3D virtual histopathology of cardiac tissue from Covid-19 patients based on phase-contrast X-ray tomography

  1. Marius Reichardt
  2. Patrick Moller Jensen
  3. Vedrana Andersen Dahl
  4. Anders Bjorholm Dahl
  5. Maximilian Ackermann
  6. Harshit Shah
  7. Florian Länger
  8. Christopher Werlein
  9. Mark P Kuehnel
  10. Danny Jonigk  Is a corresponding author
  11. Tim Salditt  Is a corresponding author
  1. Georg-August-Universität Göttingen, Germany
  2. Technical University of Denmark, Denmark
  3. University Medical Center of the Johannes Gutenberg University Mainz, Germany
  4. Medical University Hannover, Germany

Abstract

We have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross section of 3:5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.

Data availability

The tomographic datasets recorded in WG configuration as well as the PB datasets used for the segmentation of the vascular system and the respective laboratory datasets were uploaded to https://doi.org/10.5281/zenodo.4905971.Additional data (raw data, PB and laboratory reconstructions, structure tensor analysis) is curated here at University ofGöttingen and at DESY can be obtained upon request from the corresponding author (tsaldit@gwdg.de); due to the extremely large size >15TB it cannot presently be uploaded easily to a public repository.The implementation of the structure tensor analysis is provided in https://lab.compute.dtu.dk/patmjen/structure-tensor.The neural network code used for the segmentation of the vasculature was uploaded to GitHub (github.com/patmjen/blood-vessel-segmentation)

The following data sets were generated

Article and author information

Author details

  1. Marius Reichardt

    Institut für Röntgenphysik, Georg-August-Universität Göttingen, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Patrick Moller Jensen

    Technical University of Denmark, Kopenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  3. Vedrana Andersen Dahl

    Technical University of Denmark, Kopenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  4. Anders Bjorholm Dahl

    Technical University of Denmark, Kopenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  5. Maximilian Ackermann

    Institute of Anatomy and Cell Biology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9996-2477
  6. Harshit Shah

    Medical University Hannover, Hannover, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Florian Länger

    Medical University Hannover, Hannover, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Christopher Werlein

    Medical University Hannover, Hannover, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Mark P Kuehnel

    Medical University Hannover, Hannover, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Danny Jonigk

    Medical University Hannover, Hannover, Germany
    For correspondence
    Jonigk.Danny@mh-hannover.de
    Competing interests
    The authors declare that no competing interests exist.
  11. Tim Salditt

    Institut für Röntgenphysik, Georg-August-Universität Göttingen, Göttingen, Germany
    For correspondence
    tsaldit@gwdg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4636-0813

Funding

Bundesministerium für Bildung und Forschung (Max Planck School Matter to Life)

  • Marius Reichardt
  • Tim Salditt

Bundesministerium für Bildung und Forschung (05K19MG2)

  • Tim Salditt

Deutsche Forschungsgemeinschaft (EXC 2067/1-390729940)

  • Tim Salditt

H2020 European Research Council (XHale,771883)

  • Danny Jonigk

Deutsche Forschungsgemeinschaft (KFO311 (project Z2))

  • Danny Jonigk

Hanseatic League of Science

  • Patrick Moller Jensen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: Formalin-fixed paraffin-embedded tissue blocks of control hearts, influenza and coxsackie virus myocarditis hearts were retrieved from archived material from the Institute of Pathology at Hannover Medical School in accordance with the local ethics committee (ethics vote number: 1741-2013 and 2893-2015). Formalin-fixed paraffin-embedded tissue blocks of COVID-19 autopsy cases were retrieved after written consent in accordance with the local ethics committee at Hannover medical school (ethics vote number: 9022 BO K 2020)

Reviewing Editor

  1. Hina Chaudhry, Harvard University, United States

Publication history

  1. Received: June 17, 2021
  2. Preprint posted: September 18, 2021 (view preprint)
  3. Accepted: December 10, 2021
  4. Accepted Manuscript published: December 21, 2021 (version 1)
  5. Version of Record published: January 10, 2022 (version 2)

Copyright

© 2021, Reichardt et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Marius Reichardt
  2. Patrick Moller Jensen
  3. Vedrana Andersen Dahl
  4. Anders Bjorholm Dahl
  5. Maximilian Ackermann
  6. Harshit Shah
  7. Florian Länger
  8. Christopher Werlein
  9. Mark P Kuehnel
  10. Danny Jonigk
  11. Tim Salditt
(2021)
3D virtual histopathology of cardiac tissue from Covid-19 patients based on phase-contrast X-ray tomography
eLife 10:e71359.
https://doi.org/10.7554/eLife.71359
  1. Further reading

Further reading

    1. Epidemiology and Global Health
    Ceereena Ubaida-Mohien et al.
    Research Article Updated

    Background:

    Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.

