Continuous Acyl-Ghrelin Infusion in ARC

Mice infected with AAV-GFP or AAV-LEAP2 in the ARC were implanted with Alzet osmotic mini-pumps (model 1002, DURECT Corporation, Cupertino, CA) for 14-day continuous infusions. The mini-pumps were filled with sterilized 100 µl acyl-ghrelin or vehicle (saline), and the solution was delivered at a rate of 0.25 µl/h. For subcutaneous acyl-ghrelin treatment, pumps were implanted subcutaneously in the interscapular region to deliver continuous acyl-ghrelin infusion to achieve a cumulative daily dose of 4 mg/kg of body weight, as previously described (34 (link)). For intracerebroventricular acyl-ghrelin treatment, a cannula (0008851, Alzet brain infusion kit 3) was implanted into the lateral ventricle (anteroposterior, −0.6 mm; mediolateral, −1.3 mm; and dorsoventral, −2.3 mm), and a pump was implanted subcutaneously connected to the cannula via a 2-cm-long catheter. Acyl-ghrelin concentration in the pumps for intracerebroventricular infusion was 3 µg/day, as previously described (35 (link)). Food intake and body weight were measured daily for 13 days following pump implantation.