L-lactate induced proteomic changes in rat hippocampus

Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxic instrument (Kopf instrument). After exposing the skull, bilateral craniotomy (0.5 - 0.8 mm holes, 4 mm posterior to bregma and 2.6 mm lateral from midline) was done. In one group of rats (Lactate group), each hippocampus (3.5 mm ventral from the surface of the skull at the craniotomy site) was infused with 100 nmol of L-lactate dissolved in 1µl of ACSF at a flow rate of 0.1 µl/min (controlled by microinjection pump; World Precision Instruments, USA). In another group of rats (ACSF group), 1 µl of artificial cerebrospinal fluid (ACSF; NaCl 124 mM, KCl 3 mM, CaCl2 2.4 mM, MgSO4 1.3 mM, glucose 10 mM, and HEPES 10 mM, pH = 7.3) was infused per HPC. Needle was kept at injection site for additional 5 minutes for the proper dispersion of solution after L-lactate/ACSF infusion. Then rats were kept into their cages for approximately 60 minutes. After that higher dose of sodium pentobarbital was given, and decapitation was done quickly. Following decapitation, the dura mater was removed carefully, and the brain was flipped out from skull into ice cold PBS using spatula. The right and left hippocampus were isolated on an ice-cold plate using sterile forceps and were stored immediately at -80°C until further use. Later, the right hippocampus was used as sample for mass spectrometry (MS).