Mitoq

All animal procedures were approved by the Animal Care and Use Committee of the National Institute on Aging Intramural Research Program. Seven-week-old C57BL/6N mice weighing 23–26 g were purchased from Army Medical University. The animals were randomly divided into different experimental groups.
The animals were anesthetized with halothane (70% N₂O and 30% O₂; 4% induction, 2% maintenance, China), immobilized on a stereotactic instrument (RWD Life Sciences Ltd. China), and injected with 25 μl of autologous blood into the right caudate nucleus. The following coordinates were used, as described previously, from bregma: 0.8 mm anteriorly, 2.5 mm laterally, and 3.0 mm deep [23 (link)]. The craniotomy was finished with bone wax, and sutures were applied to the scalp. During the entire experiment and recovery, the body temperature of the animals was maintained at 37 ± 0.5°C. Sham-operated mice were subjected to needle insertion only.
MitoQ was purchased from BioVision (B1309, USA, dissolved in a 1 : 1 ratio of ethanol to water and dissolved in 1 mL 0.9% sterile NaCl at a final concentration of 1 mg/mL) and administered intraperitoneally (i.p.) 1 hour and 24 hours after ICH (4 mg/kg). The ICH+vehicle group received an equal volume of solvent at the corresponding time point as the ICH+MitoQ group [20 (link), 24 (link)].

The antibodies used were as follows: rabbit antibody against human E-cadherin (24E10; Cell Signaling Technology, Danvers, MA, United States); rabbit antibody against human N-cadherin (GTX127345; GENETEX, Inc., Irvine, CA, United States); rabbit antibody against human vimentin (EPR3776; Abcam, Cambridge, United Kingdom); rabbit antibody against human β-actin (4967; Bioss Antibodies, Woburn, MA, United States); and HRP-conjugated secondary antibody against rabbit IgG (7074; Cell Signaling Technology). The inhibitors used were as follows: deferoxamine (DFO; D9533; Sigma-Aldrich, St. Louis, MO, United States), ferrostatin-1 (SML0583; Sigma-Aldrich), and MitoQ (B1309; Biovision, Milpitas, CA, United States).

The extension of semen samples was performed with an SL nanoparticle-based extender consisting of 3.6342 g Tris, 1.8252 g citric acid, 0.5044 g glucose, and 6 mL glycerol in 100 mL of distilled water containing 2% SL nanoparticles. The concentration of SL nanoparticles was selected based on the beneficial effects reported by our previous research [3 (link)]. In the treatments, five extenders were supplemented with MitoQ (BioVision, Catalogue No. B1309-5) at various concentrations: 0 nM (M0, as control), 50 nM (M50), 100 nM (M100), 150 nM (M150), and 200 nM (M200). The pH and osmolality of the extenders were 7.05 and 340 mOsm/kg, respectively.
Semen cryopreservation was processed as described previously, with some modifications [12 (link)]. Briefly, semen samples were diluted to a final concentration of 50 × 10⁶ spermatozoa/mL with the extenders (1:20 v/v) at room temperature. Samples were cooled at 5 °C for 15 min and then loaded into 0.25 mL plastic straws. After 20 min of exposure to liquid nitrogen vapors, the straws were placed for at least one month in liquid nitrogen. The frozen straws were thawed in a 37 °C water bath for 20 s; then, various sperm functions and oxidative stress parameters were assessed.