We used 406 male and female mice (7–8 weeks old). Mice were allowed to acclimate for seven days before beginning the experiments. They were housed in individually ventilated cages, up to 5 mice per cage on 12-h dark/light cycle and allowed free access to standard dry rodent diet and water, supplemented with 1g/l L- cysteine (AppliChem, Darmstadt, Germany) [11]. Analgesia was achieved with paracetamol suppositories, in order to avoid interactions with the immune system.

H2S synthesis is catalyzed by three different enzymatic systems, cystathionine γ- lyase (CSE), which is the key enzyme for H2S synthesis in the liver and kidney, cystathionine β- synthase (CBS), which is overexpressed in brain tissue and 3-mercaptopyruvate sulfurtransferase (3MST) which is ubiquitously expressed [12]. We investigated the role only of CSE and 3MST, due to the fact that homozygotic CBS deficiency is related to a neonatal mortality of over 90%. [13,14]. Cse -/- mice were created after breeding of Cse +/- mice (Friedrich Shiller University Hospital, Jena, Germany). The Cse+/- mice were generated as previously described [13]. In short chimeric male Cse +/- mice were produced after injection of Cth+/- stem cells into C57BL/6 blastocysts. These were then crossed with C57BL/6 female mice to obtain Cse +/- pups. Cse +/- pups were backcrossed multiple times to achieve high genetic homogenicity on C57BL/6 background. The Cse +/- males and females produced were finally bred to obtain wild- type (Cse +/+), Cse +/-, and Cse -/- littermates. 3Mst+/+ and 3Mst-/- mice were created, as described previously [15].

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