Name: assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy
Uploaded: 2018-04-09T14:45:00
Description: Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

The authors thank the Danish Molecular Biomedical Imaging Center at the Faculty of Natural Sciences, University of Southern Denmark, for the use of laboratories and microscopes. Kristoffer Rosenstand and Ulla Melchior are acknowledged for excellent technical assistance with the pressure myography and imaging.

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...

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 ...

The pathogenic contribution and effects of resistance artery remodeling are documented in essential hypertension, diabetes and the metabolic syndrome1,2,3,4,5. 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 disease related remodeling of the arterial wall.
Prior studies focused in understanding how the microarchitecture of the arterial wall relates to arterial wall mechanics by incorporating mechanical measures and the microarchitecture of the extracellular matrix (ECM) are almost exclusively performed on large, elastic conduit arteries from mice or swine6,7,8,9,10,11. Imaging of the microstructures of the wall is typically performed using nonlinear optical techniques, taking advantage of the autofluorescence of elastin and second harmonic generation by collagen. This allows spatiotemporal imaging of the two major components of the extracellular matrix, elastin and collagen, without a need for staining. Imaging of the arterial wall in full thickness is a challenge in large conduit arteries due to scatter of the light in the thick tunica media. However, to determine how the microarchitecture of the structural components of the arterial wall relate to the observed mechanical properties, three-dimensional information must be obtained during the mechanical testing. For large arteries like the human aorta, this requires biaxial mounting, mechanical testing and imaging of regions of interest in 1-2 cm2 pieces of the arterial wall7,9,10,12. Only part of the wall can be imaged and mechanically tested.
For smaller arteries of any species (e.g., human pericardial13, pulmonary14 and subcutaneous15 arteries, rat mesenteric arteries16,17,18,19,20, mouse cremaster, mesenteric, cerebral, femoral and carotid arteries21,22,23,24,25,26,27) imaging of the entire wall thickness is possible and can be combined with mechanical testing. This allows simultaneous recording of the mechanical properties and the structural arrangements within the wall. However, a direct mathematical modeling of the relationship between the observed alterations in the three-dimensional structure of the ECM and changed mechanical properties of the resistance arterial wall, has to the best of our knowledge only been reported upon recently in human resistance arteries13,15.
In this work, an ex vivo method for passive mechanical testing and simultaneous three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries is described. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for obtaining measures of internal elastic lamina branching angles and adventitial collagen straightness13 using Fiji28. Collagen and elastin volume densities are determined using Ilastik software29 and finally, the inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed.
The goal of describing the imaging and image analyses techniques in combination with mathematical modeling is to provide investigators a systematical approach to describe and understand observed pressure induced changes in the ECM of resistance arteries. The described method is focused in quantifying the changes in the ECM in a vessel during pressurization, by comparing the structure of the ECM at 20, 40 and 100 mmHg. These pressures were chosen for determining the structure of the arterial wall at its more compliant (20 mmHg), stiff (100 mmHg) and intermediate (40 mmHg) state, respectively. However, any process in the vascular wall of live arteries, including changes induced by vasoactive components, hysteresis and flow, can be quantified, depending on the research hypothesis in question by the investigator.
The use of two-photon excitation fluorescence microscopy (TPEM) in combination with a pressure myograph for studying pressure (or other) induced changes in the ECM of live arteries is emphasized. First, because this allows simultaneous acquisition of the overall three-dimensional structure of the arterial wall (diameter and wall thickness) along with three-dimensional label-free acquisition of high quality, detailed images of the collagen and elastin microarchitectures as described13 by taking advantage of the elastin autofluorescence and the collagen second harmonic generation signal (SHG)30. Second, TPEM allows use of low-energy near-infrared excitation light, minimizing photodamage of the tissue and thus, repeated imaging at exactly the same position within the vascular wall is allowed, permitting repeated-measurements analyses of observed changes.
The use of an alternative approach using confocal imaging of pressure fixed arteries is discussed to allow users without access to TPEM an opportunity to use the described method as well. Information on ECM structure and volume densities can also be retrieved from two-dimensional analyses of tissues sectioned in serial, e.g. as described by31,32. However, due to the lack of possibility to retrieve three-dimensional structural information over the length scales of the artery as well as during changing conditions using this method, it is not recommend using this approach for investigations of pressure and treatment induced three-dimensional changes in the ECM.
The minimum requirement for the investigator to apply the herein described method is the access to a setup for cannulation and pressurization of arteries in combination with a confocal or two-photon excitation fluorescence microscope. The setup described in the following protocol is a custom-built pressure myograph with a longitudinal force transducer, built to fit on a custom built inverted two-photon excitation fluorescence microscope.

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assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy

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.

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 &#215; 24 mm #1.5 glass coverslip (replaceable). The pressure controller was built from a standa...

Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

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

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.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

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