INTRODUCTION: The ketogenic diet, a high-fat, low-carbohydrate regimen, has gained attention for its neuroprotective potential in neurological disorders. β-Hydroxybutyrate (BHB), a ketone body, serves as an alternative energy substrate and modulates inflammation.

AIM(S): This study aimed to evaluate the impact of BHB on the cellular response to scratch injury in rat cortical and hippocampal primary cultures.

METHOD(S): The cortices and hippocampi were isolated from the brains of rat pups, and mixed neuronal-glial cultures were established. On days 14–21 in vitro (DIV), the medium was supplemented with 5 mM BHB. On DIV21, a scratch wound was introduced using a pipette tip to simulate brain injury. Cell migration was monitored using the CytoSMART™ Lux2 Live Cell Imager for 24 hours. The expression of GFAP and NeuN (astrocytic and neuronal markers) was assessed via Western blot in cell lysates at 6 and 24 hours post-injury. The expression of inflammatory markers was assessed using antibody arrays 6 hours post-injury. At 24 hours post-injury, cells were fixed and prepared for electron microscopy.

RESULTS: BHB treatment prior to injury reduced migration speed in cortical cultures and altered the inflammatory response—reducing the expression of IL-1β while increasing levels of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF). In hippocampal cultures, BHB had no effect on migration speed but increased the expression of neuropilin-1. Moreover, changes in GFAP expression were observed in BHB-supplemented hippocampal cultures—a decrease in GFAP levels 6 hours post-injury compared to non-injured controls, followed by an increase at 24 hours post-injury. Ultrastructural analysis revealed a large number of lipid droplets and active lipophagy in BHB-treated cells.

CONCLUSIONS: In summary, BHB alters the cellular response to injury and may have region-specific effects on brain cells.

FINANCIAL SUPPORT: Funding: National Science Centre Preludium21 Grant 2022/45/N/NZ4/03028