Study On The Neurotoxicity Mechanism Of RhoA-ROCK Signaling Pathway In Hippocampal Neurons During Ketamine Development

1 The cultivation,identification and morphological investigation of rat primary hippocampal neuronsObjective: The new born 24 h SD rat hippocampal neurons were cultured in order to master the cultivation method of rat primary hippocampal neurons in vitro and observe the morphological characteristic of primary hippocampal neurons.Immunofluorescence chemistry was uesd to identify the neurons.Methods:The SD rats were sacrificed with 75% ethanol,cut off the head,cut the skin and skull,cut the brain with bent tweezers,placed in the pre-cooled DMEM solution,quickly stripped out of the hippocampus on both sides,The blood vessels and membranes of the hippocampus were removed under a dissecting microscope.The tissues were cut with micrographs and transferred into a mixed digestion solution of papain and DNase I and incubated in a 5% CO2 incubator for 30 min.The tissue was transferred to the DMEM containing 10% fetal bovine serum for 5 min,and then the tissue was transferred into the inoculation medium.The cells were weighed with 1 ml long gun and gently repeated until the liquid became turbid and no bulk,Followed by a 200 mesh cell screen.A viable cell count was made under a microscope to prepare a cell suspension of 1 x 106 / ml density and grown in a poly-lysine-treated 6-well culture plate.2.5 ml per well was placed at 37?,5% CO2 Of the incubator.6h after the full replacement of serum-free maintenance medium.After the day,half the amount of fluid change once.On the fourth day,5fu and uracil at a final concentration of 10 mM were added to inhibit the growth of glial c ells.The growth of neurons was observed under a microscope.After 7 days of hippocampal neuron culture,the nuclei of all cells were labeled with fluorescently labeled MAP-2 neurons and DAPI us ing immunofluorescence technique.Under the fluorescence microscope,5 holes were randomly selected and divided into 5 groups.The images were observed under low magnification(4 × 10)and high power field(10 × 20 times).Count the number of neurons and all nuclei,and calculate the percentage of neurons,taking the mean as the purity of neurons per well.Results:MAP-2 antibody and nuclear antibody DAPI were stained and stained under fluorescence uptake microscope to calculate the purity of hippocampal neurons,and the purity of hippocampal neurons was about(91.52 ± 1.70)%.Summary:In vitro culture of primary SD neonatal hippocampal neurons typical morphology,and hippocampal neurons purity of 90% or more,to meet further experimental requirements.2 Effects of different concentrations of ketamine on hippocampal neurons dendritic spinesObjective: Observe the effects of different concentrations of ketamine on dendritic spines of hippocampal neurons during development.Methods:The hippocampal neurons were cultured in the first 5 days.The rats were randomly divided into 4 groups:(1)C group(control group): adding equal volume of maintenance medium(2)K1 group: adding ketamine,the final concentration of 100?M(3)K2 group: adding ketamine,Final concentration of 300?M(4)K3 group: adding ketamine,the final concentration of 500?M.Each group was cultured for 6 hours and then fixed with fluorescent dye V22888.After that,the fluorescence images were taken at random under confocal microscopy,and the density and average length of dendritic spines were calculated.Results:1 Dendritic spine density(F=13.298,P=0.002):There was significant difference between K2 group and K3 group and C group(P<0.05).There was no significant difference between K1 group and C group.Compared with K2 group,the number of K3 group decreased,the difference was statistically significant(P<0.05);2 The average length of dendritic spines(F=30.440,P=0.000): K2 group and K3 group compared with C group,the value decreased,the difference was statistically significant(P<0.05);K1 group and C group,the difference was not statistically significant.K3 group and K2 group,the difference was not statistically significant.;Summary: The effect of ketamine on hippocampal neuronal dendritic spines was dose-dependent.With the increase of ketamine concentration,the more obvious damage to dendritic spines,the stronger the neurotoxicity effect.3 Study on the neurotoxicity mechanism of RhoA-ROCK signaling pathway in hippocampal neurons during ketamine developmentObjective:1 Western blot was used to detect the changes o f RhoA and its downstream ROCK expression after using ketamine and the changes of RhoA and ROCK protein expression after Y-27632.2 The effect of RhoA-ROCK signaling pathway on the toxicity of ketamine to primary cultured hippocampal neurons was elucidated by immunofluorescence cytochemistry.Methods:The hippocampal neurons were cultured in the first 5 days.The rats were randomly divided into four groups:(1)C group(blank control group): adding equal volume of maintenance medium(2)Y group: adding Y-27632 and final concentration of 10?M(3)group: Ketamine at a final concentration of 300 ?M(4)K + Y group: ketamine and Y-27632 were added at a final concentration of 300 ?M and 10 ?M,respectively.1 After 6 hours of incubation,the protein was extracted and the expression of RhoA and ROCK protein was detected by Western blot.2 After 6 hours of incubation,the rats were stained with V22888,and the changes of dendritic spines in each group were observed under confocal microscope.Results:1 RhoA(F=404.774,P=0.000):There was no significant difference in the expression of RhoA protein between group Y and group C.The expression of RhoA protein was significantly higher in group K than that in group C(P<0.05).There was no significant difference in the expression of RhoA protein between K + Y group and K group.2 ROCK(F=721.413,P=0.000):There was no significant difference in the expression of ROCK protein between group Y and group C.The expression of ROCK protein in group K was significantly higher than that in group C(P<0.05).K + Y group compared with K group,ROCK protein expression was significantly reduced,and the difference was statistically s ignificant(P<0.05);3 The density of dendritic spines(F=11.383,P=0.003):There was no significant difference between group Y and group C.There was significant difference between group K and group C(P<0.05).Compared with K group,the number of K + Y group increased,the difference was statistically significant(P<0.05).4 The average length of dendritic spines(F=26.152,P=0.000):There was no significant difference between group Y and group C.There was significant difference between group K and group C(P<0.05).Compared with K group,the number of K + Y group increased,the difference was statistically significant(P<0.05).Summary:1 Ketamine can activate the RhoA-ROCK signal transduction pathway to damage neuronal dendritic spines and exert its neurotoxicity.2 Y-27632 can reduce the damage of ketamine on dendritic spines,weaken the neurotoxicity of ketamine,and thus play a neuroprotective effect.Conclusions:Ketamine can play a neurotoxic role through the RhoA-ROCK signal transduction pathway.