The hippocampal CA1 samples were immediately homogenized with 0.6 M perchloric acid. The homogenates were centrifuged at 5000 × g for 10 min at 4 °C and neutralized with 1 M KOH and then centrifuged at 5000 × g for 10 min at 4 °C. Filtered supernatants (0.22 μm filter, Millipore) were stored at −80 °C until processing. A 20 μl supernatant was injected to determine ATP levels by HPLC (Hypersil ODS2 column: 4.6 mm × 150 mm, 5 μm; Elite Analytical Instruments Co. Ltd, Dalian, China) using 254 nm wavelength; Mobile phase was 84 mM phosphate buffer (61 mM NaH2PO4 and 23 mM Na2HPO4 buffer solution, PH 6.5) and methanol (99.9: 0.1) and flow rate was 1.0 ml/min. Column temperature: 30 °C. ATP levels were determined by ATP standard curve (ATP standard, Roche, Mannheim, Germany) and presented as fold changes relative to the sham group.
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