Descriptive oxidative and lipid-related fluorescence observations associated with Esketamine treatment in a high-concentration NMDA-exposed HT-22/C8-D1A co-culture model.

Background: In HT-22 cells, 500 μM NMDA was used as a high-concentration oxidative stressor rather than a classical NMDA receptor-mediated excitotoxic stimulus. Because HT-22 cells lack functional ionotropic NMDA receptors, this model cannot test NMDA receptor antagonist-mediated pharmacology.

Methods: HT-22 cells and C8-D1A astrocytes were examined in a Transwell co-culture system. CCK-8 viability readout, ApoE Western blot signal, lipid peroxidation, BODIPY C12-associated fluorescence, TOMM20-region BODIPY C12 fluorescence, and MitoSOX fluorescence were assessed.

Results: NMDA exposure was associated with a lower CCK-8 viability readout and higher lipid peroxidation fluorescence. Esketamine treatment was associated with a higher CCK-8 viability readout and lower lipid peroxidation fluorescence under these oxidative stress conditions. In co-cultured astrocytes, NMDA-challenged HT-22 cells were associated with increased lipid peroxidation, altered BODIPY C12-associated fluorescence, and elevated MitoSOX fluorescence, whereas these signals were lower in the esketamine-treated condition. ApoE changes were descriptive and were not used to infer mechanism. The source and molecular identity of astrocytic BODIPY C12-associated fluorescence remain uncertain.

Conclusions: Esketamine-associated effects in this model should be interpreted as receptor-independent, oxidative-stress-associated viability and fluorescence observations. The findings are descriptive and hypothesis-generating and do not establish direct lipid transfer, causal metabolic coupling, mitochondrial fatty-acid utilization, or a defined lipid-metabolic mechanism.