| 1. The cultivation, identification and morphological investigation of ratprimary hippocampal neuronsObjective: The new born24h SD rat hippocampal neurons were culturedin order to master the cultivation method of rat primary hippocampal neuronsin vitro and observe the morphological characteristic of primary hippocampalneurons. Immunofluorescence chemistry was uesd to identify the neurons.For the sake of the cell viability of developing hippocampal neurons, thegrowth curve of hippocampal neurons were detected.Methods: New born24h SD rats were sacrificed by decapitation, and thebrains were carefully removed and transferred into the fluid of icecoldD-hanks. Two coronal cuts were then made to expose the anterior horn of thelateral ventricles, and hippocampi were microdissected. Divested themeninges and vessel,the small tissue pieces obtained were dissociatedmechanically and digested with ACCUTASE for15min at37°C,5%CO2inthe incubator. The reaction of ACCUTASE was stopped with DMEM involvedin10%FBS, and cells were recovered after5min of centrifugation at800r/min. Single cell suspension was obtained by svsieving cells with a200mesh cell sieve. Cells were washed three times with culture medium andtransferred to plastic culture flasks into the incubator for1h to harvest theneurons which the speed of adherence were slower then glial cells beforeplating onto metrigial-coated in glass covers for different experiments at adensitiy of1×106/mL cells. The serum containing plating medium werereplaced by a serum-free Neurobasal medium supplemented with2%B27,1%N2and100mM L-glutamine within24h after plating. Half of the culturemedium was changed every2days thereafter. With7d neurons4%PFA fixed,0.3%Triton15-20min,10%bovine serum albumin(BSA) for30min at37°C, incubated overnight at4°C with a mouse against rat antibody againstβ-tublinШ, FITC labeled goat anti mouse antibody and50μg/mL Hoechst33258, kept at room temperature without light for1h. Investigate β-tublinШexpression with light microscopy, choose5visual field randomly and countthe positive ratio and photography. Cells were cultivated in96well plates,detected the absorbance every2d, total detected5times.Results:1The newly inoculated hippocampal neurons were round, small,translucent, and in a single suspension uniform distribution. After vaccinationwith3h, the cells started adhere on the culture plates, and most of neurons hadadhered after20h. Most of the cell morphologies were fusiform, triangular,with different lengths of slender protrusions. On the3th day, cells aggregated,and the prominences were elongated and enlarged those could form sparseconnection. On the7th to10th day, the cell body became chubbiness. Withtime extending, cell body enlarged furthermore, prominence enlarged andelongate, and then connected with each other. The cell body was multipolar.There were many kinds of shapes, such as pyramid, triangle, spindle andellipse. After cultivated for20d, cells began degenerate. There were a numberof granule vacuoles in the neurons, and the phenomenons of prominenceregression, nuclus pycnosis and disintergration could be observed.2Theprimary cultivated hippocampal neurons were verified by positive staining ofβ-tublinШ against Hoechst33258. The positive cell ratio is94.2%±3.6%.3The primary cultivated hippocampal neurons undergone the latency period(2-4d), the exponential growth phase(4-14d), in which period the rate of cellgrowth was extremely fast, and then the growth plateau phase(14-16d).Conclusions:1The primary cultivated neurons were matureon the10d.The cell bodies were triangle, spindle and oval under the phase invertedcontrast microscope.2The immnunocytochemistry of cultivated hippocampalneurons was showed with the positive of β-tublinШ against Hoechst33258.The positive cell ratio was94.2%±3.6%.3The cultivated neurons wouldundergone a latency period, exponential growth phase and plateau phase invitro. 2. The neurotoxicity effect of ketamine on developing hippocampalneuronsObjective: To evaluate the effects of increasing concentrations anddurations of ketamine exposure on the differentiation and survival of immatureprimary hippocampal neurons in culture, MTT assay was used to filter theconcentration and duration with which the neurons were significantly inhibit.Morphology and apoptosis were observed to evaluate the neurotoxicity ofketamine. In order to observe whether NMDA can reduce the neurotoxicity ofketamine, MTT and Hoechst33258immunofluorescence staining were used.Immunohistochemistry and Western blot were used to observe the effect ofcPKCγ-GAP-43signaling pathway in the ketamine neurotoxicity.Methods: The immature hippocampal neurons which were cultivated for4-5d days in vitro were added different concentrations of ketamine(0.1uM,1uM,100uM,300uM,1mM), and then cultivated for3h,6h,12h and24hrespectively. In order to correct the MTT value, blank control groups weredesigned in each experiment. The neurons were randomly grouped