Zolazepam

On P17, the mice in each group were anesthetized with zolazepam (Zoletil, Virbac, Carros, France). PBS containing fluorescein-dextran (FD40S, Sigma, MO, USA).was subsequently circulated through the left ventricle. The retinas were dissected, flat mounted onto glass slides and viewed using fluorescence microscopy (BX51, Olympus, Tokyo, Japan). The non-perfusion area in the retina were measured in flat mounts labeled with fluorescein-dextran The neovascular tufts in the retina were measured in flat mounts labeled with tetramethylrhodamine isothiocyanate (TRITC)-conjugated isolectin B4 (Sigma, MO, USA). The retinas were incubated with 1 % bovine serum albumin and 5 % Triton X-100 in PBS for 3 hours at room temperature.The retinas were washed 3 times with PBS and incubated overnight at 4 °C with Bandeiraea simplicifolia isolectin B4 (Sigma-Aldrich, MO, USA) diluted 1:50 in PBS. The retinas were washed with 0.05 % Tween 20 in PBS, followed by incubation with streptavidin TRITC (1:500, Serotec, Oxford, UK) for 4 h at 37 °C. The neovascular tufts was viewed with an Olympus BX51 microscope (Olympus, Tokyo, Japan). Quantification of the non-perfusion area and neovascular tufts in the retina was quantified using ImageJ software (NIH, MD, USA).

Ten to 12-week-old male pigs weighing 20–30 kg were fasted for 12 h, but supplied with water ad libitum, before anesthesia. The pigs were premedicated with tiletamine–zolazepam (Zoletil forte veterinary use; Virbac, Carros, France) at a dose of 5 mg/kg (2.5 mg/kg of zolazepam and 2.5 mg/kg of tilazamine) and atropine (0.1 mg/kg) given intramuscularly before the anesthesia. Anesthesia and neuromuscular blockade were induced with pentobarbital sodium (10–18 mg/kg h⁻¹), fentanyl (10–26 mg/kg·h⁻¹), and cisatracurium (0.1 mg/kg·h⁻¹). A PiCCO catheter (PULSION Medical Systems, Munich, Germany) was inserted into the right femoral artery and was used for the measurement of cardiac parameters and pulmonary edema. A Swan–Ganz catheter (Edwards Lifesciences, Irvine, CA, USA) was wedged into a pulmonary arteriole through the central venous catheter for mean pulmonary arterial pressure and PCWP measurements [36 (link)].

Anesthetic preparation containing 50 mg/ml tiletamine and 50 mg/ml zolazepam was purchased from Virbac (France). Xylazine hydrochloride was bought from Bioveta (Czech Republic). Fluorescein sodium solution was from Novartis (Switzerland). Ultragrade Tris was purchased from Amresco (USA). Molecular biology grade phosphate buffer saline (PBS) was bought from Gibco. Hemoglobin, luminol, hydrogen peroxide solution, and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were from Sigma-Aldrich (USA). Other chemicals used in this study were from Sigma-Aldrich, Amresco, or Serva (Germany) and were at least of reagent grade. All buffers and other solutions were prepared using ultrapure water. The Schirmer test tear strips were from Contacare Ophthalmics & Diagnostics (India). SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium) was synthesized and provided by the Institute of Mitoengineering of Moscow State University (Moscow, Russia).

Cefazolin (Huarun, Shenzhen, China) was procured from Suzhou Municipal Hospital. Titetamme and Zolazepam (Zoletil 50; Virbac, Carros, France), idazoxan hydrochloride 60 mg in 2 mL; Baite), and Xylazine hydrochloride (0.2 g in 2 mL; Baite, Chang Sha, China)were purchased from Chow Fung Veterinary Hospital.

The Institute of Mitoengineering of Moscow State University (Moscow, Russia) provided SkQ1 (10-(60-plastoquinonyl)-decyltriphenylphosphonium). Nepafenac (Nevanac), Alcaine and Betadine Ophthalmic were from Alcone (New York, NY, USA). Tiletamine and zolazepam were from Virbac (Carros, France). Xylazine hydrochloride was from Nita-Farm (Saratov Oblast, Russia). Phosphate buffer saline (PBS) was from Thermo Fisher Scientific (Waltham, MA, USA). Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) was from Sigma–Aldrich (St. Louis, MO, USA). TNF-α assay kit was from Cloud-Clone Corp. (Katy, TX, USA). Reagents for histological examination were from Biovitrum (Moscow, Russia). Malondialdehyde and bicinchoninic acid assay kits were from Sigma-Aldrich (USA). Glutathione peroxidase and superoxide dismutase assay kits were from Randox (Crumlin, UK). Oasis® PRIME HLB cartridge (60 mg, 3cc, cat.no. 186008056) were obtained from Waters (Eschborn, Germany). Other reagents and supplies were from Sigma–Aldrich, Amresco (St. Louis, MO, USA), and Serva (Heidelberg, Germany). All of the buffers were prepared while using ultrapure deionized water.