NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons

Abstract

N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to acute brain insults such as stroke and brain trauma. Understanding the mechanisms underlying NMDAR-mediated excitotoxicity may lead to development of novel therapeutics for treating neurological diseases. Stimulation of primary cultured neurons with excessive NMDA is widely used as an in vitro model for studying NMDAR-mediated excitotoxicity, which allows careful dissection of the detailed cellular mechanisms underlying excitotoxic neuronal death.Lactate dehydrogenase (LDH) is a cytoplasmic enzyme which can convert NAD into NADH. LDH is released from cells into culture medium when the plasma membrane integrity is compromised. Therefore, the amount of released LDH represents the degree of cell death. In our current study, the extracellular LDH level was measured using an in vitro Toxicology Assay Kit obtained from Sigma-Aldrich. The basis of this LDH assay is: 1) LDH reduces NAD into NADH, 2) the resulting NADH is then utilized in the stoichiometric conversion of a tetrazolium dye, and 3) the resulting colored compound is measured by a spectrophotometric microplate reader at a wavelength of 490 nm. The cell death rate was expressed as a percentage (%) between the absorbance of treated group and that of control group.