as controlgroup and different concentrations of ketamine groups. Each group wascultured with neurobasal medium for3h,6h,12h and24h respectively. Thenthe MTT data of different groups were dected. Cell viability rate was derivedfrom the formula as Rate=(contorl group-blank group)/(experimental group-blank group). According to the results of MTT, neurons were randomlydivided into the normal control group, different concentrations of ketaminetreatment groups which were treated with the concentration of the firstexperiment. The neurons of the longest neurite(LPD), branch number(PDs)and neurite length(TDL)were observed under aninverted phase contrastmicroscope after cultured for12h. Total dendritic length(TDL) was measureddrawing all visible processes with Image-Pro Plus6.0. And then the neuronalapoptosis of each experimental group was photographed after staining with Hoechst33258. According to the results of morphological observation andHoechst33258immunofluorescence staining experiments, the neurons weredivided into four groups, the contronl group(C),300μM ketamine group(K),100μM NMDA group(N) and300μM ketamine plus100μM NMDA group(K+N). Using the method of MTT to evaluate the cell viability in each groupand the immunofluorescence staining with Hoechst33258to evaluateapoptosis in each group; Immunohistochemistry and Western blot analysiswere used to decte the expression levels of cPKCγ, GAP-43and p-GAP-43ineach experimental group.Results:1Effect of ketamine on the hippocampal neurons viability hadtime and concentration interaction (F=6.227, P<0.01). The neural vitality hada significant difference among different concentrations groups in that neuronswere exposed to different durations of ketamine (F=6.750P<0.01), of which300uM and1mM ketamine for different durations had a significant difference(F=4.192, P<0.05and F=44.444, P<0.01),while no significant difference wasobserved among other concentrations to deal with different durations. On theother hand, the neural vitality had a significant difference among differentdurations groups in that neurons were exposed to different concentrations ofketamine (F=136.068P<0.01), and in every same duration, compared tocontrol group, the effect of300uM and1mM ketamine on hippocampalneurons vitality had a significant difference. The inhibition of neurons vitalitywas the most serious when1mM ketamine cultured for24h, and neuronsvitality was down to40%. Interestingly,0.1and1uM ketamine at24h and12hhad an positive effect of hippocampal neurons vitality respectively (P<0.05).2In the duration of12h, TDL and LPD of0.1uM and1uM groups, comparedwith the control group were statistically significantly increased(P<0.01), butPDs was no significant difference in0.1uM group. On the contrary the TDL,LPD and PDs of100μM,300μM and1mM, compared with the control group,were decreased significantly(P<0.01), especially in300μM and1mM(P<0.001). PDs of1mM group was significantly decreased from4.66±0.59to1.82±0.53(P<0.01).3Staining with Hoechst33258, the nucleus of the control group showed homogeneous blue light. Apoptosis in0.1uM,1uM and10uMgroups was not obvious, but the apoptotic cells would be observed in100μM,300μM and1mM especially in300μM and1mM groups.4In the duration of12h, compared with the control group, N group could significantly improvethe neuron activity(P<0.05), the neuron activity of300μM which wasinhibited by ketamine could significantly be improved when100μM NMDAwas jioned in(P<0.05). Apoptosis in N and K+N group was not obvious, butthe phenomenon of apoptotic could be observed in K group.5Theimmunocytochemical analysis, compared with C group, showed that adding300uM ketamine could significantly reduced the expression of cPKCγ ofGAP-43and p-GAP-43protein(P<0.01), adding NMDA could mitigatesignificantly in the inhibition of ketamine on the expression of cPKCγ(P<0.01), GAP-43(P<0.01) and p-GAP-43(P<0.05) protein, but there werestill significant differences compared with C group.6Western blot analysisshowed that: the expression of cPKCγ, GAP-43and p-GAP-43in N group hadno significant differences compared with C group(P>0.05). And adding300uM ketamine could significantly reduce the expression level of the abovethree proteins(P<0.01), but joined100uM NMDA could inhibit the reductioneffect of K group on these proteins expression levels.Conclusions:1Ketamine could induce concentration-and time-dependent apoptosis in the immature hippocampal neurons. With theconcentration of ketamine increasing, the cell viabilities of neurons werenotablly decreased, especially at the concentration of1mM.2Ketamine couldpromote hippocampal neuron survival when they were exposured at theconcentration of0.1μM and1uM.3The amount of cPKCγ, GAP-43andp-GAP-43protein expression could be decreased by ketamine.4100uMNMDA played a significant neuroprotective effect when the immaturehippocampal neurons were exposured to300uM ketamine. |
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