*2.3. Animals*

All of the procedures used in this study were approved by the State Veterinary and Food Administration of the Slovak Republic in accordance with the European Union Directive 2010/63/EU.

Rats were divided into two groups: control (Cont.) and the group treated with PEG-coated ultra-small superparamagnetic iron oxide (Fe3O4) nanoparticles (USPIONs). Control rats were given 10-min infusions of saline, starting approximately 30 min from the beginning of the experiment. USPION-treated rats were given 10-min infusions of USPIONs at the dose of 1 mg Fe/kg.

Wistar-Kyoto (WKY) male rats, 12–16 weeks old, were used in this study. Rats were housed under standard conditions at 22–24 ◦C in a 12-h light/dark cycle and fed with pelleted diet Altromin formula 1324, variant P (Altromin Spezialfutter, Lage, Germany) and tap water ad libitum.

One day before the experiment, all of the rats had two catheters implanted under 2.5–3.5% isoflurane anaesthesia, as described previously [26]. All of the rats were also pre-treated with meloxicam (Meloxidolor, Le Vet Beheer B.V., Oudewated, Nederland) at 2 mg/kg intramuscularly before surgery to prevent post-surgical pain. Fine-bore polyethylene catheters (Smiths Medical International Ltd., Kent, UK) were inserted into the left carotid artery (internal diameter 0.28 mm) for i.v. administration of USPIONs (suspended in saline) or saline (in control), respectively. Catheters were exteriorised in the interscapular region, and rats were allowed to recover from anaesthesia for approximately 20–24 h. During the experiments, the conscious rats were placed into a plastic box with dark walls and transparent lid (27 cm × 14 cm × 9 cm in size), which allowed the rats free movement. At the end of the experiment, rats were exposed to brief CO2 anaesthesia and decapitated 100 min post USPION-infusion. The samples of the liver, left heart ventricle, kidney, aorta and blood were collected for the determination of the magnetic characteristics, histochemical determination of the iron and superoxide production. Tissues were dissected using ceramic scissors and ceramic or plastic forceps. After dissection, the tissues were cleaned out of the connecting tissue, washed in the saline solution and dried of saline solution using filtration paper. Trunk blood was collected into Eppendorf test tubes. Fresh tissues were collected for determination of superoxide and for histochemical analyses. For determination of USPIONs by SQUID, the tissues and blood were frozen in the liquid nitrogen and kept at –80 ◦C until further analyses.
