Abstract
Purpose :
Evaluation of metabolites for cell survival and neurite outgrowth of neuronal and/or retinal ganglion cells (RGCs). In combination, both analyses allow for selectivity of the most viable metabolites with potential to treat neurodegenerative diseases.
Methods :
From a combination of literature review and UHPLC metabolite profiling from murine models of glaucoma, metabolites were selected for their potential in promoting neuronal cell survival and neurite outgrowth. The following metabolites were tested: Taurine, Histidine, Allantoin, alpha-Glycerophosphocholine (alpha-GPC), and Acetyl-L-carnitine. In preparation for the experiment, the metabolites were solubilized in phosphate-buffered saline (PBS) to obtain a 50 and 100 picoMolar concentration.The SH-SY5Y is a well-established cell line to test for cell death. Twenty-four hours after seeding, normal media was switched with metabolite-specific media. The effects of each metabolite were recorded at day five. Cell survival was evaluated with a staining process using three dyes: Propidium Iodide (PI), Nuc Blue (NB), and calcein AM. PI is an impermeable dye that stains for nuclear cell death, NB is a permeable dye that stains all cell nuclei, and calcein AM stains for live cells. To measure neurite outgrowth, RGCs were first isolated from a murine globe, seeded into a 96 well plate, and left to incubate. The metabolite-specific media was then administered. After three days, the RGCs were stained with β tubulin 3, an antibody that acts as a neuronal marker. Both cell types were imaged in serum- and phenol red-free media using Leica Stellaris 5 confocal microscope. The images were analyzed by ImageJ.
Results :
Several metabolites were found to promote neurite outgrowth in RGCs. However, many metabolites that promote neurite outgrowth in dorsal root ganglion cells, hippocampal cells or PC12 cells were found not to promote neurite outgrowth in RGCs.
Conclusions :
Between both concentrations (50 and 100 pM), all metabolites showed increased neurite outgrowth. Understanding these effects further allows one to compare cell viability to future neurite outgrowth experiments on RGCs. A combined analysis of cell survival and neurite outgrowth can provide guidance to design further in vivo studies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.