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

Abstract

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.

In Vivo Ultrafast Doppler Imaging Combined with Confocal Microscopy and Behavioral Approaches to Gain Insight into the Central Expression of Peripheral Neuropathy in Trembler-J Mice

“…This is a particularly critical context in the SNP where PMP22 shows its maximum expression. However, the biological consequences of the micromutation generate a complex phenotype which, as we have shown, also manifests in the CNS [47,48]. In addition, its presence in other cell types, and at the nuclear level, augurs other unelucidated roles with more systemic characteristics in the expression of the TrJ phenotype.…”

“…The cut-off values used in the SVD clutter filter were selected based on achieving on the best signal-to-noise ratio. Further information regarding this experimental procedure can be found in [48].…”

“…achieving on the best signal-to-noise ratio. Further information regarding this experimental procedure can be found in [48].…”

“…Vibratome sections of 60 µm thickness were obtained in a Leica, VT 10000S vibratome. For vessel visualization, brain sections containing the hippocampal head regions were stained as previously described using Isolectin GS-IB4 Alexa Fluor 488 conjugate (Cat#: I21411, ThermoFisher Scientific, Waltham, MA, USA) in 1:100 concentration (Figure 1c) [48].…”

“…This technique allows us to observe the cerebral blood volume (CBV) with high sensitivity. Precisely, using this technique in association with Scanning Laser Confocal Microscopy (SLCM), we developed a method for quantifying the blood flow and its corresponding vascular structure, differentiating the CBV in quartiles according to the different vessel sizes, and finding that the CBV and the vascular structure vary with age [48].…”