V1534 000

Male and female Ffar2/Ffar3^{-/-41 (link)}, Csf2^-/- (Jackson Laboratory), and Ccr2^-/- (Jackson Laboratory) mice on C57BL/6 background used for comparative analysis and experiments were housed & bred in the same facilities (animal husbandry, Charité) with identical specific pathogen-free conditions under 22 °C, 50 − 55% relative humidity, and 12 h/12 h light/dark cycle conditions as C57Bl/6 WT control animals, with free access to food (rat/mouse maintenance food V1534-000, Ssniff) and water. Sex was not considered in this study. Mice were chosen by fitting age ranging from 8 to 16 weeks.

Pregnant Wistar rats were delivered by Charles-River (Sulzfeld, Germany) on gestational day (GD) 13 for the purpose of MIA at GD15. Prior to iTBS and behavioral testing the dams and their offspring were housed within the central experimental animal facility of the medical faculty with free access to food pellets (V1534-000, Ssniff Spezialdiäten GmbH, Soest, Germany) and tap water and with a light-dark cycle of 12/12 h (light on at 6 am). On postnatal day 21, offspring were separated from their mothers with females and males housed separately in groups of four per cage (Macrolon type IV). To avoid additional variability due to varying hormone status of the females and for better comparability to other NIBS studies on male MIA offspring, we included only males in this study. One week before starting the experimental procedures the rats were moved to ventilated cabinets within the department and randomly allocated to the different experimental groups (see below) while still housed in the same groups of 4 animals per cage (Macrolon type IV). All experiments were performed in compliance with German laws and the directive of the European Community (2010/63/EU, Sept. 22th, 2010) for the use of animals in research and were approved by the local ethics committee (State Office for Nature, Environment and Consumer Protection, LANUV, Section 81-Animal Welfare, Az. 84-02.04.2014.A294).

All animal experiments were approved by the animal care and use committee of local government authorities (Regierung von Mittelfranken, Ansbach, Germany; Az 54–2532.1-11/13) and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Twenty-four male C57BL/6NCrl mice, aged between 12 and 15 weeks weighing 25 to 30 g, were obtained from Charles River (Sulzfeld, Germany) and acclimated for at least 7 days prior to study initiation. They were housed in groups of four animals per cage under a 12:12 hour light-dark cycle at constant temperature (22±1°C) and humidity (55²5%) with free access to food and tap water. Mice were randomly assigned into two groups, including the control (Ctrl) group (n = 12) fed with standard rodent chow (V1534-000, ssniff Spezialdiäten, Soest, Germany) and the CKD group (n = 12) receiving a diet supplemented with 0.2% (w/w) adenine. Body weight and clinical status of mice were monitored daily. After 3 weeks mice were sacrificed by exsanguination under deep isoflurane anesthesia, and their kidneys were harvested. Kidneys from 8 control and 8 adenine-fed mice were fixed with paraformaldehyde for 16 hours before imaging and (immuno-) histological studies. Kidneys from 4 control and 4 CKD mice were subjected to MRI directly after removal and 16 hours after paraformaldehyde fixation.

All experimental treatments of animals were approved by the German Animal Welfare Authorities (approval no. G0016/11). All mice were maintained under conventional conditions and a 12:12 h light:dark cycle (lights on at 6:00am) at a temperature of 22 ± 2°C. Animals had ad libitum access to food and water. To perform fine mapping of the obesity quantitative trait locus (QTL; Arends et al. 2016 (link)), generation 28 was fed with a rodent high-fat diet containing 19.5 MJ/kg of metabolizable energy, 45% from fat, 24% from protein, and 31% from carbohydrates (E15103-34, ssniff EF R/M; Ssniff Spezialdiäten GmbH, Soest, Germany). All other generations used in this study were fed a standard breeding diet (V1534-000, ssniff EF R/M; Ssniff Spezialdiäten GmbH, Soest, Germany).

All animal experiments were performed with permission of the Landesamt für Verbraucherschutz, Landwirtschaft und Flurneuordnung, Referat 23, of the State of Brandenburg (file number 32-44,456+1). Male Sprague Dawley rats were obtained from the Charles River Laboratory (Sulzfeld, Germany). The animals were housed individually. They were maintained at a day/night cycle of 12 h/12 h at 22 ± 2 °C. We noticed that old animals habituated to standard laboratory chow only consumed modest amounts of broccoli. However, consumption could be strongly enhanced, when weaning animals were accustomed to fresh food. Therefore, 3-week-old rats received green salad, cucumber and apple in addition to standard rat diet (V1534-000, ssniff, Soest, Germany) ad libitum for a few weeks before the start of the actual experiment.