Bsmas001 1

Mice were intravenously injected with a lethal dose of pentobarbital (100 mg per kg body weight) before transcardial perfusion with 15 ml of ice-cold 1× PBS followed by 15 ml of ice-cold artificial cerebrospinal fluid (87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 75 mM sucrose, 20 mM glucose, 1 mM CaCl₂, 7 mM MgSO₄). The brain was removed, placed into a mouse brain matrix slicer (Zivic Instruments, BSMAS001-1), 1 mm slices were immediately snap-frozen and the region of interest was manually dissected into a frozen Eppendorf tube. Tissue samples were kept at −80 °C.

Brains for lesion size determination (veh = 15, sitagliptin = 11) were sectioned (1 mm) in a coronal matrix (BSMAS001-1; Zivic Instruments, Pittsburgh, PA, USA), and incubated in 1% 2,3,5-triphenyltetrazolium (TTC, in saline; Sigma-Aldrich, Brøndby, Denmark) for 30 min at 37°C. TTC is a functional mitochondrial stain. Planimetry was performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) blinded to treatment groups comparing ipsilateral and contralateral hemispheres. Sections where lesions extended into adjacent sections but did not pass entirely through the 1 mm were excluded due to imprecision of the depth dimension.

Collected brains were stored frozen in –80 °C. For protein isolation brains were taken out from storage and placed in the chilled mouse brain matrix (Zivic Instruments, #BSMAS001-1) and cut into 1 mm coronal sections. Striatum was isolated from two forebrain sections from both hemispheres under the stereotactic microscope, placed in the dissection buffer (Sucrose 300 mM, Imidazole 25 mM, EDTA 1 mM, pH 7.2 with the addition of proteinase inhibitors 0.4 μg/ml leupeptin, 0.1 mg/ml pefabloc) and homogenized with for 15 s. Resulted homogenate was centrifuged 4000 g/15 min at 4 °C and the amount of proteins was assessed with BCA kit (Thermo Fisher Scientific, #23225) in supernatant containing cytosolic fraction.

The day after the end of each experiment, the mice were sacrificed under CO₂ anesthesia. Plasma was collected from abdominal blood by centrifugation at 3000×g for 15 min. After removing brain tissue immediately, each brain region was isolated using a coronal mouse brain matrix (1 mm, BSMAS001-1; Zivic Instruments, PA, USA) and biopsy punch (1 mm, BP-10 F, Kai Medical, Japan). For biochemical analysis, the brain tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer (R0278, Sigma, MO, USA) supplemented with protease and phosphatase inhibitor cocktails (#1861284, Thermo Scientific, MA, USA). For subcellular protein analysis, the cytoplasm, membrane and nucleus were extracted using a subcellular protein fractionation kit (#87790, Thermo Scientific, MA, USA). The total protein concentration was measured using a bicinchoninic acid protein assay kit (BCA1 and B9643, Sigma). For gene expression analysis, parts of the brain tissue were stored in RNAlater (Ambion, TX, USA). For immunohistological analysis, other mice were transcardially perfused with 0.05% heparin (10 units/mL in PBS) followed by 4% paraformaldehyde (PFA, pH 6.9), and then the brains were removed and placed in 4% PFA fixation solution.

The mice were sacrificed under CO₂ anesthesia. The serum was collected by centrifugation at 3000 × g for 15 min. The brain was immediately removed and specific regions were isolated using a coronal mouse brain matrix (1 mm, BSMAS001-1; Zivic Instruments, Pittsburgh, PA, USA) and biopsy punch (1 mm, BP-10F, Kai Medical, Seki, Japan). The tissues of each region were homogenized in a radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. For the immunohistological analysis, the mice were subjected to transcardial perfusion with heparin (10 units/mL) and 4% paraformaldehyde (PFA) solution, and their brains were fixed with 4% PFA. The total protein concentrations were measured using a bicinchoninic acid protein assay kit (Sigma). The absorbance at 560 nm was measured using a UV spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, USA).