Mouse brain slicer matrix

Plasma was prepared from peripheral blood collected in EDTA-treated tubes. Midbrain tissue containing SN and forebrain tissue containing striatum were dissected out in 1.0 mm thick coronal slices using a mouse brain slicer matrix (Zivic Instruments, Pittsburgh, PA, USA) according to the mouse brain atlas. The brain tissue slices were collected in cryovials, quickly frozen in liquid nitrogen and stored at −80°C until analysis. The frozen tissue samples were homogenized in tissue lysis buffer containing 137 mM NaCl, 20 mM Tris (pH 8.0), 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 1 μg/mL leupeptin, and 0.5 mM sodium vanadate at a tissue concentration of 30 mg/mL and centrifuged at 20,000 × g for 10 min at 4°C. The GDNF concentrations in plasma, SN, and striatum were measured using a commercially available GDNF E_max ImmunoAssay System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. The assay detects both human and rodent GDNF with pg/mL level sensitivity.

A 0.5 mm thick slice of coronal forebrain containing striatum was dissected out using a mouse brain slicer matrix (Zivic Instruments, Pittsburgh, PA, USA) and stored at −80°C until analysis. The concentrations of DA, DOPAC, and HVA were determined by high performance liquid chromatography (HPLC) combined with electrochemical detection. The frozen tissues were sonicated in 0.2 mol/L perchloric acid (20% wt/vol) containing 100 ng/mL 3,4-dihydroxybenzylamine (internal standard) and centrifuged at 15,000 × g for 7 minutes. The supernatant was filtered using a 0.2 μm Nylon-66 filter and a 25 μL aliquot of the filtrate, equivalent to 2.5 mg of tissue, was injected directly into the HPLC/electrochemical system. The mobile phase consisted of 0.07 mol/L potassium phosphate, pH 3.0, 8% methanol, and 1.02 mmol/L 1-heptane sulfonic acid. The concentrations of DA, DOPAC, and HVA were calculated using a curve generated with authentic standards.

Mice were sacrificed by cervical dislocation 24 h after the last behavioral test (n = 3 for each group). Hippocampus, medial prefrontal cortex (mPFC), and striatum were dissected using a brain matrix (Mouse Brain Slicer Matrix) and sectioned at 1.0-mm coronal slice intervals (Zivic Instruments Inc., Pittsburgh, PA, USA) on a cold plate and immediately frozen on dry ice. The three brain regions and the remaining brain sample were transferred to individual tubes, weighed, and stored at –80 °C until assay. For metabolite analyses, the tissue sample was submerged in 1.35 mL of a water-methanol-chloroform (2:1.5:1, v/v/v) solution and homogenized using a pestle with the addition of glass beads. The mixture was sonicated on ice and then centrifuged at 16,000×g for 10 min at 4 °C. The tubes were subsequently transferred to a –20°C freezer and kept there overnight to allow residual chloroform to separate from the aqueous methanol phase. The two liquid phases were transferred to separate 1.5 mL microcentrifuge tubes and lyophilized under vacuum (Thermo Scientific Savant SPD 1010 SpeedVac, USA). Only the methanol phase was used for the current analysis. The dry residue was reconstituted in 20μL cold methanol and diluted to a final volume of 200μL with the initial LC mobile phase (0.1% formic acid in water).

At 28 dpi, mice were sacrificed by ketamin/xylazin overdose, followed by transcardial perfusion with 20 ml PBS and extracted brains were cut in 3 * 2 mm thick brain slices (Mouse Brain Slicer Matrix, # BSMAS001-1, Zivic Instruments, USA). Slices were immediately collected into cold PBS and scanned. Atrophy was calculated as: Atrophy [%] = 100 – ((V_total28dpi/V_total3dpi) × 100, where V_total28dpi represents the total brain volume on day 28 (overlay scanner) and V_total3dpi represents the total brain volume on day 3 (MRI images). Loss of tissue mass is expressed as percentage compared to initial values at treatment start for each individual animal.