Mechanisms Underlying The Effects Of Bupivacaine On Glutamate-induced Intracellular Calcium Increase In Primary Cultured Hippocampal Cells From Rat Fetuses

Bupivacaine is a local anesthetic used for caudal,epidural and spinal anesthesia and is widely used in clinics to manage acute and chronic pain.However,bupivacaine is more cytotoxic than other local anesthetics.Systemic exposure to excess levels of bupivacaine mainly produces toxic effects in central nervous system and cardiovascular system.The toxic effects of bupivacaine on the central nervous system usually occur at lower blood plasma concentrations than its cardiovascular toxicity.The detailed mechanisms of neurotoxicity induced by bupivacaine and other local anesthetics have not yet been clarified.Local anesthetics have been reported to inhibit hippocampal long-term potentiation,the form of synaptic plasticity underlying learning and memory,in rat hippocampal slices.Although the hippocampus is a crucial structure involved in learning and long-term memory functions,the hippocampus was previously reported to be highly sensitive to the convulsive activity of local anesthetics.However,little published information is available concerning the effects of local anesthetics on primary cultures of hippocampal cells.Glutamate is the most important synaptic transmitter responsible for higher brain functions in humans.Therefore,we investigated the effects induced by bupivacaine in primary cultures of hippocampal neurons and astrocytes.And in the subsequent studies,digitized video fluorescence microscopy and two-photon excitation laser scanning microscopy were used to investigate bupivacaine-induced changes in intracellular free Ca²⁺concentration([Ca²⁺]_i),mitochondrial membrane potential and reactive oxygen species?ROS?generation in primary cultures of hippocampal neurons and astrocytes from rat fetuses to clarify the underlying mechanisms.Part one Effects of bupivacaine on glutamate-induced[Ca²⁺]_i increase in rat hippocampal cellsObjective:To investigate the effects of bupivacaine on the glutamate-induced[Ca²⁺]_i increase in hippocampal cells.Methods:Cultures of primary hippocampal neurons and astrocytes were prepared from Sprague-Dawley rat fetuses.The immunofluorescence technique was used to identify the purity of pure cultures of neurons and astrocytes.The calcium imaging technology was applied to record the changes in glutamate-induced[Ca²⁺]_i increase as well as the effect of bupivacaine on glutamate-induced[Ca²⁺]_i increase in cultures of hippocampal cells.Results:1.The difference of glutamate-induced[Ca²⁺]_i increase between co-cultures and pure cultures of hippocampal cellsThe peak value of[Ca²⁺]_i increase induced by the first exposure of glutamate at 1 mM in the pure culture of astrocytes was 239±40%which was not significantly different from that in astrocytes co-cultured with neurons?233±42%;P>0.05?.However,the peak value of[Ca²⁺]_i increase?240±81%?induced by the first exposure of glutamate at 1 mM in the pure culture of neurons was significantly greater than that in neurons co-cultured with astrocytes?169±20%;P<0.01?.2.The effects of bupivacaine on glutamate-induced increases in[Ca²⁺]_i in co-cultures or pure cultures of hippocampal cellsThe glutamate-induced increase in[Ca²⁺]_i in astrocytes co-cultured with neurons was significantly attenuated by a 5 min treatment with 300?M bupivacaine.The peak value decreased from 229±55%?first glutamate exposure?to 178±41%?second exposure?with a decrease of 40%?P<0.01?.In contrast,the glutamate-induced[Ca²⁺]_i response in neurons co-cultured with astrocytes was significantly potentiated by the same treatment with bupivacaine,and its peak value increased significantly from 168±21%?first glutamate exposure?to 231±37%?second exposure?with an increase of102%?P<0.01?.The[Ca²⁺]_i response to the second glutamate exposure in pure astrocyte cultures was significantly reduced by an incubation with 300?M bupivacaine.The peak value decreased from 239±40%?first glutamate