Dysregulated Hippocampal Fatty Acid Metabolism Following Intermittent Hypoxemia-induced Neonatal Brain Injury is Rescued by Treatment with Acetate
Description
Targeted lipidomics of frozen mouse microdissected hippocampal tissue. Using an established model of neonatal brain injury due to intermittent hypoxemia, we examined the extent of changes in the brain’s lipid profile. Mice underwent intermittent hypoxemia (IHx) during postnatal days (P) 1-11 and were compared to normoxia controls (Nx). We measured hippocampal lipids at several time points (P11, P17, P22, and P30). Our results show changes in hippocampal lipid composition.
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Frozen mouse microdissected hippocampal tissue or human hippocampal tissue were homogenized with PBS buffer at a concentration 100 mg/ml. The total lipid extracts of the brain tissue were prepared using a revised MTBE lipid extraction method68 . Briefly, 10ul of brain homogenate, 400ul of ice-cold methanol, and 10ul of internal standard (EquiSPLASH, Avanti Polar Lipids, Alabaster, AL) were combined, vortex mixed for 30s, and then incubated at 4°C for 10 min with 650 rpm shaking. Next, 500ul of ice-cold MTBE was added, vortex mixed for 30s and incubated at 4°C for 1 h with 650 rpm shaking. After incubation, 500ul of ice-cold H2O was added, vortex mixed for 30 s and incubated at 4°C for 15 min with 650 rpm shaking. The mixture was then centrifuged at 8,000 g for 8 min at 4°C, and the top organic layer was collected and stored on ice. An additional 200ul of MTBE was added to the aqueous phase, vortex mixed for 30s, incubated at 4 ℃ for 15 min, and centrifuged at 8,000 g for 8 min at 4°C. The organic layer was collected and combined with the previous extract. The collected organic layer was dried under N2 gas and re-suspended in 200ul of CHCl3/MeOH (1:1, v/v) containing 200 uM of butylated hydroxytoluene (BHT) and stored at −20°C until analysis. Samples were further diluted with IPA/ACN/H2O (2:1:1 v/v) prior to analysis. The total protein content was measured with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) according to the manufacturer’s protocol, and used for the data normalization. The liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis was conducted on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph (UHPLC) coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). Chromatographic separation was achieved with an ACQUITY Amide BEH column (1.7 μm, 2.1 × 100 mm) maintained at 45°C (Waters, Milford, MO). Mobile phase compositions for solvents A and B consisted of ACN/H2O (95:5, v/v) and (50:50, v/v) respectively, with 10 mM ammonium acetate. The gradient profile had a flow rate of 0.6ml min−1 and ramped from 0.1 to 20% B in 2 min, from 20 to 80% B in 3 min, dropped from 80 to 0.1% B in 0.1 min, and held 0.1% B for 2.9 min. Total chromatographic run time was 8.0 min. The injection volume was 2ul. All analytes were detected using negative or positive ionization mode and SRM library were adopted from Medina et al69. Collision energies and RF lens voltage were optimized for each reference standard.
Institutions
- University of Maryland Baltimore