J. F. Lenz

Oncotic pressures opposing filtration across non-fenestrated rat microvessels

We hypothesized that ultrafiltrate crossing the luminal endothelial glycocalyx through infrequent discontinuities (gaps) in the tight junction (TJ) strand of endothelial clefts reduces albumin diffusive flux from tissue into the ‘protected region’ of the cleft on the luminal side of the TJ. Thus, the effective oncotic pressure difference (σ□π) opposing filtration is greater than that measured between lumen and interstitial fluid. To test this we measured σ□π across rat mesenteric microvessels perfused with albumin (50 mg ml−1) with and without interstitial albumin at the same concentration within a few micrometres of the endothelium as demonstrated by confocal microscopy. We found σ□π was near 70% of luminal oncotic pressure when the tissue concentration equalled that in the lumen. We determined size and frequency of TJ strand gaps in endothelial clefts using serial section electron microscopy. We found nine gaps in the reconstructed clefts having mean spacing of 3.59 μm and mean length of 315 nm. The mean depth of the TJ strand near gaps was 67 nm and the mean cleft path length from lumen to interstitium was 411 nm. With these parameters our three‐dimensional hydrodynamic model confirmed that fluid velocity was high at gaps in the TJ strand so that even at relatively low hydraulic pressures the albumin concentration on the tissue side of the glycocalyx was significantly lower than in the interstitium. The results conform to the hypothesis that colloid osmotic forces opposing filtration across non‐fenestrated continuous capillaries are developed across the endothelial glycocalyx and that the oncotic pressure of interstitial fluid does not directly determine fluid balance across microvascular endothelium.

Quantitative Laser Scanning Confocal Microscopy on Single Capillaries: Permeability Measurement

Objective: We tested the hypothesis that there is significant solute loss to the superfusate during microvessel solute permeability measurement in frog mesentery which leads to underestimation of permeability coefficient. This is the first application of laser‐scanning confocal microscopy to measure microvessel permeability.

Low density lipoprotein transport across a microvascular endothelial barrier after permeability is increased.

We investigated the pathways for low density lipoprotein (LDL) transport across an endothelial barrier in individually perfused microvessels before and after an increase in permeability. The divalent cation ionophore A23187 (5 microM) was used to increase microvessel permeability. LDL permeability coefficients (PsLDL) were measured using quantitative fluorescence microscopy. In the control state, PsLDL measured after 10-23 minutes of accumulation of fluorescent-labeled LDL outside the microvessel wall was 4.8 x 10(-8) cm/sec. The transvascular vesicular exchange of approximately 50 vesicles/sec would account for the measured flux. The flux of LDL across the microvessel wall increased as much as 170-fold at the peak of the permeability increase (2-4 minutes after ionophore infusion). Permeability returned toward control values 10 minutes after ionophore infusion but remained elevated for as long as ionophore was present in the perfusate. The effective PsLDL was similar in magnitude to the Ps for fluorescent-labeled dextran (MW 20,000) when permeability was increased. To investigate the nature of pathways for LDL in the high-permeability state, PsLDL was measured at a series of microvessel pressures. LDL transport increased as microvessel pressure increased, demonstrating coupling of LDL flux to transvascular water flow. Solvent drag accounted for more than 95% of the increased flux of LDL in the period 2-10 minutes after permeability increased. Our results conform to the hypothesis that porous pathways between adjacent endothelial cells contribute to LDL transport across an endothelial barrier when permeability is increased.

Application of image restoration and three-dimensional visualization techniques to frog microvessels in-situ loaded with fluorescent indicators

In situ experiments on microvessels require image sensors of wide dynamic range due to large variations of the intensity in the scene, and 3D visualization due to the thickness of the preparation. The images require restoration because of the inherent tissue movement, out-of- focus-light contamination, and blur. To resolve the above problems, we developed an imaging system for quantitative imaging based on a 12 bits/pixel cooled CCD camera and a PC based digital imaging system. We applied the optical sectioning technique with image restoration using a modified nearest neighbor algorithm and iterative constrained deconvolution on each of the 2D optical sections. For the 3D visualization of the data, a volume rendering software was used. The data provided 3D images of the distribution of fluorescent indicators in intact microvessels. Optical cross sections were also compared with cross sections of the same microvessels examined in the electron microscope after their luminal surfaces were labeled with a tracer which was both electron dense and fluorescent. This procedure enabled precise identification of the endothelial cells in the microvessel wall as the principal site of accumulation of the fluorescent calcium indicator, fura-2, during microperfusion experiments.

Application of two dimensional and three dimensional imaging techniques to frog microvessels in-situ loaded with fluorescent indicators

A limitation of the photomultiplier tube (PMT) based quantitation systems in fluorescence microscopy is the lack of spatial information. In this paper, a cooled CCD based imaging system for quantitative imaging in fluorescence microscopy and two applications in single capillaries is presented. Two dimensional (2D) ratio imaging revealed uniform distribution of intracellular ion concentration in the endothelial cells along a capillary. Optical sectioning and image restoration showed that the observed non-homogeneity of indicator loading into the microvessel wall was due to variation in cell thickness.