Figure 2. TRF delays vascular density loss across cortical and hippocampal regions during aging.

A-D) Representative images of vascular density in the brain measured by ICONEUS functional ultrasound, which was binarized and overlayed with the Allen brain atlas to identify specific neuroanatomical regions. E-H) Vascular coverage in the whole brain, cortex, hippocampus, and white matter before starting TRF regimen (18 months of age) and at the experimental endpoint (24months of age) after 6 months of ad libitum diet for controls or TRF regimen for aged TRF mice. I) Representative confocal microscopic images of cortical and hippocampal regions in embedded brain sections stained with DAPI (blue) and CD31/Endomucin (red). J) Quantification of vascular coverage in cortex and hippocampus from immunohistochemical staining. All data are shown as mean ± SEM. Statistical significance was calculated using unpaired t tests (E-H) and two-way ANOVA with Tukey’s post hoc test to determine differences among groups (J). *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.