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Ca^<2+>浓度升高是否会产生神经毒性，我们用Fura-2成像技术比较了培养的滞水蛙神经元的形态学变化和Ca^<2+>浓度。我们使用了BAPTA-AM（一种Ca^<2 +>螯合剂）和钙离子载体Calcimycin A23187（一种Ca^<2+>载体）来防止细胞内Ca^<2 +>浓度的升高，确定在无利多卡因的情况下细胞内Ca^<2+>浓度升高与神经元损伤之间的关系。用台盼蓝染色证实细胞形态学改变，增加利多卡因的剂量可增加细胞内Ca^2+浓度，然而，在3 × 10 ^ M的利多卡因中细胞没有形态学损伤<-3>。3 × 10 ^ M的利多卡因<-2>使生理盐水（从238±63 nM增加到1038±156 nM）和无Ca^2+培养基（从211±97 nM增加到1046±169 nM）中的细胞内Ca^2+浓度增加，并产生形态学损伤和具有粗糙表面的收缩。通过加入BAPTA-AM，3 × 10 ^ M的利多卡因<-2>适度增加了细胞内Ca^&lt;2+&gt;浓度（从150±97 nM增加到428±246 nM），并产生了形态学损伤。这些形态学改变的细胞用台盼蓝染料染成深蓝色。Ca^2+载体可使细胞内Ca^2+浓度显著升高，但未观察到细胞形态学的损伤，提示细胞形态学的损伤是由利多卡因本身引起的，而不是细胞内Ca^2+浓度升高引起的。控制细胞内Ca^2+浓度的增加不足以防止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.