Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks - PubMed (original) (raw)

Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks

Walid S Kamoun et al. Nat Methods. 2010 Aug.

Abstract

Not all tumor vessels are equal. Tumor-associated vasculature includes immature vessels, regressing vessels, transport vessels undergoing arteriogenesis and peritumor vessels influenced by tumor growth factors. Current techniques for analyzing tumor blood flow do not discriminate between vessel subtypes and only measure average changes from a population of dissimilar vessels. We developed methodologies for simultaneously quantifying blood flow (velocity, flux, hematocrit and shear rate) in extended networks at single-capillary resolution in vivo. Our approach relies on deconvolution of signals produced by labeled red blood cells as they move relative to the scanning laser of a confocal or multiphoton microscope and provides fully resolved three-dimensional flow profiles within vessel networks. Using this methodology, we show that blood velocity profiles are asymmetric near intussusceptive tissue structures in tumors in mice. Furthermore, we show that subpopulations of vessels, classified by functional parameters, exist in and around a tumor and in normal brain tissue.

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Conflict of interest statement

Authors’ Disclosures of Potential Conflicts of Interest

Rakesh K. Jain has an advisory role in the following companies: SynDevRx, AstraZeneca, Dyax, Millenium and Honoraria role in AstraZeneca

Figures

Figure 1

Figure 1. Description, validation and application of RTLS and RVFS

(a) Representative MPLSM angiogram of a tumor blood vessel. x vs t plots were generated by scanning along the centerline of the vessel with ALS and perpendicular to the vessel with RTLS. Analysis of flow velocity is based on the slope of the RBC signal (Δx/Δt) for ALS and on residence time (rt) for RTLS. RBC velocity and flux measured along the vessel cross-section using RTLS are compared to ALS-based analysis of flow (mean ± s.e.m.). (b) A single vessel scanned with a range of scanning velocities in two opposing scanning directions (scanning velocity - Vs). Scanning from top to bottom with velocities from 1.1 to 1.5 mm s−1 caused "velocity-matched" red blood cells with higher residence times (rt) and a measurable traveled distance (d), which can be used to measure velocity. (DiD, 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate) (c) 3D MPLSM angiogram of the brain of a tumor-bearing mouse illustrating the position of the line scans performed at several z planes. Comprehensive velocity and flux 3D maps were generated using RTLS (d) 3D Velocity map of a glioma network analyzed by RTLS and RVFS. Vessels in which RVFS velocity measurement differs from RTLS due to low RBC flux (closed arrowheads). Scale bars, 100 µm (a), 50 µm (b), 100 µm (c), 100 µm (d).

Figure 2

Figure 2. Analysis of cross-sectional flow profiles within tumor vessels undergoing intussusceptive angiogenesis

Cross-sectional velocity, flux, hematocrit shear rate profiles and raw data maps upstream and at the level of a single (a) or multiple (b) intussusceptions (yellow arrow heads Scale bars, 50 µm (a), 50 µm (b). Bigger images are scanned areas of the plane where the cross-sectional line scanning was done.

Figure 3

Figure 3. Multiparametric phenotipic vessel clustering to compare vessels within tumor, peritumor and contralateral brain regions in a glioma model

(a) All the vessels analyzed in U87-bearing animals (n = 6) are plotted. Scatter plots of velocity vs flux and diameter vs hematocrit showing the gating applied to analyze three different vessel sub-types (hypoperfused, transport, and hemodiluted).(b) Box plots for vessel diameter from clustered vessels. (* P < 0.05). (c). U87 MPLSM micrographs. Rhodamine BSA was used for the angiographic contrast and fluorescent RBCs (green) were injected 1 day prior to imaging. Glioma cells expressed GFP which allowed accurate localization of the tumor and determination of the tumor edge (light blue dashed line). Tumor vessels are heterogeneous with high (closed arrow head) and low (open arrow head) hematocrit vessels. Peritumor vessels are morphologically normal and have low (open arrow head) hematocrit when compared to contralateral brain. Scale bars, 100 µm (c).

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