exposure?to 189±34%?second glutamate exposure??P<0.01?.However,the[Ca²⁺]_i response to the second glutamate exposure in pure neuronal cultures was not significantly changed by the incubation with bupivacaine compared with the first exposure?P>0.05?.3.The concentration-dependent effects of bupivacaine and ropivacaine on glutamate-induced increase in[Ca²⁺]_i in co-cultures of neurons and astrocytesIn the astrocyte/neuron co-culture system,bupivacaine and ropivacaine regulated the glutamate-induced increases in[Ca²⁺]_i in the two cell types in an opposite,concentration-dependent manner.Bupivacaine initiated its effect on the glutamate-induced increase in[Ca²⁺]_i at 0.3?M and peaked at 300?M.The EC₅₀ values calculated from the peak increase in[Ca²⁺]_i were 1.64?M in astrocytes co-cultured with neurons and 1.76?M in neurons co-cultured with astrocytes.Ropivacaine,another local anesthetic,had the same effects as bupivacaine,but its EC₅₀ values were 15.51?M and 16.18?M in the two types of cells,respectively.Summary:Amide local anesthetics significantly inhibited the glutamate-induced increases in[Ca²⁺]_i in rat hippocampal astrocytes co-cultured with neurons but potentiated the glutamate-induced increases in[Ca²⁺]_i in neurons co-cultured astrocytes.The toxic effect of bupivacaine on rat hippocampal neurons(significantly potentiating glutamate-induced[Ca²⁺]_i increase)may be the result of its direct inhibitory effect on astrocytes.Part two Effects of bupivacaine on the mitochondrial membrane potential and ROS generation in hippocampal cells from rat fetusesObjective:To investigate the effects of bupivacaine on the mitochondrial membrane potential and ROS generation in hippocampal cells.Methods:Cultures of primary hippocampal neurons and astrocytes were prepared from Sprague-Dawley rat fetuses.The JC-1 fluorescent probe and two-photon microscopy were used to measure the change in mitochondrial membrane potential in hippocampal cells.The change of intracellular ROS generation was record with the ion imaging technology using DCFH-DA fluorescent dye and intracellular ROS kit.Results:1.Effects of bupivacaine on the mitochondrial membrane potential of co-cultured hippocampal cellsIn neurons/astrocytes co-culture system,the administration of 300?M bupivacaine significantly decreased the mitochondrial membrane potential in astrocytes co-cultured with neurons by 60%?from 1 to 0.40±0.05?,but it did not affect the mitochondrial membrane potential in neurons co-cultured with astrocytes.2.Effects of bupivacaine on the mitochondrial membrane potential of pure cultures of astrocytes and the reversal effects of cyclosporin A?Cs A?At 300?M,bupivacaine still did not affect the mitochondrial membrane potential of pure neuronal cultures.On the other hand,bupivacaine significantly decreased the mitochondrial membrane potential by 61%in pure astrocyte cultures.Bupivacaine?0.3-300?M?dose-dependently decreased the mitochondrial membrane potential of pure astrocyte cultures,with a maximum inhibition of 61%and an IC₅₀ of 1.64?M.Pretreatment with 1?M CsA,an inhibitor of the mitochondrial permeability transition pore,significantly inhibited the bupivacaine-induced decrease in the mitochondrial membrane potential in pure astrocyte cultures.3.Effects of bupivacaine on ROS generation in pure astrocyte cultures and the reversal effects of CsABupivacaine?300?M?did not affect ROS generation in pure neuronal cultures loaded with the DCFH-DA dye,but significantly and dose-dependently increased ROS generation by 130%in pure astrocyte cultures.A pretreatment with 1?M CsA for 5 min significantly inhibited bupivacaine-induced ROS generation by 81%in pure astrocyte cultures.Summary:Bupivacaine only significantly decreased the mitochondrial membrane potential and increased ROS generation in rat hippocampal astrocytes.But bupivacaine did not influence the mitochondrial membrane potential or ROS generation in neurons.Treatment with CsA suppressed the decrease in the mitochondrial membrane potential and the increase