Mass spectrometry

Mice with spinal cord compression were assessed (n = 4). SCI was induced using a disposable straight clip (AM-1; Natsume Seisakusho) for 15 s. At 20 min after SCI, mice were anaesthetized and sacrificed. Spinal cords with spinal bones were quickly removed and immediately immersed in liquid nitric oxide. Thin sections (8 μm thick) were cut with a cryomicrotome (CM3050; Leica Microsystems). To prepare thin slices of the entire spinal column, Kawamoto’s method, which preserves the morphology of the soft and hard tissues during sectioning, was used.12 The sections obtained on Kawamoto’s film were fixed on indium-tin-oxide-coated glass slides (Bruker Daltonics, Billerica, MA, USA) using double-sided conductive adhesive tape to facilitate electrical conduction.12 The sections were then spray-coated with 2,5-dihydroxybenzoic acid as a matrix (40 mg/ml, dissolved in 80% methanol) using an artistic airbrush (Procon Boy FWA Platinum 0.2-mm calibre airbrush; Mr Hobby, Tokyo, Japan). Imaging measurements were performed using an orbitrap mass spectrometer (QExactive Focus; Thermo Fisher Scientific, Waltham, MA, USA) coupled with an atmospheric pressure-scanning microprobe matrix-assisted laser desorption/ionization ion source (AP-SMALDI10; TransMIT GmbH, Giessen, Germany). The raster step size was set at 50 μm. Signals within a mass range of 80–900 were acquired with a mass resolving power of 70 000 at m/z 200. Thereafter, the spectral data were transformed to image data and analysed using the ImageQuest 1.0.1 (Thermo Fisher Scientific) and SCiLS 2019a (Bruker Daltonics) software programs.