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 ...

The overall goal of this methodology is to provide a standardized procedure for obtaining images of live human resistance arteries for analysis of the relationship between the microarchitecture of the extracellular matrix and the mechanics of the arterial wall. This method can help answer key questions in vascular biology and mechanics such as how pressure induces changes in the overall geometry as well as the microarchitecture of the vascular wall. The main advantage of the technique is that you don't have to label your sample for the imaging.Though this method can provide insight into resistance artery mechanics, a modified version of the protocol can be applied to other tube-like organs like the bronchi or to strips from human bladder, or the intestinal wall. To begin, collect tissue samples of interest immediately upon their excision during surgery and prepare the vessels as described in the accompanying text protocol. Pressurize a cannulated artery to five millimeters mercury.Then set up the optical path for the imaging by activating the two-photon laser and setting the excitation wavelength to 820 nanometers. Insert a 460 nanometer long pass dichroic mirror to split the emission light between the photo multipliers and collect the emission simultaneously in two channels. Use 30 to 60 nanometer wide bandpass filters centered at 520 nanometers to collect the elastin autofluorescence and another centered at 410 nanometers to collect the collagen second harmonic generation signal.Now set the laser dwell time and pixel to 10 microseconds. Set the resolution to 512 by 512 pixels for 100%field of view. Enable continuous scanning and use as low excitation light power and pixel dwell time as possible to avoid photo damaging the live artery.Then manually scan through the artery to find the plane with the maximum diameter. Next, choose a rectangular slice covering the artery's widest diameter and scan a single frame with a pixel size of 300 nanometers per pixel or less. Save this image for analysis at a later time.To determine lumen diameter and wall thickness at the maximum diameter of the artery, first load the image in Fiji. Set the scale by clicking on main menu and then go to analyze followed by set scale. Here enter the micrometer per pixel and pixel ratio.Next, choose the Fiji line tool and draw a line between the two internal elastic lamina at each side of the arterial lumen. Then click Control M to report the length in the results table. Continue to draw more lines to measure the thickness of each wall and save the final measurement.Change to a 60X objective with a numerical aperture of one or greater. Image the collagen and elastin microarchitecture at five millimeters mercury by scanning the entire thickness of the arterial wall. Next, define the Z stack start and end depth and the Z step spacing and pixel density.When you're designing your Z stacks, make sure your region of interest is small enough to avoid any inference of curvature on your subsequent analysis. When complete, set each channel to be saved separately. Lastly, find one to three different regions of interest and obtain 3D image stacks of good quality.If your elastin autofluorescence is bleaching, you are using too high excitation light power. This should be avoided. In order to finish your ongoing experiment, you may add a dilute concentration of eosin to enhance the elastin autofluorescence.When finished, repeat the entire process at pressures varying up to 100 millimeters mercury, replacing the buffer in the myograph chamber with fresh 37 degrees Celsius HBS after each pressure step. To begin image analysis, open the elastin image stack in Fiji. Then go to image, click on stacks, and select Z project to choose the images to work with and choose max intensity for projection type.Save the max intensity projection as a TIFF image. Then choose as many internal elastic lamina fiber branching points as possible and measure them using the angle tool. Systematically work through each image and when finished, save the Fiji results sheet.To measure the waviness of the adventitial collagen, open the collagen image stack in Fiji. Next, go to image down to stacks and select Z project to choose images to work with and to set the max intensity projection. Now change the image type to an eight-bit TIFF image by clicking image, type, and then eight-bit.Save the max intensity projection as a TIFF image. Open the Neuron J plugin by clicking on Fiji and selecting Neuron J under plugins. Then open the eight-bit TIFF file in Neuron J.Once open, click on add tracings and select fibers to analyze by clicking on the start and the end of each fiber.Next, go to measure tracings and choose display tracing and vertex measurements in the dialogue box. To calculate collage fiber straightness, first copy the values in the Fiji tracings window to Excel. The length values in the tracings window are measures of the full lengths of the analyzed collagen fibers Lf.Second, copy for each collagen fiber bundle analyzed the first and last XY coordinates of the Fiji vertices window to Excel.Then calculate the length of the straight line connecting each end of the analyzed collagen fiber L0 using Pythagoras Theorem. Collagen fiber straightness is finally calculated as L0/Lf. Following the protocol just shown, the widest part of the artery was identified and at this point the artery was scanned using label-free fluorescence microscopy to measure the maximum vessel diameter and vessel wall thickness.Utilizing a higher magnification to high numerical aperture objective, not only can the vessel diameter and wall thickness be measured, but also the microarchitectures of collagen and elastin. The microarchitectures of elastin fiber branching angles and collagen straightness can be determined in consecutive image sections using the appropriate image analysis software. This allows a direct analysis of the relationship between the pressure induced changes in the microarchitectures of collagen and elastin and the incremental elastic modulus at different pressures.Following this procedure, other information can be extracted from the images as well. This could be the orientation angels of adventitial collagen along the vessel longitudinal axis or it could be the microarchitecture of the extracellular matrix in tunica media. The quantitative measures extracted from the images using this method can be fed into mathematical models in order to elucidate how the healthy and deceased human circulation respond to different conditions.After watching this video, you should have a thorough understanding on how to design your imaging experiments and subsequent image analysis in order to extract information on how pressure induces changes in the microarchitecture of the resistance artery wall. Remember that you have to avoid any exposure to visible and invisible light when working with two-photon excitation fluorescence microscopy.

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