Effects Of Propofol On Ca²⁺ Mobilization Of The Rat Developing Cortical Neurons

Could general anesththetics cause developmental toxicity central nervous system.This issue was brought into sharp focus in recent years.Propofol is a commonly used drug in clinical intravenous anesthetic. It have advantages of rapid onset,short duration of action,rapid recovery,easy to contral and low side effects,so that it is widely used pediatric.anesthesia and intensive care unit.But a growing number in vivo or in vitro anmimal experiments confirmed that intravenous anesthetic propofol is toxical on the developing nervous system.Propofol toxicity mechanism on developing brain is unclear,one possibility is that it caused the disorder of neuron calcium homeostasis.Developing nuurons have their special physiological characteristics,by culturing developing neurons in vitro, this study is to evaluate the effect of different concentrations of propofol and nifedipine on Ca2+mobilization induced by KCI of the rat developing cortical neurons.The experiment mainly has two parts: frist,to optimize and improve methods of culturing primary cortical neurons in vitro,observation and identification of deceloping neurons,and to cultivate neuron of high purity and good vitality to meet the requirements of the next experiment.Second,to observe morphological changes of cortical neurons on development stage after Fluo-4 am cell permeant under the microscope, to evaluat changes in calcium concentration of developmental stage of rat within cirtical neurons under different concentrations of propofol(10,30,100μmol / L) and to evaluate the role of L-type calcium channels in propofol effecting high K+-trigged intracellular Ca2+ overload.Part 1: A method for primary culture of postnatal rat cortical neurons with high viabilityObjective : Establish a stable,efficient,convenient and practicable method to primary culture neonate rat cerebral cortical neurons. Methods:Take out SD rats born within 24 hours,separate cortex, use a cultural method: digesting with papain, Plating Poly-L-lysine before experiment, using mild mechnical triturating and after 2 hour’s cell inoculation, the whole volume of culture medium was replaced with Neurobasal A medium added B-27, The survival rates of neurons was detected by MTT assay. The morphological changes of neuron cells were observed by light microscope at different time. Immuno-staining of microtubule-associated protein 2(MAP2) was performed to identify the purity of neurons. Results Useing papain resulted in a higher neuronal survival in comparison to trypsin(P < 0.01). Using mild mechnical triturating, Plating Poly-L-lysine before experiment and after 2 hour’s cell inoculation, the whole volume of culture medium was replaced with Neurobasal A medium added B-27, there were no significant effect on the viability of neurons( P>0.05). Most cortical neuron had typical neuron morphology. Fluoresence staining with MAP2 showed that the purity of neurons was about 95%. Conclusion The present protocol is a simple,efficient,convenient and reliable method for cultureing rat cortical neurons.Part2: Effects of propofol on Ca2+ mobilization induced by KCl of the rat developing Objective: To evaluate the effect of different concentrations of propofol and nifedipine onCa2+mobilization induced by KCI of the rat developing cortical neurons,and to evaluate the role of L-type calcium channels in propofol effecting high K+-trigged intracellular Ca2+overload. Methods: Primary cultured cortical neurons day in vitro 7 obtained from neonatal SD rats, stained with fluo-4AM. The effects of propofol(0、10、30、100μmol/L) and nifedipine on changes in intracellular Ca2+([Ca2+]i) fluorescent intensity induced by KCI were determined with laser scanning confocal microscope.Results:The [Ca2+]i in vitro 7 in each group of basic value and propofol pretreatment were not statistically significant difference(P> 0.05); The[Ca2+]i After propofol pretreatment were not statistically significant difference compared to baseline values(P> 0. 05); The [Ca2+]i in cortical neurons after addition of KCl were increased(P< 0. 01), Group P30 and P100 were lower than group Control( P <0. 01), respecticely decreased by 26.3% and 28.6%. Role of LCCs in proppofol suppressing high K+-trigged intracellular Ca2+ overload.In control neurons, depolarization stimulation by high K+evoked an acute elevation of intracellular Ca2+level(P< 0.01). In propofol pretreated neurons, high K+ stimulation evoked significantly less elevation of the intracellular Ca2+ level than in control neurons(P< 0.01).After exposure to nifedipine, an LCC antagonist, for 25 min,high K+stimulation resulted in a reduction of intracellular Ca2+overload compared with control neurons lacking nifedipine exposure(P< 0.01). The inhibitory effect of nifedipine on high K+-induced intracellular Ca2+ overload did not have a notable difference in the presence and the absence of propofol(P> 0. 05). Conclusion:Propofol can decrease [Ca2+]i in Primary cultured cortical neurons day in vitro 7 probably by decreasing Ca2+ infiux via the L-type voltage dependent Ca2+ channel.