Rotenone

On Day 9, the differentiation medium was replaced by a medium containing the external factors. The wells were divided into five groups. The groups were as follows: control group, rotenone 1 nM (#45656, Sigma-Aldrich), progesterone 10 nM (#P8783. Sigma-Aldrich), rotenone 1 nM + progesterone 10 nM, rotenone 1 nM + progesterone 10 nM + AG205 5 nM (#A1487, Sigma-Aldrich) and AG205 5 nM only. All wells contained a final concentration of 1 µL/mL propidium iodide (PI) (#P4864, Sigma-Aldrich). rotenone and AG205 were diluted in DMSO until the desired concentration was reached. progesterone was diluted in EtOH. Note that the final concentration of DMSO and EtOH in the medium was never higher than 0.01% respective 1%, which has no influence on cell integrity [20 (link)]. All external factors were stored at −20 °C. The cell culture was then incubated for 24 h and fixed with 4% PFA in PBS for 15 min under dark conditions, as PI is light sensitive.

After confirmation of the safety of Y. aloifolia extract, rats were randomly divided into four groups as follows (n = 8 per group): group 1; NC group: received 2 mL/kg 0.9% saline by oral gavage followed 20 min later by 1 mL/kg 2.5% DMSO (Sigma-Aldrich, MO, USA), subcutaneous (s.c.) for four weeks; group 2: rotenone group received 2 mL/kg 0.9% saline by oral gavage followed 20 min later by rotenone (Sigma-Aldrich, St. Louis, MO, USA) suspended in 2.5% DMSO (1.5 mg/kg/day, s.c.) for four weeks; group 3 and 4: received active Yucca extract (50 and 100 mg/kg, respectively) dissolved in 0.9% saline by oral gavage, then 20 min later, they received rotenone (as the second group) for four weeks.
Twenty-four hours following the last dose, rats were tested for neurobehavioral and locomotor abnormalities in addition to depression. They were sacrificed after being anesthetized with ketamine hydrochloride (30 mg/kg IM) and xylazine hydrochloride (5 mg/kg IM). Then blood was collected from the abdominal aorta, and their brains were dissected. The isolated striata from one hemisphere were stored at -80°C until further processing for biochemical analysis. The second striata were fixed in either 10% neutral formalin or 2.5% glutaraldehyde for subsequent light and electron microscopic histopathological examination.

Neuronal cultures at 12 days in vitro (DIV12) were subjected to mitochondrial stress by adding different concentrations of the respiratory chain complex I inhibitor rotenone (Sigma–Aldrich), diluted in DMEM containing 1 g/L glucose (Gibco), supplemented with 2 mM glutamine, 1% penicillin/streptomycin, 2% B-27, 1% N2 supplements (Gibco). Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma–Aldrich, 100 μM, 4 h) was used as a positive control of mitochondrial respiration failure, while treatments with N-acetylcysteine (NAC, Sigma–Aldrich, 100 μM, 24 h) and hydrogen peroxide (H₂O₂, Sigma–Aldrich, 100 μM, 30 min-2 h) were used to modulated ROS amounts. To inhibit the protease activity, carbobenzoxy-Leu-Leu-leucinal (MG132, Sigma Aldrich, 10 µM), was added for 4 or 24 h, according to rotenone treatment duration.

After acclimatization for 10 days, animals were allocated into four groups, n = 5. We purchased rotenone (Sigma-Aldrich, MO, USA) and solubilized it in sunflower oil.

• The vehicle group (vehicle control) received nine subcutaneous [s.c.] injections of sunflower oil (10 mL/kg) every other day.

• The PD control group received 9 injections of rotenone [1 mg/kg, s.c.] every other day. This schedule of injecting rotenone has displayed minimal lethality when applied for induction of the PD model in mice.

• PD + crude M-Oil (M-oil) group mice received rotenone [1 mg/kg, s.c.] every other day and oral gavage of 0.2 mL of M-Oil per mouse.

• The PD + M-NE4 group received rotenone [1 mg/kg, s.c.] every other day and oral gavage of 0.2 mL of M-NE4 per mouse.

Oils were given daily at 11:00 a.m. from day 1 to day 17.

Rats were divided into four treatment groups: (1) control+ vehicle, (2) calpeptin, (3) Rotenone, and (4) Rotenone plus calpeptin. Rotenone (Sigma, St. Louis, MO, USA) was injected subcutaneously (s.c.) at a dose of 2 mg/kg body weight. Groups 3 and 4 received Rotenone s.c. daily for four days, and then every other day for 6 days. Group 4 also received calpeptin (Sigma, St. Louis, MO, USA) intraperitoneally (i.p.) daily at a dose of 25 µg/kg body weight. The calpeptin injections started one day after the 1st injection of Rotenone (nine injections total) and were injected 1 h after Rotenone injection.
Stock solution of Rotenone was prepared in dimethylsulfoxide (DMSO, Sigma). The working emulsified dilution was made in sunflower oil. The dose was prepared fresh every other day and stored in amber-colored glass vials. The calpeptin stock solution was prepared in DMSO, and working dilution was prepared in sterile saline. The working dilution was prepared fresh each time before use. The animal’s body weight was measured before starting the treatments and at the end of the treatments.