Alzet 2006

Osmotic minipumps (ALZET^® 2006, DURECT Corporation, Cupertino, CA, USA; pumping rate 0.15 µL/h) were filled and connected to a hook-delivery device (HDD) consisting of a commercially available silicone catheter (ALZET^® rat jugular catheter, DURECT Corporation, USA; 0.94 mm OD; 0.51 mm ID) and a small hook-shaped stainless-steel tip (Nordson Optimum^® #7018433, Nordson Deutschland GmbH, Erkrath, Germany) with an outer diameter of 0.31 mm [43 (link)]. Pumps for control animals were filled with artificial perilymph (AP; 145 mM NaCl, 2.7 mM KCl, 2.0 mM MgSO₄, 1.2 mM CaCl₂, 5.0 mM HEPES, pH = 7.4 with 0.1% guinea pig serum albumin) [96 (link)]. The pumps of the treatment group were filled with IGF-1 (Recombinant Human IGF-1, #100-11, PeproTech^® Germany, Hamburg, Germany) in a concentration of 2 μg/mL diluted in AP. The pumping rate of the osmotic pump results in a release of 0.3 ng IGF-1 per hour. During the priming time of 60 h each pumps’ function was macroscopic controlled by fluid drain out of the catheters’ tip [43 (link)]. After the in vivo experiment, the pumping of the explanted pumps was re-checked the same way.

3-to-5-month male WT and P2rx7^−/− rats were anesthetised with 3% isoflurane inhalation in 100% O₂, and an osmotic minipump (Alzet 2006, Durect, Cupertino, CA) was implanted subcutaneously using aseptic techniques. ANGII was infused at a rate of 250 ng/kg/min for ~ 6 weeks. Naive age-matched rats were used as controls. Preliminary experiments revealed that ANGII infusion was poorly tolerated by F344 rats, as shown by signs of illness including body weight loss, loss of grooming, reluctance to move, and abnormal posture. Following advice of our veterinary team, blood pressure was not recorded to avoid excessive handling of the animals that may cause stress, and mashed food was provided throughout the entire experiment to facilitate feeding and avoid excessive body weight loss. At the end of ANGII infusion period, rats were culled by CO₂ asphyxiation. Bladder urine was collected for urine analyses, and kidney and heart were weighed and processed for histological analyses. Tibia length was measured using a digital calliper.