Effects And Mechanisms Of Propofol And Dexmedetomidine On Developing Hippocampal Neurons Of Rats In Vitro

PartⅠ The culture and identification of primarily cultivated hippocampal neurons from SD rats Objective: The hippocampal neurons of newborn SD rats were cultivated in order to master the methods of primarily culture. Immunofluorescence technique was used to identify the neurons in vitro. Methods: Newborn(within 24 h) SD rats were disinfected with iodine and 75% alcohol,then sacrificed by decapitation.The brains were carefully transferred into the icecold DMEM solution with curved forceps. Hippocampus were separated and dissected. The vessels and meninges of hippocampus were divested under microscope.Then hippocampus were transferred into another DMEM solution involved papain, digested for 30 min at 37 °C in the incubator. The digestion was stopped with DMEM that involved in 10% fetal bovine serum.The solution was collected and extracted 0.9 ml for counting cells by Typan staining assay. Cells were planted onto metrigial-coated plate in the density of 1×106/ml. Neurons were observed by inverted microscope. The serum containing plating medium was replaced by serum-free neurobasal medium( supplemented with 2% B27, 1% N2) after 4~6 h. Half of the culture medium was changed every 2 or 3 days thereafter. Applying immunofluorescence technique labeled neurons and astrocytes respectively in three randomly wells,and captured three photos from every well randomly. The number of neurons, astrocytes and all nucleus were counted separately.The purity of neurons in each well were indicated by the mean percentage of neurons. Results: 1 The primarily cultivated hippocampal neurons grew in good condition with typical morphology. 2 The purity of neurons was(94.81 ± 1.90)%, no significant difference between groups(P = 0.81).Conclusions: The primarily cultivated neurons can meet the requirements of further experiments.Part ⅡEffects of propofol and dexmedetomidine on axons, dendrites and synapses of developing hippocampal neurons Objective: To evaluate the effects of propofol and dexmedetomidine on axons, dendrites and synapses of developing hippocampal neurons. Methods: The immature hippocampal neurons which were cultivated for 6 days were divided into five groups(group C,F,P,D,PD)randomly. Group P was added propofol to a final concentration of 100 μmol/L; Group F was added fat emulsion in the same volume; Group D was added dexmedetomidine to a final concentration of 100 nmol / L; Group PD was added propofol and dexmedetomidine to a final concentration of 100 μmol/L and 100 nmol / L respectively; Group C was added saline in the same volume as blank control group.Each group was washed twice with PBS and changed new neurobasal medium 8 h after drug treatment. 24 h after that,using microscope shot photos for neurons in each group randomly.The number of neurons and the total length of neurites within each view were measured with software. Statistical indicators were the mean total length of neuritis(L) in each view. The fluorescence images of synaptophysin were obtained by immunofluorescence technique. The amount of synaptophysin was indicated by the mean optical density(MOD) of synaptophysin in each group. Results: 1 The mean total length of neuritis(L):compared with group C, the value decreased in group P and PD,and the differences were statistically significant(P <0.05); There were no significant differences among group F, group D and group C; Compared with group P, the value increased in group PD,and the difference was statistically significant(P <0.05). 2 MOD:Compared with group C, the value decreased in group P and PD,and thedifferences were statistically significant(P <0.05); There were no significant differences among group F, group D and group C; Compared with group P, the value increased in group PD,and the difference was statistically significant(P <0.05). Conclusions: 1 Propofol inhibitted the development of axons,dendrites and synapses in developing primarily cultivated hippocampal neurons; 2 Dexmedetomidine extenuated the inhibition of propofol on developing axons,dendrites and synapses of hippocampal neurons in vitro.Part Ⅲ The possible mechanism of propofol and dexmedetomidine effecting the development of hippocampal neurons Objective: To detect the possible molecular mechanisms of propofol and dexmedetomidine effecting the development of hippocampal neurons. Methods: The immature hippocampal neurons which were cultivated for 6 days in vitro were divided into six groups(group C,F,P,D,PD, Y)randomly. Group P was added propofol to a final concentration of 100 μmol/L; Group F was added fat emulsion in the same volume; Group D was added dexmedetomidine to a final concentration of 100 nmol / L; Group PD was added propofol and dexmedetomidine to a final concentration of 100 μmol/L and 100 nmol / L respectively; Group Y was added yohimbine to a final concentration of 2 μmol/L first, then added propofol and dexmedetomidine to a final concentration of 100 μmol/L and 100 nmol / L respectively;Group C was added saline in the same volume as blank control group. Each group was washed twice with PBS and changed new neurobasal medium 8 h after drug treatment. Neurons of each group were collected separately 24 h after that, and used for western blot.Applying gel image analysis V 2.02 software analysed the gray values of every protein bands. The relative amount of target proteins(BDNF, Trk B and CRMP-2) were indicated by the ratio of the target proteins with β-actin.Results: 1 BDNF:Compared with group C, the value decreased in group P and PD,and the differences were statistically significant(P <0.05); Compared with group P, the value increased in group PD,and the difference was statistically significant(P <0.05); Compared with group P, the value decreased in group Y,and the difference was statistically significant(P <0.05); There were no significant differences among group F, group D and group C; 2 Trk B: Compared with group C, the value decreased in group P, group D and group PD,and the differences were statistically significant(P <0.05); Compared with group P, the value increased in group PD and group D,and the differences were statistically significant(P <0.05); Compared with group P, the value decreased in group Y,and the difference was statistically significant(P <0.05); There were no significant differences between group F and group C;3 CRMP-2: Compared with group C, the value decreased in group P and PD,and the differences were statistically significant(P <0.05); Compared with group P, the value increased in group PD and group Y,and the differences were statistically significant(P <0.05); There were no significant differences among group F, group D and group C. Conclusions: 1 Propofol reducing the expression of BDNF,Trk B and CRMP-2 may be one of the possible molecular mechanisms for propofol effecting the development of primarily cultivated hippocampal neurons; 2 Dexmedetomidine enhancing the expression of BDNF,Trk B and CRMP-2 in propofol treated hippocampal neurons may be one of the possible molecular mechanisms for dexmedetomidine extenuated the inhibition of propofol on developing hippocampal neurons in vitro.