ABSTRACT: Cerebral small vessel disease has been linked to cognitive, psychiatric and physical disabilities, especially in the elderly. However, the underlying pathophysiology remains incompletely understood, largely due to the limited accessibility of these small vessels in the live brain. Here, we report an intravital imaging and analysis platform for high-resolution, quantitative and comprehensive characterization of pathological alterations in the mouse cerebral microvasculature. By exploiting multi-parametric photoacoustic microscopy (PAM), microvascular structure, blood perfusion, oxygenation and flow were imaged in the awake brain. With the aid of vessel segmentation, these structural and functional parameters were extracted at the single-microvessel level, from which vascular density, tortuosity, wall shear stress, resistance and associated cerebral oxygen extraction fraction and metabolism were also quantified. With the use of vasodilatory stimulus, multifaceted cerebrovascular reactivity (CVR) was characterized in vivo. By extending the classic Evans blue assay to in vivo, permeability of the blood-brain barrier (BBB) was dynamically evaluated. The utility of this enabling technique was examined by studying cerebrovascular alterations in an established mouse model of high-fat diet-induced obesity. Our results revealed increased vascular density, reduced arterial flow, enhanced oxygen extraction, impaired BBB integrity, and increased multifaceted CVR in the obese brain. Interestingly, the 'counterintuitive' increase of CVR was supported by the elevated active endothelial nitric oxide synthase in the obese mouse. Providing comprehensive and quantitative insights into cerebral microvessels and their responses under pathological conditions, this technique opens a new door to mechanistic studies of the cerebral small vessel disease and its implications in neurodegeneration and stroke.
