Brain matrix

We collected interscapular brown adipose tissue (iBAT) and hypothalamus tissues between 9 and 12 am (3 to 6 h after lights on) from mice 3 weeks after HFD feeding and stored at -70°C until analysis. To collect the hypothalamus, the brain was placed ventral side up in brain matrix (World Precision Instruments, Sarasota, FL, USA), and a 2 mm-thick coronal slice was dissected out by an anterior coronal cut in the middle of the optic tract, just rostral to infundibulum, and a posterior coronal cut, at the posterior border of the mammillary bodies. The slice was then dissected laterally up to the hypothalamic sulcus and dorsally just above the third ventricle. For microdissection of the PVH, the brain was placed ventral side up in the brain matrix (World Precision Instruments, Sarasota, FL), while two 1 mm-thick sagittal slices (right and left side from the centerline of the brain) were dissected. The slices were observed under a stereoscopic microscope, and the VMH was microdissected based on the key visible anatomical structures [24 (link)]. All procedures were conducted rapidly on ice to prevent RNA and protein degradation.

Rats were euthanized via decapitation under isoflurane anesthesia after 24 h of reperfusion, and their brains were removed and cut into 2 mm coronal slices using a brain matrix (World Precision Instruments, Inc., Sarasota, FL, USA). The infarct size was evaluated using TTC staining. In brief, coronal brain sections were immersed in 2% TTC at 37 °C for 6 min and were fixed with 4% paraformaldehyde at 4 °C for 24 h. Infarct areas were identified via imaging analysis software (Image J 1.52v; National Institutes of Health, Bethesda, MD, USA). The following formula determined the volume of each coronal section: V = 2/3 (area of infarction on the rostral side + area of infarction on the caudal side + √ area of infarction on the rostral side × area of infarction on the caudal side). The total volume of infarction was obtained according to the following formula: volume of infarct area (%) = [left hemisphere volume − (right hemisphere volume − the infarct volume)] × 100/left hemisphere volume.