Abstract
Our previous work has demonstrated efficacy of a new chemical class of the slow γ-aminobutyric acid type A receptor anesthetics that produce minimal effects on breathing and hemodynamics in rats. To advance pre-clinical testing, we screened one member of our class of compounds, KSEB 14 - 01, for mitochondrial toxicity in primary neuronal and astrocyte cultures from mice. Prior to treatment, cell cultures were incubated with: Mitotracker GreenTM to assess mitochondrial density, tetramethylrhodamine ethyl ester to assess mitochondrial membrane potential, dihydroethidium to assess reactive oxygen species (ROS), and 4',6-diamidino-2-phenylindole for cell counting. Cultures were treated with either propofol or KSEB 14 - 01 (dissolved in dimethylsulfoxide) at concentrations of 5, 10, or 30μM. ROS and mitochondrial membrane potential were measured for 6h after which mitochondrial density and cell proliferation were quantified. In parallel experiments, continuous oxygen consumption rates (OCR) were measured during glucose deprivation (GD) in astrocyte cultures. No significant differences were observed in cell count or between treatments. In both neurons and astrocytes, mitochondrial membrane potential was decreased at 6h with 5μM propofol and KSEB 14 - 01 treatment, but remained stable with higher doses of propofol and KSEB 14 - 01. Neuronal ROS generation increased in all groups by 6h but was significantly lower with 10 and 30μM propofol and KSEB 14 - 01. In all treatment groups, astrocyte ROS generation was significantly increased after 6h, with no differences between groups. After 48h GD astrocyte OCR was maintained with all doses of KSEB 14 - 01 and with the highest dose of propofol. In conclusion, this study demonstrates that KSEB 14 - 01 does not exhibit any overt mitochondrial toxicity relative to propofol in either neurons or astrocytes. Future in vivo work is warranted to explore the mechanisms and applications for maintenance of bioenergetic equivalents during cell stress with KSEB 14 - 01.