Abstract
Background: Indomethacin (INDO) is used for of intraventricular hemorrhage prophylaxis and closure of ductus arteriosus in premature infants. Cellular and molecular actions of INDO on brain cells remain unclear. We examined INDO effects on cellular parameters (growth, proliferation, respiratory burst).
Methods: Assays for MTT proliferation, proteasome function (chymotrypsin activity), viability testing, and respiratory burst (flow cytometric analysis) were performed on cell lines (N2a neuroblastoma and N9 microglia) and primary neuronal and astrocyte cultures treated with INDO at 0, 100, 250, 500, 750 and 1000uM.
Results: Percent viability of N2a cells was significantly higher at 250uM and lowest at 1mM. Percent viability of N9 microglia significantly decreased from baseline (91%) to 71% at 250uM (p<0.0001), precipitously decreasing to 10% at 500uM. Proliferation assays of primary neonatal cells indicate 250–500uM INDO treatment increased astrocyte proliferation while attenuating neuronal proliferation in a dose-dependent manner. Proteasomes play an important role in intracellular degradation of aging and oxidized proteins and in activating signaling proteins like NF-kB. Normal proteasomal function of N2a neuronal cells or N9 microglia was maintained up to 250uM INDO treatment. N9 cells exhibited marked loss of activity at 500uM and 1000uM while N2a maintained activity up to 500uM. Respiratory burst assays on N9 microglial cells showed INDO pretreatment and cotreatment with LPS/IFN-gamma caused a 10% reduction in reactive oxygen species (ROS)-release by N9 cells compared to N9 with LPS/IFN-gamma treatment. Estrogen (1nM)-pretreated N9 cells decreased ROS release by 30%.
Conclusion: Cell function and integrity assays indicate a narrow dosage window (250–500uM) where INDO can preserve cellular growth, viability, respiratory burst, and proteasomal function. That estrogen is more efficient than INDO in decreasing ROS production may explain suggested gender differences in efficacy. Results indicate differential susceptibilities of CNS cells, with microglia being most sensitive while neurons and astrocytes tolerate INDO treatment better.
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Dimayuga, F., Dimayuga, E., Keller, J. et al. 95 Indomethacin Affects Cell Proliferation and Proteasome Activity in Neuronal Cultures. Pediatr Res 58, 371 (2005). https://doi.org/10.1203/00006450-200508000-00124
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DOI: https://doi.org/10.1203/00006450-200508000-00124
