Scr sirna

Intracerebroventricular (i.c.v) drug administration was performed as previously described (He et al., 2015 (link)). Rats were placed in a stereotaxic apparatus under anesthesia with 2.5% isoflurane in in 70/30 % medical air/oxygen. The needle of a 10 μL Hamilton syringe (Microliter701; Hamilton Company, Reno, NV, USA) was inserted into the right lateral ventricle through a burr hole using the following coordinates relative to bregma: 1.5 mm posterior, 0.9 mm lateral, and 3.3 mm below the horizontal plane of the bregma. Drugs and siRNA were infused directly into the lateral ventricles at a rate of 1 μL/min by a pump. The needle was retracted 5 minutes after the injection when the burr hole was plugged with bone wax immediately. LY294002 (Selleck Chemicals, Houston, USA) was prepared at 50 mmol/L in PBS (contains 25% DMSO), and 5 μl of the DMSO and LY294002 were infused 30 minutes before SAH induction in the SAH+Ex-4+DMSO and SAH+Ex-4+LY294002 rats, respectively. GLP-1R siRNA and scrambled RNA (OriGene Technologies, Rockville, MD, USA) were prepared at 500 pmol in RNAse free suspension buffer and were infused (5 μl of the siRNAs) 48 hours before SAH modeling in the SAH+Ex-4+GLP-1R siRNA and SAH+Ex-4+Scr siRNA rats, respectively.

For knockdown experiments, differentiated 3T3-L1 adipocytes were transfected with 50 nM synthetic predesigned siRNA targeting Nat1 or non-silencing siRNA (scr siRNA) (Origene) using lipofectamine 2000 transfection reagent (Life Technologies) following the manufacturer’s recommended protocol. After 48 h of transfection, the media was changed.