Impairment And Mechanism Induced By Chronic Morphine In Primary Cultured Hippocampal Neuron Of Rat

Objective:Morphine is the most important opioid analgesic drug. As analgesic effect of morphine is intense, it is widely used in the long-term clinical application. However, morphine could produce strong euphoria, resulting in morphine dependence, psychiatric disorders, cognitive problems and behavior disorder.Thehippocampus is an important brain region in the central nervous system participating in the occurrence and development of various nervous system diseases. It is a significant method to explore the mechanism of drug addiction and damage inhippocampus for research object. The previous study found that chronic morphine treatment resulted in δ opioid receptor cell redistribution ofhippocampus, and reduced δ opioid receptor cell density, which may be major reasons for changes inhippocampal physiological function and long time cognitive disorders.Apoptosis is a form of cell death, with DNA degradated specificly and morphology changes including that nuclear pyknosis, cell membrane vacuolation and apoptos body, which is regulated by specific genes without obvious cell lysis. It is generally believed that there are three pathways of apoptosis: mitochondrial pathway, death receptor pathway and endoplasmic reticulum pathway, among which the former two are classical apoptotic pathways, and the latter one is an apoptotic pathway in recent years. The decrease of mitochondrial membrane potential is an important marker of the early stage of apoptosis. TUNEL assay can reflect the typical biochemical and morphological characteristics of apoptosis. Annexin V is recognized as one of the sensitive indicators for early detection of apoptosis. Caspase-3 plays an important function in many pathways of apoptosis signal transduction. By the detection of the above indicators, apoptosis is determined.A large number of studieshave indicated that morphine caused damage to the nervous system, and the different levels of damage are caused by different treatment methods of morphine, dosages, times and brain areas. Bajic found that cortex and amygdalahad obvious neuronal apoptosis by testing the levels of caspase-3 of newborn rat which was continuous injected morphine for six and ahalf days. A series of studieshave been done on the dependence, injury induced by morphine and its mechanism in our laboratory. The content of cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate(cGMP) were measured in various regions of the brain of morphine dependent rats by radioimmunoassay, and Gu found that cAMP content significantly was increased and cGMP content were significantly decreased in striatum, deutencephalon, mesencephalon, pons andhippocampus. This suggested that the change of cAMP and cGMP signaling system played an important role in morphine dependence. Another study found that rats after administration of morphine, glutamate(Glu) content was significantly increased in pons, but was not changed significantly in striatum, deutencephalon, mesencephalon andhippocampus; it was also found gamma aminobutyric acid(GABA) content was increased in striatum, deutencephalon, mesencephalon and pons. The results suggested amino acid neurotransmitters were participated in the process of morphine dependence. Shi Weibo found that after morphine long-term treatment, dopaminergic neurons in the ventral tegmental area and black substance was showed apparent degeneration and necrosis. The result demonstrated that morphine produced toxic effects. Other experiments using MTT assay found that morphine could reduce the survival rate of PC12 and SH-SY5Y cells in a dose dependent manner, and detected thehalf inhibitory rate were 2520μmol/L and 680μmol/L, and microscope observed cell synapse shortened or disappeared, the original cells form lost.In view of the above research background, this experiment was based on rat primary culturedhippocampal neurons, and by MTT assay to determine the suitable concentration of morphine, and by TUNEL assay, flow cytometry, mitochondrial membrane potential detection(JC-1) method and Western Blot to observe apoptosis of rat primaryhippocampal neuron induced by chronic morphine and analysis the mechanism of neuronal apoptosis.Methods:1 The experiment was based on the SD neonatal rat, and then carried on primary culturedhippocampal neurons.2 Neurons were identified by immunofluorescence, and the morphology of cells was observed by laser scanning confocal microscopy.3 MTT assay was used to detect the relationship between morphine and the survival rate of rat primaryhippocampal neurons, then selected appropriate morphine concentration for subsequent experiments.4 The TUNEL assay was used to observe the changes of the primaryhippocampal neurons in rats with morphine, and the apoptosis rate was calculated.5 To detect apoptosis rate of cells in each group by flow cytometry Annexin V-FITC/PI double staining assay.6 To observe the changes of mitochondrial membrane potential of the primary culturedhippocampal neurons under the administration of morphine by using the mitochondrial membrane potential detection assay.7 The expression level of Bax and caspase-3 protein was detected by Western blot.The data were analyzed with one way ANOVA,presented as mean±SD and compared with Tamhane’s T2 by SPSS 17.0 statistical program. A level of P<0.05 was supposed to be statistically significant.Results:1 As mature primaryhippocampal neurons, synaptic extended andhad branches, woven into the network; cell bodies were oval, conical, triangular and plump, apertures around; a few neurons appeared agglomerate growth phenomenon.2 Using immunofluorescence to identify mature primaryhippocampal neurons, with microtubule associated protein 2(MAP2) as the first antibody, Tritc labeled second antibody staining and DAPI counterstain. Using confocal laser scanning microscope to observe neurons, the nucleus was blue and bulky, synaptic red and interwoven into a network, stereo sense was particularly strong.3 Compared with the control group, Mor(0.156 mM~1.25mM) in rat primaryhippocampal neuron 6h, 12h, 24h, 48h, neuronal growth inhibitory ratehad no significant change, light microscope observation of the morphology of neurons was no significant change; Compared with the control group, Mor(2.5mM~10mM) in rat primaryhippocampal neurons 24h, 48h, Mor 5mM in rat primaryhippocampal neurons 12h, Mor 10 mM in rat primaryhippocampal neuron 6h, 12h, neuronal growth inhibitory rate increased significantly, and with the morphine concentration increased and more elevated, also in a time dependent manner, using light microscope could observe neuronal vacuolus-like structure and rupture, synapse shortened or disappeared, the original cells form lost.4 Compared with the control group, 1mM, 2mM, 3mM morphine in rat primaryhippocampal neurons for 48h, neuronal apoptosis rate increased significantly and in a concentration dependent manner, and observed the number of TUNEL positive increased with the increase of morphine concentration.5 Compared to the control group, Mor 1mM, Mor 2mM, Mor 3mM group, the total apoptosis rate was significantly increased, and the increase in the early stage of apoptosis, and showed a concentration dependent manner.6 Compared with the control group, Mor 1mM, Mor 2mM, Mor 3mM group red fluorescent intensity gradually decreased with the increase of the concentration, the green fluorescent intensity gradually increased with the increase of the concentration. This shows the decrease of mitochondrial membrane potential and the occurrence of early apoptosis.7 Compared with the control group, Mor 1mM, Mor 2mM, Mor 3mM group primaryhippocampal neurons Bax, caspase-3 protein expression levels were significantly increased, and showed a concentration dependent manner. This suggested the apoptosis of neurons was related to the increase of Bax and caspase-3 protein expression.Conclusion:1 Low concentration of morphine could promote rathippocampal primary neuronal growth, whereashigh concentrations of morphine inhibited the growth of rat primary neurons.2 High concentration of morphine can cause degeneration and necrosis and apoptosis of cultured rat primary neurons, and the activation of Bax and caspase-3 may be one of the molecular mechanisms of apoptosis in rat primary neurons.