Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

Summary

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

Abstract

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

Introduction

The pathogenic contribution and effects of resistance artery remodeling are documented in essential hypertension, diabetes and the metabolic syndrome^1,2,3,4,5. Deciphering the relationship between the mechanical and microarchitectural properties of the microvascular wall is essential for developing mathematical models of this association. Such models will improve understanding the remodeling process and will support the development of in silico models useful for testing pharmacological strategies targeting ....

Protocol

Collection of biopsies of the human parietal pericardium for use in this work was performed after written informed consent, as previously described³³. The study of human tissues conform to the principles outlined in the Declaration of Helsinki³⁴ and was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20100044 and S-20140202) and the Danish Data Protection Agency.

1. Collect Tissue and Isolate (Human) Resistance Artery

1. Collect tissue samples of interest immediately upon the excision during a surgery. Transfer the tissue to 4 °C HEPES buffer....

Results

The custom-built pressure myograph for imaging used in this work is shown in Figure 1. Special attention for the design of the myograph was paid to i) the chamber with a small volume (2 mL) and ii) the possibility for positioning the cannulae close to, and parallel with the glass bottom (Figure 1B). The bottom of the chamber fits a 50 × 24 mm #1.5 glass coverslip (replaceable). The pressure controller was built from a standard 1 .......

Discussion

This work represents our suggestion for a standardized, combined imaging and pressure myography approach, valuable for simultaneous assessment of the mechanical properties of resistance arteries and pressure-related changes in the structure of the arterial wall over a pressure range from 0 to 100 mmHg. The presented approach was developed using custom built equipment, however, any pressure myograph that fits on a two-photon excitation fluorescence microscope can be used, when the design of both equipments allows imaging .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fine Science Tools	15401-12
Fine Science Tools	11251-23
Nikon	SMZ800N
Sigma-Aldrich, Brøndby, Denmark.	761028	for dissection purpose
Vitrex Medical A/S, Herlev, Denmark	1.63, 2.13, 210mm
Smiths medical Intl, UK
Ethicon	Ethilon 11-0
Custom built		DK patent number 201200167, University of Southern Denmark, J. Schoubo V. Jensen, F. Jensen. T.R. Uhrenholt
Mettler toledo
Sigma-Aldrich, Brøndby, Denmark.	B3259
Sigma-Aldrich, Brøndby, Denmark.	A7030
Sigma-Aldrich, Brøndby, Denmark.	C5670
Sigma-Aldrich, Brøndby, Denmark.	G7021
Sigma-Aldrich, Brøndby, Denmark.	E3889
Merck Millipore, Hellerup, Denmark	1.00496.9010	Phosphate buffered (pH 6.9) 4% formaldehyde solution
Sigma-Aldrich, Brøndby, Denmark.	H3784
Sigma-Aldrich, Brøndby, Denmark.	P9666
Sigma-Aldrich, Brøndby, Denmark.	P5655
Sigma-Aldrich, Brøndby, Denmark.	M2643
Sigma-Aldrich, Brøndby, Denmark.	S2002
Sigma-Aldrich, Brøndby, Denmark.	S5886
Sigma-Aldrich, Brøndby, Denmark.	S5761
Sigma-Aldrich, Brøndby, Denmark.	1.06462
Gibco, ThermoFisher Scientific	10010015
Sigma-Aldrich, Brøndby, Denmark.	PHR1423
Sigma-Aldrich, Brøndby, Denmark.	Z370525
Tocris Bioscience, Bristol, UK	538944
Nikon	Custom built
Spectra Physics, Mountain View, CA
Nikon	CFI Plan Apo IR SR 60XWI NA 1.27
Nikon	CFI Plan Fluor 20XMI (multi-immersion) NA 0.75
Hamamatsu, Ballerup, Denmark	H7422P-40
AHF analysentechnik AG (Tübingen, Germany).	ChromaET 460 nm long pass dichroic
AHF analysentechnik AG (Tübingen, Germany).	Semrock FF01-520/35-25 BrightLine filter
AHF analysentechnik AG (Tübingen, Germany).	Chroma ET402/15X
Scotch TM
	coverslip thickness should match used objective on microscope (#1 or #1.5), alternatively, set adjustment collar to match coverslip