Reduction of single cell microglial glucose uptake upon isolation from regular brain environment

Einleitung In neurodegenerative diseases, alterations of microglial energy demand and metabolism have attracted high attention. In vitro methods such as the seahorse assay are used in this context, but unclear remains, if energy metabolism of in brain highly motile microglia is impaired, when not acting in their regular environment. Therefore, we performed a direct comparison of in vivo and in vitro glucose uptake of microglia by scRadiotracing.

Material und Methoden Using CD11b/ACSA-2 immunomagnetic cell sorting (MACS) after in vivo ¹⁸F-FDG injection microglia and astrocytes were isolated from mouse brains, followed by activity measures in a gamma counter and cell counting by flow cytometry. Enriched cell fractions were then incubated with excessive ¹⁸F-FDG (50000-fold), washed, to remove non-cellular bound tracer, with equivalent gamma counting and flow cytometry. Radioactivity per cell for all fractions normalized to injected or incubated radioactivity and ratios of glucose uptake in microglia relative to the uptake in astrocytes and the CD11b/ACSA2-negative fraction were calculated. The experiment was repeated with glucose-free buffer and cross-validated by sole in vivo injection and sole in vitro incubation, to exclude influences of sobriety and glucose injection. Moreover, we compared cell culture ¹⁸F-FDG incubation against scRadiotracing data.

Befunde After in vivo injection, glucose uptake was higher per microglia cell when compared to single astrocytes (50.4-fold, p < 0.0001) and non-microglia/non-astrocyte cells (10.6-fold, p < 0.0001). Microglia still comprised the highest in vitro glucose uptake per cell fraction, but with a distinct reduction of microglia-to-astrocyte (Ratio 50.4 vs 5.7, p < 0.0015) and microglia-to-CD11b/ACSA2-negative (Ratio 10.6 vs 1.7, p < 0.0001) ratios. Sobriety, sole in vivo and in vitro experiments, validated the results. Compared to cell culture neurons (1:100) or astrocytes (1:10) cell culture microglia indicated only low ¹⁸F-FDG uptake.

Schlussfolgerungen Microglia show a marked reduction of glucose uptake relative to the other cells in vitro when compared to regular in vivo glucose uptake. This has to be taken into consideration when studying microglial energy metabolism extracorporal usingt in vitro assays.

Publication History

Article published online:
13 June 2025