| Objective: It is well known that 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. Continuous morphine intake can cause varying degrees of harm to each system of the body, and the most significant toxicity is on nervous system. The pathological damage on many regions of the brain including the cerebral cortex induced by chronic morphine dependence, cause acute or chronic brain ischemia and hypoxia, and further induce a series of pathological changes in nerve cells and fibers. Recent research on morphine dependence has almost been confined to function and metabolism, while pathological morphology has not been investigated well.Previous studies indicated that morphine dependence can cause nerve damage. However, it has not been detailedly reported which kind of proteins involved in nerve injury, protection and remodeling of neural structure. Among transcription factors involved in regulation of survival, maintenance and injury of neurons, microtubeasso ciated protein-2(MAP2), signal transduction and transcription protein-3(STAT-3), apolipoprotein E(APOE), 3-nitrotyrosine(3-NT) and C-fos may play a critical role. What is more, it is not clear that what changes of those factors after different duration of morphine dependence. The present study was to investigate the expression changes of MAP2, STAT-3, APOE, 3-NT and C-fos after different morphine exposure. The findings were meaningful to provide morphological evidence for studying the mechanism of nerve injury induced by morphine toxicity.Method:1 Adult female Wistar rats, weighing 300 ± 20 g, were used to study. The rats were randomly divided into the following groups: control, 1 week, 2 weeks, 4 weeks and 6 weeks morphine dependent groups(n = 8). The model of morphine dependence was established by increasing subcutaneous injections of morphine hydrochloride. The four 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 was10mg/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, 2, 4 or 6 weeks post-establishment of dependence. 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. ①Hematoxylin eosin(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 Fluoro-Jade B staining was used to detect degeneration and necrosis, and terminal deoxynucleotidyl transferase-mediated d UTP nick-end-labeling(TUNEL) detected apoptosis of nerve cells. ④ 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.Results1 Results of HE stainingStructure 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 dependence at 1week and 2 weeks, 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 4 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.2 Results of thionine stainingNissl body in neurons was easily identified in the control rats. There was no significantly difference between morphine dependent rats at 1-2 weeks and control rats. By contrast, the number of nissl body was decreased and structure was unclear in the morphine dependent rats at 4 weeks. Furthermore, nissl body was disappeared and neurons were shrunken after 6 weeks of morphine exposure.3 Degeneration and necrosis of neurons in the cerebral cortexDegeneration and necrotic neurons were not found in the control and 1week as well as 2 weeks morphine dependent groups. After 4 weeks of morphine dependence, degenerating, necrosis positive cells were randomly observed. After 6 weeks, a few scattered degenerating, necrosis positive cells were observed consistently.4 TUNEL stainingApoptotic cells were not found in the control and morphine dependent groups, suggesting that morphine treatment for varying times in our experiments may not cause obvious apoptosis in cerebral cortex.5 Immunohistochemical staining5.1 Results of MAP2 immunohistochemical stainingThe protein of MAP2 was strongly observed in cytoplasm and synapses of cerebral cortex neurons in the control group. While MAP2 expression was decreased with prolonged morphine exposure.5.2 STAT-3 and APOE immunohistochemical staining resultsThe positive expression of STAT-3 and APOE in cerebral cortex neurons were increased in the morphine dependent 1week and 2 weeks rats compared with those in the control group. However, with the duration of morphine exposure further prolong, two proteins level showed a trend of decrease.5.3 Immunohistochemisty of 3-NT3-NT proteins in cerebral cortex neurons from the morphine dependent group at 1week and 2 weeks were insignificantly different when compared with those in the control group. While the proteins level was increased after 4 weeks and 6 weeks morphine dependence.5.4 Immunohistochemisty of C-fosC-fos positive cells were not found in the control group. While in morphine dependent group of 1week, there were few of positive cells. However, with the duration of exposure prolonged, they were not found in any other morphine dependent groups.Conclusion:This study successfully established rat models of different duration of morphine dependence.With conventional histopathology, histochemistry special dye as well as immunohistochemistry,we get the conclusion as follows: degeneration and necrosis of neurons in the cerebral cortex have been shown after long-term of morphine dependence, meanwhile MAP2,STAT-3,APOE,3-NT,C-fos involved in the degeneration and necrosis of the nerve cells. |
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