| Abstrakt: |
Background: Microglia play an important role in the early onset of Alzheimer's disease pathology1. They are responsible for maintaining brain homeostasis, where changes in energy metabolism alter microglial metabolic profile and function2. The majority of cell culture‐based studies have relied on growing microglia under optimal media conditions, not reflecting the brain's physiological levels of key metabolites. This study aims to define a suitable experimental culture system that reflects the brain's physiological glucose level, approximately 1mM3, so the effects of altered glucose concentration can be tested. Method: SIM‐A9 mouse immortalised microglial cells were initially grown in DMEM/F‐12 medium (17.5mM glucose) containing 10% FBS and 0.1% Penicillin/Streptomycin (Gibco, UK). After 20 hours cells were re‐seeded in Neurobasal medium containing 1x B‐27 supplement (Gibco, UK) and either 1mM or 5mM glucose and grown for 20 hours. Cell proliferation and viability was assessed over 40 hours using an Incucyte imaging system (Essen BioScience, UK) and Trypan Blue assay. Media glucose concentration, cellular glucose uptake (Promega, UK), microglia activation and metabolic marker expression using qRT‐PCR were measured following stimulation of cells with Lipopolysaccharide (LPS) (2.5ng/ml) Result: Maintaining SIM‐A9 cells in 1mM and 5mM glucose medium showed static proliferation but similar viability over 20 hours compared to growth in DMEM/F12. LPS treatment increased TNFa and IL1b expression in SIM‐A9 cells in all glucose conditions, indicating the cells' competency to produce a pro‐inflammatory response. No change in pro‐inflammatory activation markers (TNFa, IL1b) was seen after maintenance in 1mM, but in 5mM glucose for 20 hours, compared to LPS treated cells. Media glucose concentration was 0.7mM over 20 hours in 1mM and 1mM in 5mM glucose media. Expression of glucose transporters Glut1 and Glut3 and glycolysis marker PFKFB3 under these glucose conditions was investigated. Conclusion: Until now microglial metabolic studies have not always reflected the physiological concentrations of glucose in the brain. The results show that culturing microglia in 1mM glucose medium does not induce pro‐inflammatory activation, while maintaining minimal cell death and unvaried confluency. References: Yoshiyama Y et al (2007) Neuron 53(3): 337‐351. 2. Bernier L et al(2020) Trends in Neurosci43(11):854‐869. 3. Van de Ven K et al (2012) Diabetes 61 [ABSTRACT FROM AUTHOR] |
