Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease
February 8th, 2022
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Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.
Overview
This protocol provides high-resolution micro computed tomography imaging of healthy and pathological large mammalian hearts. It includes collagen-selective contrast enhancement to visualize cardiac microstructure and associated diseases.
Key Study Components
Area of Science
- Cardiac imaging
- Micro computed tomography
- Pathological analysis
Background
- Understanding cardiac microstructure is crucial for diagnosing heart diseases.
- Pathological conditions can lead to life-threatening arrhythmias.
- High-resolution imaging techniques are essential for detailed analysis.
- This protocol utilizes large mammalian models for translational research.
Purpose of Study
- To optimize imaging techniques for whole heart samples.
- To enhance visualization of structural diseases using contrast agents.
- To provide insights into the distribution of cardiac fibrosis.
Methods Used
- Preparation of cardioplegic solution and PBS/EDTA.
- Use of PMA contrast agent for enhanced imaging.
- Micro CT scanning with specific imaging parameters.
- Sequential ethanol perfusion for tissue preservation.
Main Results
- Successful imaging of cardiac microstructure in large mammalian hearts.
- Demonstrated the effectiveness of collagen-selective contrast enhancement.
- Provided detailed visualization of fibrosis and myocardial organization.
Conclusions
- The protocol allows for high-resolution imaging of cardiac tissues.
- It enhances understanding of structural changes in heart diseases.
- This method can aid in the development of therapeutic strategies.
Frequently Asked Questions
What is the main goal of this protocol?
The main goal is to obtain high-resolution images of large mammalian hearts to study structural diseases.
How does collagen-selective contrast enhancement work?
It enhances the visibility of collagen structures in the heart, aiding in the analysis of fibrosis.
What are the key components of the cardioplegic solution?
The cardioplegic solution contains sodium heparin and is stored at 4 degrees Celsius.
What imaging technique is used in this study?
Micro computed tomography (micro CT) is used for imaging the heart samples.
Why is ethanol perfusion important?
Ethanol perfusion helps in tissue preservation and prepares the samples for imaging.
Who demonstrates the procedure in the protocol?
The procedure is demonstrated by Nestor Pallares-Lupon, a postdoc and PhD graduate.
Executive Industry Relevance
This protocol enables high-resolution, non-destructive imaging of large mammalian hearts to visualize disease-related structural remodeling at microscale resolution. By selectively enhancing collagen-rich fibrotic tissue, it supports target validation and mechanistic de-risking in cardiovascular drug discovery. The approach bridges preclinical imaging with histopathological confirmation, improving predictive confidence in disease model relevance.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by visualizing fibrosis distribution and myocardial organization in disease models.
- Operational Value: Provides quantitative, spatially resolved structural data to clarify pathophysiological pathways.
- Predictive Value: Supports biological de-risking through direct visualization of target engagement with disease-modified tissue.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems with stabilized tissue conformation for reproducible downstream imaging workflows.
- Operational Value: Ensures assay standardization via air-dried, rigid heart samples suitable for longitudinal micro-CT monitoring.
- Scalability: Enables platform reuse across disease models (infarction, fibrillation) and species (pig, sheep) for comparative screening.
Translational & Preclinical Research
- Scientific Value: Maintains disease relevance by preserving endogenous collagen distribution for biomarker-aligned structural analysis.
- Operational Value: Facilitates translational continuity from discovery through preclinical validation via correlative histology compatibility.
- Risk Mitigation: Supports risk-adjusted advancement decisions by enabling serial, non-destructive monitoring of structural disease progression.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target validation through preclinical assessment, enabling iterative evaluation of structural disease modifiers in large mammalian models.
- Discovery Biology: Supports hypothesis testing and pathway clarification by mapping fibrosis-surviving myocardium interfaces in chronic disease models.
- Screening: Delivers assay readiness through ethanol fixation and HMDS reinforcement, ensuring dimensional stability for high-resolution imaging.
- Analytics: Generates quantitative 3D volumetric readouts of contrast agent deposition, enabling objective comparison of fibrotic burden across conditions.
- Translational Research: Connects to preclinical validation via direct correlation with histological staining (e.g., Mason’s trichrome) of the same air-dried samples.
- Enterprise Reuse: Establishes a reusable imaging capability for longitudinal studies across multiple cardiac disease models and compound testing cycles.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence by reducing mechanistic ambiguity in structure-function relationships underlying arrhythmogenic substrates.
- Operational Value: Delivers standardization and reproducibility through fixed tissue preparation and invariant imaging parameters (20.7 µm isotropic resolution).
- Strategic Value: Improves go/no-go decisions by providing early, translatable structural biomarkers of disease severity and regression.
- Portfolio Impact: Enables risk-adjusted prioritization through objective, imaging-based fibrosis quantification in preclinical models.
Implementation Considerations
- Requires expertise in cardiac dissection, perfusion techniques, and contrast agent handling.
- Depends on access to micro-CT scanners capable of sub-25 µm resolution and vacuum-compatible contrast loading.
- Necessitates cross-team standardization between imaging, histology, and pathology teams for correlative analysis.
- Involves adaptation considerations for varying heart sizes and disease-induced tissue fragility across models.
- Limited by perfusion duration requirements for complete ethanol exchange and contrast agent diffusion in large tissues.
Biopharma FAQs
Why does selective collagen enhancement matter for target validation?
Selective collagen enhancement via phosphomolybdic acid enables visualization of fibrotic scar regions, which is critical for validating targets involved in extracellular matrix remodeling. This supports mechanistic de-risking by confirming target engagement with disease-altered tissue architecture.
How does isolation of the perfusion system support discovery pipeline integrity?
Isolating the coronary vasculature via cannulation and controlled perfusion ensures precise delivery of contrast agents and fixatives, minimizing variability. This isolation supports reproducible structural imaging across samples, which is essential for reliable target validation in discovery workflows.
What quantitative outputs enable fibrosis assessment in this method?
The method generates isotropic 3D image volumes with 20.7 µm resolution, allowing quantification of contrast agent density in fibrotic versus healthy myocardium. These volumetric readouts provide objective metrics for comparing disease burden and treatment effects across models.
Why are replication requirements important for cross-functional collaboration?
Replication across healthy and diseased hearts (pig, sheep) establishes baseline variability and disease-specific contrast patterns, which is necessary for consistent interpretation. This reproducibility enables alignment between imaging, histology, and pharmacology teams on structural endpoints.
What statistical capabilities are needed before implementing this imaging workflow?
Implementation requires capability to analyze 3D volumetric data, including intensity thresholding and regional contrast measurements to distinguish fibrotic from non-fibrotic tissue. These analytical functions are essential for deriving statistically robust endpoints from micro-CT datasets.
Chapters in this video
0:04
Introduction
0:54
Solution Preparation
2:05
Tissue Preparation
5:21
Tissue Dehydration and Drying
6:57
MicroCT
9:20
Results: Contrast-Enhanced Air-Dried Tissue Preparations Enabled Microscale Imaging of Intact Large Mammalian Cardiac Models with Chronic Disease
11:36
Conclusion
