A novel method for quantifying the permeability of isolated small venules
Authors: Koller Akos , Deng Wensheng , Szenasi Annamaria , Zhang Bin , Kandhi Sharath , Huang An , Sun Dong 
Published: April 2020

Background

The wall of venules is the primary site for the regulation of the transfer of water, ions and larges molecules in order to maintain tissue homeostasis. The impairment of venular permeability can cause tissue edema, followed by organ dysfunction. To date, although some methods are available to visualize the permeability of vessels, the quantification of the results remains elusive. Thus the aim of the present study was to develop a novel method to quantify the permeability of small isolated venules.

Methods

Mouse mesenteric venules (160–200 μm diameter at 8.0 mmHg of perfusion pressure) were isolated, cannulated in a vessel chamber and perfused with MOPS‐PSS (pH=7.4 at 37°C). The perfusion chamber contained two glass cannulas (inflow and outflow). A PE tubing of 15 mm in length with a rectangular window (2 × 7 mm) carved in the center is pre‐inserted onto the inflow pipette. After a venule was cannulated, the PE tubing was pushed forward (directed from inflow to the outflow pipette) to create a small sealed pool between the two cannulas (see the illustration). The chamber solution was then aspirated, the micro‐pool was washed and filled with 30 μl fresh MOPS‐PSS through the opened window. The inflow and outflow pressures were set at 8 cmH2O to maintain a constant intravascular pressure. The perfusion apparatus was then placed into a 37°C humidified incubator to equilibrate for 30 minutes. After equilibration, a one‐cmH2O pressure gradient was established by an increasing the inflow pressure and a decreasing the outflow pressure equally to create a constant intraluminal flow composed of MOPS‐PSS containing 5×10−4 M Evans blue dye (EBD). Thus, changes in permeability of venules could be assessed by determining the EBD in the pool solution using fluorescence (620/680 nm) spectrometry method. The final level of EBD in samples was determined using an EBD standard curve (0.08–20 μg/ml) and normalized to the internal surface area of venules.

Results

Histamine (2×10−5, 2×10−4 and 2×10−3 M) elicited a dose‐dependent increase in EBD permeability from 7.63 ± 0.79 to 21.70 ± 2.28 ng/mm2/min. The histamine‐induced release of EBD was continuous and reached a peak within 15 min, responses that were significantly reduced by triprolidine hydrochloride an H1 receptor inhibitor to 14.71± 2.07 ng/mm2/min. In addition, we have found that 5% erythrocytes lysate (ECL) and 2 μg/ml lipopolysaccharide (LPS) significantly enhanced EBD permeability.

Conclusions

Using this novel micro‐chamber technique, we were able to quantify histamine, ECL, and LPS‐induced increases in permeability of isolated venules, thus this method offers a valuable tool for pharmacokinetic research of permeability at the scale of microcirculation.

Support or Funding Information

Support

NIH HL070653 (An Huang) and HL129797 (Dong Sun),Scientific Excellence Program 2019, at the University of Physical Education, Innovation and Technology Ministry, Hungary TUDFO/51757/2019‐ITM (Akos Koller).