Intracerebroventricular infusion

Regarding the animals that received additional central infusion of the test agents, rats were placed in a sealed Plexiglas box into which 4% isoflurane and 2 L/min oxygen flow were introduced for anesthesia. Then, the rats were placed in a standard stereotaxic device equipped with a gas anesthesia nose cover to maintain anesthesia throughout the surgery with 2% isoflurane and 600 ml/min oxygen flow. A drug-filled osmotic pump was surgically implanted with an ALZET Brain Infusion probe (Alzet 1002) into the right lateral ventricle with the tip of the infusion probe at the following coordinates with reference to Bregma: anterior/posterior -1.4 mm; medial/lateral 1.8 mm; and dorsal/ventral -3.0 mm (Paxinos and Watson, 2013 [31]). Intracerebroventricular infusion (icv) of 3MA (5 mM, infusion rate: 0.5 µl/hour, ALZET^® MICRO-OSMOTIC PUMP MODEL 1007D) was carried out for 7 days. An infusion of sterile saline (Sal) served as the volume and vehicle control. Only animals that showed progressive weight gain after the operation were used in the subsequent experiments.