Plasma edta and serum tubes

At the time of scarification, blood was collected by heart puncture into plasma-EDTA and serum tubes (Sarstedt, Nümbrecht, Germany) under deep isoflurane anaesthesia (Forane, Abbott, Austria). After centrifugation at 2000× g for 12 min at room temperature, plasma and serum samples were aliquoted and stored at −80°C until batched analysis. Immediately after blood collection, the following organs were explanted and snap frozen in liquid nitrogen: liver, skeletal muscle, heart, aorta, large intestine, spleen, kidney, brain, lung, visceral fat. Subsequently, all tissue samples were stored together deep-frozen at −80°C until analysis. Exclusion criteria were the development of illnesses or tumours during the intervention period.

Forty-eight female Sprague Dawley (SD) rats were purchased from Janvier Labs (Le Genest-Saint-Isle, France) at four months of age. All animals were fed a standard chow-based diet and kept on a 12 h/12 h light/dark cycle at the core facility animal housing at the Medical University of Graz (Austria). Temperature was maintained between 22 and 25°C. Humidity ranged between 55 to 58%. After one week of acclimatization, half of the animals were sacrificed at young age (n = 24). The other half was euthanatized after ten months. At the time of scarification, blood was collected by heart puncture into plasma-EDTA and serum tubes (Sarstedt, Nümbrecht, Germany) under deep isoflurane anaesthesia (Forane, Abbott, Austria). After centrifugation at 2000 g for 12 min at room temperature, plasma and serum samples were aliquoted and stored at −80°C until batched analysis. Immediately after blood collection, the following organs were explanted and snap frozen in liquid nitrogen: liver, skeletal muscle, heart, aorta, large intestine, spleen, kidney, brain, lung, visceral fat. Subsequently, all tissue samples were stored together deep-frozen at −80°C until analysis. Exclusion criteria were the development of illnesses or tumours during the intervention period. Two animals of the aged group were excluded from the study.