    Methods:

    We performed muscle biopsies in 15 octogenarian world-class track and field MAs and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mitochondrial DNA (mtDNA) copy number, and proteomics by liquid-chromatography mass spectrometry.

    Results:

    MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage-dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and that are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA.

    Conclusions:

    We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with underrepresentation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging.

    Funding:

    Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, USA.

    1. Epidemiology and Global Health
    Toby Mansell et al.
    Research Article

    Background:

    The risk of adult onset cardiovascular and metabolic (cardiometabolic) disease accrues from early life. Infection is ubiquitous in infancy and induces inflammation, a key cardiometabolic risk factor, but the relationship between infection, inflammation, and metabolic profiles in early childhood remains unexplored. We investigated relationships between infection and plasma metabolomic and lipidomic profiles at age 6 and 12 months, and mediation of these associations by inflammation.

    Methods:

    Matched infection, metabolomics, and lipidomics data were generated from 555 infants in a pre-birth longitudinal cohort. Infection data from birth to 12 months were parent-reported (total infections at age 1, 3, 6, 9, and 12 months), inflammation markers (high-sensitivity C-reactive protein [hsCRP]; glycoprotein acetyls [GlycA]) were quantified at 12 months. Metabolic profiles were 12-month plasma nuclear magnetic resonance metabolomics (228 metabolites) and liquid chromatography/mass spectrometry lipidomics (776 lipids). Associations were evaluated with multivariable linear regression models. In secondary analyses, corresponding inflammation and metabolic data from birth (serum) and 6-month (plasma) time points were used.

    Results:

    At 12 months, more frequent infant infections were associated with adverse metabolomic (elevated inflammation markers, triglycerides and phenylalanine, and lower high-density lipoprotein [HDL] cholesterol and apolipoprotein A1) and lipidomic profiles (elevated phosphatidylethanolamines and lower trihexosylceramides, dehydrocholesteryl esters, and plasmalogens). Similar, more marked, profiles were observed with higher GlycA, but not hsCRP. GlycA mediated a substantial proportion of the relationship between infection and metabolome/lipidome, with hsCRP generally mediating a lower proportion. Analogous relationships were observed between infection and 6-month inflammation, HDL cholesterol, and apolipoprotein A1.

    Conclusions:

    Infants with a greater infection burden in the first year of life had proinflammatory and proatherogenic plasma metabolomic/lipidomic profiles at 12 months of age that in adults are indicative of heightened risk of cardiovascular disease, obesity, and type 2 diabetes. These findings suggest potentially modifiable pathways linking early life infection and inflammation with subsequent cardiometabolic risk.

    Funding:

    The establishment work and infrastructure for the BIS was provided by the Murdoch Children’s Research Institute (MCRI), Deakin University, and Barwon Health. Subsequent funding was secured from National Health and Medical Research Council of Australia (NHMRC), The Shepherd Foundation, The Jack Brockhoff Foundation, the Scobie & Claire McKinnon Trust, the Shane O’Brien Memorial Asthma Foundation, the Our Women’s Our Children’s Fund Raising Committee Barwon Health, the Rotary Club of Geelong, the Minderoo Foundation, the Ilhan Food Allergy Foundation, GMHBA, Vanguard Investments Australia Ltd, and the Percy Baxter Charitable Trust, Perpetual Trustees. In-kind support was provided by the Cotton On Foundation and CreativeForce. The study sponsors were not involved in the collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication. Research at MCRI is supported by the Victorian Government’s Operational Infrastructure Support Program. This work was also supported by NHMRC Senior Research Fellowships to ALP (1008396); DB (1064629); and RS (1045161) , NHMRC Investigator Grants to ALP (1110200) and DB (1175744), NHMRC-A*STAR project grant (1149047). TM is supported by an MCRI ECR Fellowship. SB is supported by the Dutch Research Council (452173113).