4.1. Animals and MPTP Treatment

Male C57BL/6 mice (7–8 weeks old) were maintained under controlled temperature (22 ± 2 °C) and humidity (35 ± 5%) on a 12-h light/12-h dark cycle (lights on at 07:00) and allowed ad libitum access to pellet food (Crea Japan, Tokyo, Japan) and water. All procedures were performed in accordance with the Guiding Principles for the Use of Animals in Toxicology in 1989 and approved by the Animal Research Committee at Obihiro University of Agriculture and Veterinary Medicine (approval number: 19-2, Date: 01 April 2019). The animals were humanely euthanized with an overdose of the anesthetic ether at the end of the experiments.

MPTP (Sigma-Aldrich, Tokyo, Japan)-treated PD model mice were prepared as described in our previous report [8]. Eight-week-old mice were given four intraperitoneal injections of a single dose of 20 mg/kg (in 100 µL) in the first two injections and 15 mg/kg (in 100 µL) in the last two injections every 2 h. Saline was administered in a similar manner as a control. Subsequent experiments were conducted 7 days after the last MPTP or saline injection (Figure 8). This MPTP injection protocol can retain a significant reduction in the number of TH-positive cells in the SNpc at least until 16 days after MPTP injection.

A schematic figure explaining the planning of the experiments.