Effect Of Decabromodiphenyl Ether Exposed On The Hippocampal Neurons Of Primary Cultured Fetal Mice And Changes Of CHOP And Caspase-12Protein Levels

BackgroundThe results of many researches show that the neurons still can maintain or approach tothe physiological parameters of the body cells after have maturated in vitro, andPrimary cultured cells are closer to the nerve cells in the body for normal growth statewhen compared with repeated passaging cells. Moreover, they have advantages ofconditions of experimental short, easy to control, samples of the same line, goodcontrast and so on. Therefore, the establishment of isolation, culture methods forhippocampal neurons and technical support is provided for studies of hippocampalneurons in vitro.MethodsGestational day (GD)18ICR mice were used a separation of fetal mice’s hippocampus,which were planted in poly-lysine coated6-well plates after had been digested andchanged contain B27in DMEM/F12serum-free medium after24hours, differentgrowth state of the cells were observed under an inverted microscope at different times;Furthermore, hippocampal neurons were identified by immunocytochemical methodsabout structural microtubule-associated protein-2(microtubule associated protein-2,Map-2). Results2hours into adherent cell after hippocampal neurons culture, individual cells1-2growtiny bumps. Most of the cells grow three or four protrusions, length is longer, a fewcellular aggregations after24hours. After3days, neurons increase in prominence andrenewed, and form a sparse network of cells with typical morphological characteristicsof neurons. After5days, neuronal somata gradually grow protrusions graduallyextended, forming a dense network. Observation on nerve cells grow well, somatabright and surrounded by halos, Karyon is also clearly visible bumps grow longerthicker, and neural networks for formation of distinct, mature after7days. Map-2immunofluorescent cytochemical identification of neurons, resulting in visiblefragmentation of hippocampal neurons, Neuron-majority.ConclusionThe method was to obtain good growth, high purity of hippocampal neurons. It showedthat cultured neurons is a practical, resulting in advantages of stability, could serve as agood model of neurons cultured in vitro and provide the basis for the next step of thestudy. BackgroundTo research the decabromodiphenyl ether (decabromodiphenylether, BDE-209) exposedto the fetal rat hippocampal neuron, and the changes of morphology, cell viability,oxidative damage and CHOP and Caspase-12protein expression levels of thehippocampal neuron, and to explore the role of endoplasmic reticulum stress inBDE-209exposed to hippocampal neurons, provide the basis for the establishment ofthe mechanism of neurotoxicity of BDE-209in vitro studies.MethodsThe primary cultured fetal mice hippocampal neurons exposed to BDE-209aftercultured7days, experiments are divided into six groups, the control group (culturemedium containing1‰DMSO) and experimental A (6.25μg/ml BDE-209), B (12.5μg/ml BDE-209), C (25μg/ml BDE-209), D (50μg/ml BDE-209), E (100μg/mlBDE-209) group. In each group have three parallel samples. Exposed24hours toobserve the cell morphology by phase contrast inverted microscope; MTT colorimetricassay detected hippocampal neuronal survival in each group; detected the superoxidedismutase (SOD) activity and nitric oxide (NO), malondialdehyde (MDA) andglutathione (GSH) content in hippocampal neurons, and use Western blot (Western blot)to detect CHOP, Caspase-12, Bax and Bcl-2protein. ResultsUsing inverted microscope to observe hippocampal neurons in cell morphology withdifferent doses, experimental group can see neuron cell body smaller, the cell membraneintegrity but have foaming phenomenon, with increasing doses the foamingphenomenon is more obviously, neurite is shorter, observed cell shrinkage, shorten oreven disappear in the D, E group, MTT colorimetric assay to detect the survival ofhippocampal neurons, hippocampal neurons in cell viability decreased with the increaseof the concentration of BDE-209(P<0.05), experimental group compared with thecontrol group difference was significant (P<0.05). Experimental group compared withthe control group, each exposure group SOD activity decreased, NO content and MDAcontent increased, the differences were statistically significant (P<0.01), the GSHcontent was decreased in the different experimental groups with the dose of BDE-209increasing, and B, C, D, E group compared with the control group, the difference wasstatistically significant (P<0.05). Western blot analysis showed: BDE-209exposedprimary cultured fetal rat hippocampal neurons induced group C (25μg/ml BDE-209),D (50μg/ml BDE-209), E (100μg/ml BDE-209) CHOP expression increased comparedwith the control group, and the difference was statistically significant (P<0.05); group B(12.5μg/ml BDE-209), C (25μg/ml BDE-209), D (50μg/ml BDE-209), E (100μg/mlBDE-209) Caspase-12expression increased compared with the control group, and thedifference was statistically significant (P<0.05); Caspase-12expression of group A(6.25μg/ml BDE-209) lower than control group, but the difference was not statisticallysignificant (P>0.05); group B (12.5μg/ml BDE-209), C (25μg/ml BDE-209), D (50μg/ml BDE-209), E (100μg/ml BDE-209) compared with the control group, the ratio ofBax/Bcl-2increased and the difference was statistically significant (P<0.05). ConclusionBDE-209can induce fetal rat hippocampal neurons’ morphology change and survivalrate decrease, and with dose response relationship. BDE-209can cause oxidativedamage in primary cultured fetal rat hippocampal neurons, and cause endoplasmicreticulum-associated protein CHOP, Caspase-12and apoptosis-related proteins Bax andBcl-2expression.