Ischemia-Reperfusion

Animal studies were carried out in line with the principles of the Declaration of Helsinki and according to the guideline for Animal Care and Health of the State Veterinary and Food Department of the Slovak Republic, as well as ARRIVE guidelines [43]. Experiments were approved by the ethics committee of Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava (approval number EK 48/2019) and by the State Veterinary and Food Department of the Slovak Republic (approval number Ro-1360/2020 − 220). Experiments were implemented in accordance with Directive 2010/63/EU of the European Parliament and the Council for the protection of animals used for scientific purposes.

A total of 50 adult male Wistar rats (Velaz, Prague, Czech Republic) were used. All animals were maintained on a 12/12-hour light/dark cycle. Food and water were available ad libitum until the beginning of the experiments. Animal health and behaviour were monitored regularly by a doctor of veterinary medicine. Transient global cerebral ischemia was produced using the four-vessel occlusion model described previously [44]. Briefly, on day 1, both vertebral arteries were irreversibly occluded by coagulation through the alar foramina after anaesthesia with a mixture of 2% halothane, 30% O₂, and 68% N₂O. On day 2, both common carotid arteries were occluded for 15 min by small clips under anaesthesia with a mixture of 2% halothane, 30% O₂, and 68% N₂O. Two minutes before carotid occlusion, the halothane was removed from the mixture. Body temperature was maintained by means of a homeothermic blanket. Global ischemia was followed by 24 or 72 h of reperfusion induced by release of carotid occlusion. Control animals underwent the same procedure except for carotid occlusion. Immediately after the treatment, control and experimental animals were anaesthetised, perfused transcardially with ice-cold 0.1 mol/l phosphate-buffered saline (PBS, pH 7.4) and fixed by perfusion with ice-cold 4% paraformaldehyde in PBS. The brains were removed, postfixed with the same solution as above for 24 h at 4 °C, and cryoprotected by infiltration using 30% sucrose for the next 24 h at 4 °C.

The rats were randomised into the following experimental groups: (i) Control sham‑operated rats; (ii) rats that underwent a 15‑min global brain ischemia followed by 24 h of reperfusion (I24R; and iii) rats that underwent a 15‑min global brain ischemia followed by 72 h of reperfusion (I72R). Ischemic animals were included in the study if they underwent successful occlusion of both common carotid arteries defined by bilateral dilation of pupils and lack of pupillary light reflex.