Effects And Mechanisms Of Propofol On Toxity Of The Developing Hippocampal Neurons In Rats

Part Ⅰ.The cultivation, identification and morphological investigation ofrat primary hippocampal neuronsObjective: The new born24h SD rat hippocampal neurons werecultured in order to master the cultivation method of rat primaryhippocampal neurons in vitro and observe the morphological characteristicof primary hippocampal neurons. Immunofluorescence chemistry was usedto identify the neurons.For the sake of the cell viability of immaturehippocampal neurons, the growth curve of hippocampal neurons weredetected.Methods: New born24h SD rats were disinfected with75%alcoholand sacrificed by decapitation, cut the skin and skull, exposed thehemispheres, the brains were carefully removed with curved dissectingforceps and transferred into the fluid of icecold DMEM. Two hippocampiwere separated and dissected. The vessel and meninges were divested undera dissecting microscope, and transferred into anothor fluid of DMEM tinvolved in papain and digested for30min at37°C,5%CO2in the incubator.The reaction of papain was stopped with DMEM that involved in10%FBS.The tissues were blowed several times with imported spearhead and thecell suspension was collected twicely and extracted0.9ml to cells count byTypan staining assay. Cells were plated onto metrigial-coated24wellplates at a density of1×106/ml cells.Neurons were observed byinverted microscope after4-6h and the serum containing plating mediumwere replaced by a serum-free Neurobasal medium supplemented with2%B27,1%N2. Half of the culture medium was changed every2daysthereafter. With7d neurons4%PFA fixed,0.3%Triton15-20min,10%bovine serum albumin(BSA) for30min at37°C, incubated overnight at 4°C with a rabbit against rat antibody against β-tublinШ(1:200dilution),incubated2h Dylight488labeled goat anti rabbit antibody, kept at roomtemperature without light and10μg/mL Hoechst33258for20min.Investigate β-tublinШ expression with a fluorescent inverted microscopy,choose5visual field randomly and count the positive ratio and photograph.Take average as the neuronal purity. Cells were cultivated in96well plates,detected the absorbance every2days, total detected8times.Results:1The newly inoculated hippocampal neurons were round,small,translucent, and in a single suspension uniform distribution.Aftervaccination with4～6h, the cells had adhere on the culture plates and hadslender protrusions. Most of the cell morphologies were round, ellipse and afew fusiform.After24h, Most of the cell morphologies were fusiform,triangular, spindle,with different lengths of slender protrusions.Aftervaccination with48h,the number of prominences increased to2～3andelongated,few of glial cells were flat polygon. On the3th day, neuronalmorphology was typical. The protrusions were elongated and enlarged thosecould form sparse connection. On the5th day, cells aggregated andprominences form dense connection.On the7th to10th day, the cell bodybecame chubbiness and the halo around cells was obvious. The prominencesconnection were more dense. After cultivated for14d, cells aggregatedobviously and plates emerged a significant blank space between cells.Afterthis,cells began degenerate. The halo around cells was fade, the prominencebegan regression, and nuclus pycnosis could be observed.2The primarycultivated hippocampal neurons were typical, prominences formed denseconnection and nucleus were clear. Theβ-tublinШ against Hoechst33258positive cell ratio is91.21%±0.02%.3The primary cultivated hippocampalneurons undergone the latency period (2～4d), the logarithmic phase (4～12d), in which period the rate of cell growth was extremely fast, and then thegrowth plateau and degenerating phase (12～16d),in which period,cellgrowth was in a state of stagnation and decline.Conclusions:1The primary cultivated neurons were mature on the10th day.The cell bodies were triangle, spindle and oval under the phase invertedcontrast microscope.2The immnunocytochemistry of cultivated hippocampalneurons were showed with the positive of β-tublinШ against Hoechst33258.The positive cell ratio was91.21%±0.02%.3The cultivated neurons wouldundergone a latency period, exponential growth phase, plateau anddegenerated phase in vitro. PartⅡ Effects of propofol on the viability and apoptosis of thedeveloping hippocampal neuronsObjective: To evaluate the effects of different concentrations andcultivating different time of propofol exposure on developping primaryhippocampal neurons in culture, MTT assay was used to measure theviability of the neurons. Flow cytometer was used to evaluate the effects ofdifferent concentrations of propofol on the ratio of the apoptosis and cellcycle of the developing primary hippocampal neurons cultivated for24h. Inorder to observe the effects of propofol on the apoptosis of the developpinghippocampal