Effects of local anesthetics and tetrodotoxin on Cultured Neuron of Lymnaea stagnalis

为了了解利多卡因引起的细胞内钙离子浓度升高是否产生神经毒性，我们用Fura-2成像技术比较了培养的Lymneea stagnalis神经元的形态变化和细胞内钙离子浓度的变化。我们用钙离子螯合剂BAPTA-AM和钙离子载体Calcimyin A23187来阻止细胞内钙离子浓度的升高，以确定细胞内钙离子浓度升高与神经元损伤之间的关系。随着利多卡因浓度的增加，细胞内Ca~(2+)浓度增加，而3x10；~(-2)M利多卡因对细胞形态无损伤，细胞在生理盐水(从238±63 nM到1038±156 nM)和无钙培养液(从211±97到1046±169 nM)中细胞内Ca~(2+)浓度增加，细胞形态发生损伤和收缩。加入BAPTA-AM后，利多卡因浓度为3×10~(-2)~(-2)nM时，细胞内钙离子浓度由150±97增加到428±246 nM，细胞形态发生损伤。用台盼蓝染色将形态发生改变的细胞染成深蓝色。细胞内钙离子载体引起细胞内钙离子浓度的极大升高，但未观察到细胞形态的损伤，表明形态损伤是利多卡因本身引起的，而不是细胞内钙离子浓度的升高。控制细胞内Ca~(2+)浓度的升高不足以预防利多卡因的神经毒性。

To know whether the increase in intracellular Ca^<2+> concentration by lidocaine produces neurotoxicity, we compared morphological changes and Ca^<2+> concentration using fura-2 imaging in the culture neuron of Lymnaea stagnalis.We used BAPTA-AM, a Ca^<2+> chelator, to prevent the increase of the intracellular Ca^<2+> concentration and Calcimycin A23187,a Ca^<2+> ionophore, to identify the relationship between increased intracellular Ca^<2+> concentration and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells.Increasing the dose of lidocaine increased the intracellular Ca^<2+> concentration ; however, there was no morphological damage to the cells in lidocaine at 3x10^<-3> M. Lidocaine at 3x10^<-2> M increased the intracellular Ca^<2+> concentration in both the saline (from 238±63 to 1038±156 nM) and the Ca^<2+>-free medium (from 211±97 to 1046±169 nM) and produced morphological damage and shrinkage with a rugged surface. By the addition of BAPTA-AM, lidocaine at 3x10^<-2> M moderately increased the intracellular Ca^<2+> concentration (from 150±97 to 428±246 nM) and produced morphological damage. These morphologically changed cells were stained to dark blue with trypan blue dye. Ca^<2+> ionophore resulted in an extreme increase of the intracellular Ca^<2+> concentration ; however, no morphological damage was observed.These results indicated that the morphological damage was induced by lidocaine itself rather than by the increase in the intracellular Ca^<2+> concentration. Controlling the increase in the intracellular Ca^<2+> concentration is not enough to prevent the neurotoxicity of lidocaine.