Quantification of experimental venous thrombus resolution by longitudinal nanogold-enhanced micro-computed tomography

Steven P Grover, Prakash Saha, Julia Jenkins, Arun Mukkavilli, Oliver T Lyons, Ashish S Patel, Kavitha Sunassee, Bijan Modarai, Alberto Smith, Steven P Grover, Prakash Saha, Julia Jenkins, Arun Mukkavilli, Oliver T Lyons, Ashish S Patel, Kavitha Sunassee, Bijan Modarai, Alberto Smith

Abstract

Introduction: The assessment of thrombus size following treatments directed at preventing thrombosis or enhancing its resolution has generally relied on physical or histological methods. This cross-sectional design imposes the need for increased numbers of animals for experiments. Micro-computed tomography (microCT) has been used to detect the presence of venous thrombus in experimental models but has yet to be used in a quantitative manner. In this study, we investigate the use of contrast-enhanced microCT for the longitudinal assessment of experimental venous thrombus resolution.

Materials and methods: Thrombi induced by stenosis of the inferior vena cava in mice were imaged by contrast-enhanced microCT at 1, 7 and 14 days post-induction (n=18). Thrombus volumes were determined longitudinally by segmentation and 3D volume reconstruction of microCT scans and by standard end-point histological analysis at day 14. An additional group of thrombi were analysed solely by histology at 1, 7 and 14 days post-induction (n=15).

Results: IVC resident thrombus was readily detectable by contrast-enhanced microCT. MicroCT-derived measurements of thrombus volume correlated well with time-matched histological analyses (ICC=0.75, P<0.01). Thrombus volumes measured by microCT were significantly greater than those derived from histological analysis (P<0.001). Intra- and inter-observer analyses were highly correlated (ICC=0.99 and 0.91 respectively, P<0.0001). Further histological analysis revealed noticeable levels of contrast agent extravasation into the thrombus that was associated with the presence of neovascular channels, macrophages and intracellular iron deposits.

Conclusion: Contrast-enhanced microCT represents a reliable and reproducible method for the longitudinal assessment of venous thrombus resolution providing powerful paired data.

Keywords: Animal models; MicroCT; Nanogold; Venous thrombosis.

Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Figures

Fig. 1
Fig. 1
Imaging of IVC thrombi by contrast-enhanced microCT. A representative day 1 thrombus imaged by contrast-enhanced microCT shown in transverse (A), sagittal (B) and coronal (C) planes (scale bar 2 mm). Segmentation of the scan allow for reconstruction of 3D volume renders (D–F). Images have been annotated with the position of the aorta (Ao), the site of stenosis (S) and the thrombus (T).
Fig. 2
Fig. 2
Visualisation of the thrombus by histology and microCT. Thrombus was imaged at days 1, 7 and 14 post-induction by contrast-enhanced microCT (n = 6 per timepoint). (A–C) Representative transverse slices demonstrate the presence of thrombus in the IVC lumen (scale bar 2 mm), (D–F) corresponding with time-matched H&E stained thrombus micrographs (scale bar 200 μm). (J) Thrombus volume of time-matched samples determined by microCT (black bars, n = 6 per timepoint) and histology (white bars, n = 5 per timepoint) demonstrate similar temporal changes in thrombus volume (one-way ANOVA, P 

Fig. 3

Intra- and inter-observer variability of…

Fig. 3

Intra- and inter-observer variability of thrombus volume measurements by microCT. The reliability and…

Fig. 3
Intra- and inter-observer variability of thrombus volume measurements by microCT. The reliability and reproducibility of thrombus volume measurements were assessed by both intra- and inter-observer analyses of thrombi imaged at days 1, 7 and 14 post-induction (n = 6 mice per timepoint). (A) Intra-observer measurements correlated well (ICC = 0.99, P 

Fig. 4

Aurovist contrast agent extravasation. Aurovist…

Fig. 4

Aurovist contrast agent extravasation. Aurovist contrast extravasation (dark staining) was observed in (A)…

Fig. 4
Aurovist contrast agent extravasation. Aurovist contrast extravasation (dark staining) was observed in (A) H&E stained sections of day 14 thrombi. Contrast extravasation occurred in highly organised regions of the thrombus rich in (B) Mac-2 positive macrophages and (C) CD31 positive neovascular channels (arrow heads) and (D) cellular iron deposits (scale bar 40 μm). The area of thrombus containing Aurovist correlated with (E) macrophage content (r = 0.41, P 
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References
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Fig. 3
Fig. 3
Intra- and inter-observer variability of thrombus volume measurements by microCT. The reliability and reproducibility of thrombus volume measurements were assessed by both intra- and inter-observer analyses of thrombi imaged at days 1, 7 and 14 post-induction (n = 6 mice per timepoint). (A) Intra-observer measurements correlated well (ICC = 0.99, P 

Fig. 4

Aurovist contrast agent extravasation. Aurovist…

Fig. 4

Aurovist contrast agent extravasation. Aurovist contrast extravasation (dark staining) was observed in (A)…

Fig. 4
Aurovist contrast agent extravasation. Aurovist contrast extravasation (dark staining) was observed in (A) H&E stained sections of day 14 thrombi. Contrast extravasation occurred in highly organised regions of the thrombus rich in (B) Mac-2 positive macrophages and (C) CD31 positive neovascular channels (arrow heads) and (D) cellular iron deposits (scale bar 40 μm). The area of thrombus containing Aurovist correlated with (E) macrophage content (r = 0.41, P 
Similar articles
References
    1. Singh I., Burnand K.G., Collins M., Luttun A., Collen D., Boelhouwer B. Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells. Circulation. 2003;107:869–875. - PubMed
    1. Nosaka M., Ishida Y., Kimura A., Kuninaka Y., Inui M., Mukaida N. Absence of IFN-gamma accelerates thrombus resolution through enhanced MMP-9 and VEGF expression in mice. J. Clin. Invest. 2011;121:2911–2920. - PMC - PubMed
    1. von Bruhl M.L., Stark K., Steinhart A., Chandraratne S., Konrad I., Lorenz M. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J. Exp. Med. 2012;209:819–835. - PMC - PubMed
    1. Phinikaridou A., Andia M.E., Saha P., Modarai B., Smith A., Botnar R.M. In vivo magnetization transfer and diffusion-weighted magnetic resonance imaging detects thrombus composition in a mouse model of deep vein thrombosis. Circ. Cardiovasc. Imaging. 2013;6:433–440. - PMC - PubMed
    1. Saha P., Andia M.E., Modarai B., Blume U., Humphries J., Patel A.S. Magnetic resonance T1 relaxation time of venous thrombus is determined by iron processing and predicts susceptibility to lysis. Circulation. 2013;128:729–736. - PMC - PubMed
Show all 22 references
Publication types
MeSH terms
[x]
Cite
Copy Download .nbib
Format: AMA APA MLA NLM
Fig. 4
Fig. 4
Aurovist contrast agent extravasation. Aurovist contrast extravasation (dark staining) was observed in (A) H&E stained sections of day 14 thrombi. Contrast extravasation occurred in highly organised regions of the thrombus rich in (B) Mac-2 positive macrophages and (C) CD31 positive neovascular channels (arrow heads) and (D) cellular iron deposits (scale bar 40 μm). The area of thrombus containing Aurovist correlated with (E) macrophage content (r = 0.41, P 

References

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