in ROS generation in astrocytes.Bupivacaine selectively impaired the mitochondrial function of astrocytes and indirectly affected neurons.Part three Mechanisms underlying the effects of bupivacaine on the glutamate-induced[Ca²⁺]_i increase in hippocampal cellsObjective:To investigate the mechanisms underlying the effects of bupivacaine on the glutamate-induced[Ca²⁺]_i increase in hippocampal cells.Methods:Cultures of primary hippocampal neurons and astrocytes were prepared from Sprague-Dawley rat fetuses.The calcium imaging technology was used to record the changes in glutamate-induced[Ca²⁺]_i increase.Results:1.Effects of Ca²⁺-free solution or pretreatment with dantrolene on glutamate-indcuced[Ca²⁺]_i increase in pure cultures of neuronsAt 1 mM,glutamate did not produce an obvious increase in[Ca²⁺]_i in pure cultures of neurons perfused with a Ca²⁺-free solution plus 5 mM EGTA(Ca²⁺-free solution)compared to neurons perfused with a normal solution,and a pretreatment with 300?M bupivacaine did not change the action of 1 mM glutamate in pure cultures of neurons perfused with the Ca²⁺-free solution.A 10?M dantrolene treatment did not change the increase in[Ca²⁺]_i in response to 1 mM glutamate in pure neuronal cultures compared to neurons that were not treated with dantrolene?P>0.05?,and 300?M bupivacaine did not exert any effect on the 1 mM glutamate-induced increase in[Ca²⁺]_i in pure neuronal cultures that had been pretreated with 10?M dantrolene?P>0.05?.2.Effects of CsA on the bupivacaine-induced inhibition of the increase in[Ca²⁺]_i in astrocytes produced by glutamateThe inhibitory effect of bupivacaine on the glutamate-induced increase in[Ca²⁺]_i in astrocytes co-cultured with neurons and the bupivacaine-mediated potentiation of the glutamate-induced increase in[Ca²⁺]_i in neurons co-cultured with astrocytes almost completely disappeared when mixed cultures were pretreated with the combination of 1?M CsA and 300?M bupivacaine for 5 min.3.Effects of a glutamate transporter inhibitor on the opposite regulatory effects of bupivacaine in hippocampal cellsWhen 500?M dihydrokainic acid,a specific inhibitor of glutamate transporter-1,was added to the neurons/astrocytes co-culture system,the peak values of the glutamate-induced increase in[Ca²⁺]_i?first exposure?were 237±156%in neurons and 284±53%in astrocytes.In the presence of dihydrokainic acid,300?M bupivacaine did not affect the 1 mM glutamate-induced increase in[Ca²⁺]_i in neurons co-cultured with astrocytes but strongly inhibited the peak value of the glutamate-induced increase in[Ca²⁺]_i in astrocytes co-cultured with neurons.Summary:The increase in[Ca²⁺]_i induced by glutamate in neurons was mainly due to the calcium influx from extracellular fluid.The opposite regulatory effects of bupivacaine on the glutamate-induced increase in[Ca²⁺]_i were suppressed by Cs A in hippocampal astrocytes and neurons.Clinically relevant concentrations of bupivacaine directly impaired the mitochondria of astrocytes co-cultured with neurons,thereby suppressing the glutamate uptake of astrocytes,then finally induced an increase in extracellular glutamate concentration arround hippocampal cells and an increase in[Ca²⁺]_i in neurons co-cultured with astrocytes.Conclusions:Amide-typed local anesthetics potentiate the glutamate-induced increase in[Ca²⁺]_i in rat hippocampal neurons co-cultured with astrocytes,and inhibit the glutamate-induced increase in[Ca²⁺]_i in rat hippocampal astrocytes co-cultured with neurons.The opposite regulatory effects of bupivacaine on the glutamate-induced increase in[Ca²⁺]_i in the two types of cells are not the result of its sodium channel blocking activity.Clinically relevant concentrations of bupivacaine directly impaired the mitochondria of astrocytes co-cultured with neurons,thereby suppressing the glutamate uptake of astrocytes,then finally induced an increase in extracellular glutamate concentration arround hippocampal cells and an increase in[Ca²⁺]_i in neurons co-cultured with astrocytes.