Local anesthetics function by blocking voltage-gated sodium channels. Tri-cyclic antidepressants share similarities with traditional local anesthetics including molecular structure and method of nerve blockade via blockage of voltage-gated sodium channels. Tri-cyclic antidepressants harbor potential for development as novel local anesthetics because of their increased potency and duration of action when compared to current agents. Previous research has indicated that significant neurotoxicity can occur when tri-cyclic antidepressants are used as local anesthetics. The purpose of this study was to determine whether such toxicity exists in cultured rat pheochromocytoma (PC-12) cells when applied to a wide spectrum of tri-cyclic antidepressant structures. In addition, the existence of a differential level of neurotoxicity was sought in order to elucidate dose response relationships.

Studies were performed using cultured rat PC-12 cells. PC-12 cells were treated with 6 different tri-cyclic antidepressants; amitriptyline, clomipramine, desipramine, doxepin, imipramine, and nortriptyline. For all experiments, statistical analysis was performed using the GraphPad Prism™ 4.0; analysis of variance (ANOVA), Tukey's multiple comparison test, a confidence interval of 95%, and P value < 0.05. Initially, viability was assessed using the visual cell counting method. This was employed to determine if tri-cyclic antidepressants had a negative effect on PC-12 cell viability. For these experiments, tri-cyclic antidepressant treatment was applied to cultured PC-12 cells in Corning t25 flasks. PC-12 cells were removed from the t25 flasks (after 24 hours) and placed on a hemocytometer. Cells were stained with Trypan Blue and placed under a microscope for direct visual counting. The stain adhered to dead PC-12 cells, while viable cells remained stain-free. All six tri-cyclic antidepressants were tested at the following concentrations; 10uM, 30uM, 50uM, 62.5uM, 100uM, and 200uM.

In order to further elucidate dose response relationships, cell viability was then determined using fluorescence analyses. Tri-cyclic antidepressant treatment was applied to cultured PC-12 cells in 48-well plates. After incubation (24 hours), the Promega Cell Titer Blue Assay™ was applied (20uL of Cell Titer Blue reagent per 100uL of culture medium) to each well of the treated cells. Plates were wrapped in foil and placed on the Labline Max Rotator™ for 30 minutes. Relative absorbance was determined using the SynergyMX™ microplate reader (excitation = 560nm, emission = 590nm). Consistent readings among control groups facilitated comparison between various micromolar (uM) concentrations of tri-cyclic antidepressants. The six tri-cyclic antidepressants were tested using this methodology at the following concentrations; 1uM, 3uM, 10uM, 30uM, 50uM, 62.5uM, 100uM, and 200uM.

The visual cell counting method showed that with increasing concentrations of applied tri-cyclic antidepressants, PC-12 cell viability decreased. Fluorescence analysis showed similar results. The decreased PC-12 cell viability induced by the six tri-cyclic antidepressants proved to be dose-dependent. Furthermore, the dose-response relationship for each individual tri-cyclic antidepressant was found to be unique, with different dose-response curves for each drug.

KEYWORDS: local anesthetic, tri-cyclic antidepressant, pheochromocytoma cells, PC-12 cells, fluorescence analysis