neurons, Hoechst33258immunofluorescence staining wereused.Methods: The immature hippocampal neurons which were cultivatedfor4～5d days in vitro were added different concentrations of propofol(0μg/ml,10μg/ml,40μg/ml,60μg/ml,80μg/ml and100μg/ml), and thencultivated for12h,24h,48h and72h respectively. Blank control groups,that was primary culture medium were designed in each experiment. Cellviability rate was derived from the formula as Rate=(contorl group blankgroup)/(experimental group-blank group). According to the results of MTT,the immature hippocampal neurons were undergone different concentrationsof propofol as same as before and cultivated on the24well plates for24hfurther. According to formulary requirement, samples of all groups (0μg/ml, 40μg/ml,60μg/ml and80μg/ml propofol) were prepared, and then the cellcycle and apoptosis rate of all groups were analyzed by flow cytometer. Inorder to verify the results of FCM and MTT, apoptotic morphology of allgroups were detected by the Hoechst33258staining.Results:1The inhibitions of propofol on the hippocampal neuronsviability had time and concentration interaction (F=11.116, P<0.01). Theeffects of propofol on the vitality of the neurons during different action timewere significant (F=18.565, P<0.01), there were no significant difference in12h and48h exposure of propofol. On the other hand, when the exposuretime was same, the neural vitality had a significant difference amongdifferent concentrations (F=889.074, P<0.01), and in each same duration,compared with the control group, effect of10μg/ml propofol on hippocampalneurons vitality had no significant difference.2The apoptosis rate hadsignificant difference among each group（F=76.577, P<0.01). There werestatistic differences in apoptosis rate of the neurons exposed to differentpropofol groups compared with those of the control group, and the apoptoticrate of cells were dose dependent.According to cell cycle, G0/G1and S phaseof hippocampal neurons were significantly different among each group(F=138.456, P<0.01;F=114.158, P<0.01). There were statistic differences inG0/G1and S phase of40,60and80μg/ml propofol compared with those ofthe control group. With the increasing of the propofol concentration, the rateof G0/G1was declined and the rate of S was increased. There were statisticdifferences in G2/M of hippocampal neurons among each group (F=14.131,P<0.01). There were no statistic differences of40μg/ml and60μg/mlpropofol compared with those of the control group.3The results of Hoechst33258staining showed that with the increasing of propofol concentration,more and more nucleus became irregular with a slightly serrated andcorrugated border, and then the peripheral chromatins aggregated into densemasses and peripherization, and even the nucleus of100μg/ml propofolbroke into fragments.Conclusions: The inhibitions of propofol to the developing hippocampal neurons were concentration and time dependent, which hadinteraction effects. There were significantly statistic differences in apoptosisrate of the neurons exposed to different propofol concentration and theapoptosis rate was increasing with the increasing of concentration. Propofolcan promote developing neurons from G0/G1phase to S phase but not toG2/M phase. The proliferation of neurons is arrested in S phase. Part ⅢThe role of Rho signaling pathway in the neurotoxicity ofdeveloping hippocampal neurons induced by propofolObjective: To detect the effect of Rho signaling pathway in theneurotoxicity of propofol by real-time PCR.Methods: The neurons cultivated for4～5d days in vitro were dividedinto four groups, the control group(C),80μg/ml propofol group (P),80μg/ml propofol plus10μM Y27632group (P+Y) and10μM Y27632group(Y). Neurons in all groups were cultured for24h, RT-PCR were used todetect the gene expression levels of RhoA, Rac1, CDC42and PAK1in eachgroup.Results: The data derived from the analyses of RT-PCR showed that theexpressions of RhoA in group P were significantly higher than those incontrol group(P<0.01), but the expression of RAC1,CDC42and PAK1werelower(P<0.01, P<0.05).The expressions of RhoA in group (P+Y) was lowerthan that in group P (P<0.01) and significantly higher than that in groupY(P<0.01).The expressions of RAC1,CDC42and PAK1in group (P+Y)were significantly lower than those in group Y(P<0.01),and the expressionsof CDC42were significantly higher than that in group P(P<0.01), however,the expressions of RAC1and PAK1were higher but had no statisticdifferences. Compared with the control group, the expressions of RhoAin group Y was lower (P<0.05) and the expressions of RAC1,CDC42andPAK1were higher (P<0.01, P<0.05). Conclusions: Propofol could active the Rho signaling pathway, whichmight take part in the neurotoxicity of propofol. Y27632plays a significantneuroprotective role in the neurotoxicity induced by propofol.