*4.7. MPTP Mouse Model*

Animal studies were performed according to the animal welfare of the Faculty of Pharmacy, University of Lisbon, and approved by the competent national authority Direção-Geral de Alimentação e Veterinária (DAGV) and in accordance with the EU Directive (2010/63/UE), Portuguese laws (DR 113/2013, 2880/2015 and 260/2016) and all relevant legislation. To evaluate the neuroprotective effect of our hit compound—Oxa12—in the sub-acute MPTP mouse model, male 13-week-old C57BL/6N wild-type (wt) mice (Charles River Laboratories, Wilmington, MA, USA) were injected intraperitoneally (i.p.) with a unique dose of MPTP-HCl (40 mg/kg; Sigma Aldrich, St Louis, MO, USA), dissolved in sterile 0.9% saline, or vehicle only (control group). One hour after MPTP administration, mice were intraperitoneally injected with 10 mg/kg Oxa12 solubilized in 1% DMSO (Sigma-Aldrich) and 30% 2-hydroxypropyl-beta-cyclodextrin (Sigma-Aldrich) or 10 mg/kg Nec-1s (Focus Biomolecules, Plymouth Meeting, PA, USA) solubilized in 1% DMSO, 4% 2 hydroxypropyl-beta-cyclodextrin (Sigma-Aldrich), in PBS [38,41,42]. Oxa12 and Nec-1s injections were administered once every day for 30 days. Oxa12 dosage and regimen of administration were selected based on published protocols for Nec-1s [9,33]. Seven animal per group were used. After 30 days, mice were sacrificed in a CO2 chamber followed by transcardiac perfusion with ice-cold PBS. Brains were then excised, and one hemisphere was used to isolate the midbrain region, containing the SN, and the striatum, as previously described [38,41], which was rapidly frozen in liquid nitrogen and stored at −80 ◦C until processing for protein extraction. The other hemisphere was fixed in 4% paraformaldehyde for 48 h and then stored in 20% sucrose/PBS and 0.025% sodium azide, at 4 ◦C, for further immunohistochemistry analyses.
