Metabolomics analysis of dorsal root ganglion in rats of painful diabetic neuropathy
Description
DRG samples (n = 5 per group) were analyzed using mass spectrometry in both positive and negative ion modes to identify metabolic alterations. Thirty milligrams of DRG tissue were placed in 2-ml centrifuge tubes for metabolite extraction. A total of 900 μl of extraction solution (methanol: water = 4 : 1) was added to each sample. The DRG tissues were pulverized using a cold tissue grinder for 6 minutes (−10°C, 50 Hz) to ensure thorough homogenization. Following this, the samples underwent low-temperature ultrasonic treatment for 30 minutes (5°C, 40 kHz) to enhance metabolite extraction. The mixtures were left to stand at −20°C for 30 minutes and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The resulting supernatants were transferred into sample vials with insert tubes for further analysis. Metabolomic profiling was conducted using a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), as described in previous methodologies . Initial data-dependent acquisition was performed with a single full-scan at a resolution of 60,000, targeting hydrophilic metabolites with mass-to-charge ratios ranging from 60 to 900. Lipid profiling was subsequently performed within an extended mass-to-charge ratio range of 300 to 1,200. For further structural elucidation, 10 consecutive tandem mass spectrometry scans were conducted in high-energy collision dissociation mode. Stringent quality control measures were implemented throughout the metabolomic study to ensure the accuracy and reliability of the data generated.