Near-wall micro-PIV reveals a hydrodynamically relevant endothelial surface layer in venules in vivo

Abstract

High-resolution near-wall fluorescent microparticle image velocimetry (micro-PIV) was used in mouse cremaster muscle venules in vivo to measure velocity profiles in the red cell-depleted plasma layer near the endothelial lining. micro-PIV data of the instantaneous translational speeds and radial positions of fluorescently labeled microspheres (0.47 microm) in an optical section through the midsagittal plane of each vessel were used to determine fluid particle translational speeds. Regression of a linear velocity distribution based on near-wall fluid-particle speeds consistently revealed a negative intercept when extrapolated to the vessel wall. Based on a detailed three-dimensional analysis of the local fluid dynamics, we estimate a mean effective thickness of approximately 0.33 micro m for an impermeable endothelial surface layer or approximately 0.44 micro m assuming the lowest hydraulic resistivity of the layer that is consistent with the observed particle motions. The extent of plasma flow retardation through the layer required to be consistent with our micro-PIV data results in near complete attenuation of fluid shear stress on the endothelial-cell surface. These findings confirm the presence of a hydrodynamically effective endothelial surface layer, and emphasize the need to revise previous concepts of leukocyte adhesion, stress transmission to vascular endothelium, permeability, and mechanotransduction mechanisms.

FIGURE 1

Brightfield diameter versus fluorescent diameter in capillaries and venules of Tie2 GFP transgenic mice (39 vessels, 3–34 μm diameter), which specifically express GFP in vascular endothelial cells (A). Line of unity shown (dashed line). FITC-dx fluorescent exclusion zone (one-half the difference in brightfield and fluorescent diameters) in capillaries and venules (B) of control vessels (black, 31 vessels, 3.3–13 μm diameter, in two WT mice) and vessels after continuous epi-illumination (white, 22 vessels, 3.6–16 μm diameter, in two WT mice). Asterisk denotes significant difference via two-tailed t-test (p < 0.05).

FIGURE 2

Photomicrographs showing axially (A, white line) or radially aligned erythrocytes (B, black line) in a 10.3 μm venule after occlusion with a blunt micropipette (micropipette not shown). Measurements of the diameter (width) of radially (black, 45 cells in eight venules) and axially aligned (white, 45 cells in eight venules) erythrocytes in 10–14 μm diameter venules (C). Photomicrographs showing radially (D, black line) or axially aligned erythrocytes (E, white line) in a 32 μm glass capillary after perfusion with a 2.5% hematocrit solution of mouse erythrocytes in plasma. Measurements of the diameter of radially (black, 30 cells) and axially aligned (white, 30 cells) erythrocytes (F). Asterisk denotes significant difference via two-tailed t-test (p < 0.05).

FIGURE 3

Photomicrograph of the double stroboscopic image of a single microsphere in a 21 μm diameter venule, where the black and white arrows point to the first and second images of the microsphere, respectively. Scale bar = 2 μm (A). Cumulative frequency of microspheres measured in close proximity to the vessel wall in control venules (open circles) and after prolonged exposure to epifluorescence treatment (filled circles, B). Asterisk denotes significant difference via two-tailed t-test (p < 0.05).

FIGURE 4

Fluorescent intravital μ-PIV data in the plasma-rich region of mouse cremaster muscle venules from one control vessel (A and C, 32.8 μm diameter) and a different vessel (B and D, 25.4 μm diameter) after light-dye treatment to degrade the ESL showing measured (dot symbols) translational speeds of sphere centers and predicted translational speeds of fluid-particles (plus symbols) if the spheres were not present in the flow. Theoretical curves in E and F are from an analysis of the free motion of a neutrally buoyant sphere in a uniform shear flow near a Brinkman medium and were used to estimate, as a function of h/a, the translational speed, U, a sphere of radius a would have, centered a distance h from the vessel wall, relative to the speed a fluid particle would have at that distance in a uniform shear field of strength if no sphere were present in the flow (Damiano et al., 2003). Curves corresponding to U/((h − t)) in E were used to relate sphere translational speeds in the control vessel to predicted fluid-particle speeds in A assuming no flow through the layer (solid curve, K → ∞) and in C assuming a finite hydraulic resistivity (dashed, K = 10⁸ dyn-s/cm⁴). The curves in F are similarly related to B and D in the light-dye treated vessel. In every vessel we observed, linear extrapolation (dash-dotted line in A–D) of the linear fit to the estimated fluid-particle velocity data revealed a negative intercept at the vessel wall. Permeation-induced fluid drag through the glycocalyx is thought to account for the enhanced drag on the portion of the microsphere that is nearest to the glycocalyx interface. When flow through the ESL is neglected, as in A and B, layer thickness, t, is estimated as the distance from the vessel wall where the linear fit extrapolates to zero velocity, whereas for finite values of K, as in C and D, layer thickness is estimated using predicted velocity profiles in the layer (Damiano et al., 1996) as described in Materials and Methods.

FIGURE 5

Mean layer thickness of control vessels (white, 10 vessels, 24–41 μm diameter, in four WT mice) and of light-dye treated vessels (black, 10 vessels, 18–31 μm diameter, in three WT mice) obtained from the average of individual estimates of t found (as in Fig. 4) for three finite values of hydraulic resistivity (K = 10⁸, 10⁹, and 10¹⁰ dyn-s/cm⁴) and for the case of no flow through the layer (K → ∞). Asterisk denotes significant difference via two-tailed t-test (p < 0.05).