Intensity distribution segmentation in Ultrafast Doppler and correlative Scanning Laser Confocal Microscopy for assessing vascular changes associated with ageing in murine hippocampi

The hippocampus plays an important role in learning and memory, requiring high-neuronal oxygenation. Understanding the relationship between blood flow and vascular structure – and how it changes with ageing – is physiologically and anatomically relevant. Ultrafast Doppler (µDoppler) and Scanning Laser Confocal Microscopy (SLCM) are powerful imaging modalities that can measure in-vivo Cerebral Blood Volume (CBV) and ex-vivo vascular structure, respectively. Here, we apply both imaging modalities to a cross-sectional and longitudinal study of hippocampi vasculature in wild-type mice brains. We introduce a segmentation of CBV distribution obtained from µDoppler and show that this mice-independent and mesoscopic measurement is correlated with the number of vessels and Vessel Volume Fraction (VVF) distributions obtained from SLCM – e.g., high CBV relates to fewer number of vessels but with large VVF. Moreover, we find significant changes in CBV distribution and vasculature due to ageing (5 vs. 21 month-old mice), highlighting the sensitivity of our approach. Overall, we are able to associate CBV with vascular structure – and track its longitudinal changes – at the artery-vein, venules, arteriole, and capillary levels. We believe that this correlative approach can be a powerful tool for studying other acute (e.g., brain injuries), progressive (e.g., neurodegeneration) or induced pathological changes.