2.2 Animal experiment
The animals were randomly allocated into three distinct groups using a random number table: the light group (L), the dark group (D), and the acute sleep deprivation group (SD). In the SD group, the animals were subjected to 12 hours of sleep deprivation (8:00 a.m. to 8:00 p.m.) with a special sleep-deprived equipment (XR-XS108, Shanghai Xinruan Information Technology Co., Ltd.). The other operations on the animals (such as animal behaviors, metabolic kinetic study) in the L, D and SD groups were performed between 8:00 a.m. to 12:00 p.m., 8:00 p.m. to 0:00 a.m., and 8:00 p.m. to 0:00 a.m., respectively. Furthermore, after the completion of behavioral tests, all animals were allowed a week recovery in their original habitat to alleviate any potential influence of behavioral factors or other operations on the NMR experiments (Fig. 1).
2.4 1H-NMR detection
Sample preparation
Before the infusion of sodium [3-13C] lactate (99% 13C enrichment, Qingdao Tenglong Weibo Technology co., LTD, Qingdao, P.R. China), animals were fasted 16 h but had free access to tap water. On the experimental day, 0.5 mol/L solution of [3-13C] lactate was made with double-distilled water, and the pH was adjusted to 7.3. Two drops of blood were collected from a needle prick to the tip of the tail to test the blood glucose level using glucose test strips (Yuyue, China) before the tail vein catheterization. Then, a piece of PE-10 tubing (Instech PA USA) was inserted into the lateral tail vein with a 30-gauge needle for the infusion of 13C labeled lactate and was secured with the adhesive tape. After that, the other tip of PE-10 tube was connected to a swivel (Instech, PA, USA) and futher connected to an infusion pump (Fusion 100, Chemyx, TX, USA). Finally, a dose of [3-13C] lactate [18] was infused into the lateral tail vein in two minutes for each mouse.
The animals (8/group) were placed back to their home cages after injection. After a 30-min interval, the blood glucose levels were detected from the lateral tail vein, and the animals were deeply anesthetized with isoflurane immediately. Then all mice were euthanized with microwave oven (high fire, 14 seconds, Galanz, China). The brain was divided into nine different regions as described in previous studies [17, 19]: frontal cortex (FC), occipital cortex (OC), parietal cortex (PC), temporal cortex (TC), striatum (STR), hippocampus (HP), thalamus and hypothalamus (THA), midbrain (MID), and cerebellum (CE). Furthermore, a piece of liver was also retained for further detection. The tissues were weighed and immediately frozen at -80 ºC for further processing.
Tissue extraction
The tissue samples were treated with methanol-ethanol extraction method and the detailed steps are based on the previous protocol [20, 21]. Briefly, 200 µL HCl/methanol solution (0.1 M) was added to each sample and homogenized for 90 seconds at a frequency of 20 Hz with a Tissuelyser (QIAGEN company, Germany). Then 800µL ethanol (60%, vol/vol) was further added and homogenized again. The homogenate was centrifuged at 14,000 g for 10 minutes, and the supernatant was collected. The above process was repeated twice with 1200 µL of 60% ethanol for adequate extraction. Then, the supernatant was collected and lyophilized (Thermo Scientifc, Germany) after removal of organic solvents (methanol and ethanol) in a vacuum environment at 45 ºC. The dry product was dissolved with a D2O bufer solution (600 µL of D2O with 0.2 M Na2HPO4/NaH2PO4, pH = 7.2) for [1H-13C]-NMR analysis, and the chemical TMSP (3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt, 5 mM) was selected as the internal standard chemical in the buffer. A high-speed vortex was used to mix the solution fully. The mixtures were then centrifuged at 14,000 g for 15 minutes and the supernatant (about 500 µL) was collected to an NMR tube for further analysis.
NMR spectrum acquisition
The NMR spectra for all samples were collected randomly at 298 K using a BrukerAvance III 500 MHz NMR spectrometer from BrukerBiospin in Germany. The data were generated using the POCE ([1H-13C]-NMR) pulse sequence, a widely adopted method for detecting 13C enrichment in cerebral extractions [22]. This approach involves two spin-echo detections: one without a broad reverse pulse applied at the 13C frequency (the total metabolite concentrations, 12C+13C), and another with a reverse pulse (the difference of metabolites, 12C-13C). The 13C-labeled metabolites of the spectrum were obtained by subtracting the results of these two measurements. The entire process was carried out with the following parameters: an echo time of 8 ms, a sweep width of 20 ppm, a repetition time of 20 s, 64 scans, and an acquisition data size of 64K.
2.6 Statistical analysis
Data analysis was performed with the GraphPad Prism 8.0 (GraphPad, New York, USA), homemade NMRSpec in MATLAB (R2017b, Mathworks, Inc. 2017) and SPSS 21.0 (IBM, New York, USA). In order to determine the normality of data, the KolmogorovSmirnov test was used. We found that the data satisfied the assumption of normal distribution. Student’s t-test was used to compare the differences between the 2 groups. P < 0.05 was considered statistically significant. All results are expressed as the mean ± SEM.