the control group, there were insignificantly difference in the acute poisoning group.Conclusion: The study has been successfully established the model of chronic morphine exposure and acute morphine poisoning in rats. After observing by conventional histopathology, histochemical special staining and immunohistochemistry, we get the conclusion as follows: acute and chronic morphine exposure can lead to nerve injury in the brain. Proteins of GRP78?p-eIF2? ? ATF6 ? CHOP, associated with ERS, were involved in the degeneration and necrosis of neurons. Meanwhile, Ki67 and Nurr1 were decreased after nerve injury induced by morphine exposure, suggesting that the proliferation and differentiation of newborn nerve cells was influenced by morphine neurotoxicity. |
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