Assessment of the epi-pericardial fibrotic substrate by collagen-targeted probes

Figures

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  Figure 1. Atrial wall to pericardial fibrosis. Representative images of Masson's trichrome staining of atrial fibrosis in transgenic (Tg) fibrotic (A) and non-transgenic (Ntg) non-fibrotic (B) animals at 11 months of age. ×10 magnification images of atrial wall and epi-pericardial images of Tg (C) and (D) Ntg. Ishak fibrosis score of atrial wall to pericardium in Tg and Ntg (E). Mean ± SEM. **p < 0.0001 vs. Ntg by 2-way ANOVA followed by post Tukey, Tg; n = 4, Ntg; n = 2; Scale bars for (A) and (B) = 2000 µm; Scale bars for (C) and (D) = 100 µm.

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  Figure 2. Collagen types in Tg and Ntg mice. Representative atrial wall to epi-pericardial images of immunohistochemical analysis of various collagen types in fibrotic Tg (A) and Ntg (B) at 11 months of age. Mean fluorescence intensities of collagen I (C), collagen III (D) and collagen IV (E). Mean ± SEM, **p < 0.0001 vs. Ntg. 2-way ANOVA followed by post Tukey, Tg; n = 3, Ntg; n = 2 for collagen I; Tg; n = 4, Ntg; n = 2 for collagen III; Tg; n = 3, Ntg; n = 2 for collagen IV. Scale bars for (A) and (B) left 1000 µm; Scale bars for (A) and (B) right = 100 µm.

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  Figure 3. Biatrial thrombi and associated mechanism in Tg. Enlarged four chambers and biatrial thrombi in Tg but not in Ntg (A). Type I collagen and fibrin colocalisation (B). Type I collagen in association with platelet. Scale bars for (A) = 2000 µm; Scale bars for (B) and (C) = 100 µm.

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  Figure 4. Endothelial Cell Clustering and Embolic events in Tg. Overlay of PECAM-1 and platelet (A) and overlay of PECAM-1 and fibrin (B), associated with atrial emboli. Scale bars = 100 µm.

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  Figure 5. Traditional Mason’s trichrome staining (A) correlated qualitatively with T-peptide probe (B), cyclic peptide probe (C), type IV collagen immunofluorescence (D) and type collagen I immunofluorescence (E). T-peptide targets collagen IV while cyclic peptide probe targets collagen I. It is important to note that every normal atrium has intact pericardium tightly fastened to the wall of the working atrium. Tg; n = 2, Ntg; n = 2; Scale bar for (A) = 2000 µm; Scale bars for (B) and (C) = 1.5 mm. Blue arrow indicates disassociating pericardium.

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  Figure 6. Schematics illustration of near-infrared (NIR) fluorescence imaging of atrial fibrosis for in vivo and ex vivo clinical applications. (A) An in vivo method would require the injection of a targeted collagen peptide tracer into disease-affected and healthy individuals for NIR multiplex spectral imaging detection and analysis by a dedicated/customised analysis system. (B) An ex vivo approach would involve preparation of excised myocardial specimens from cardiac patients for investigation by high throughput screening, coupled with quantitative fluorescence microscopy.

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  Figure 7. Ex vivo Odyssey imaging of peptide enhancement of collagen in clarified MAL. Representative well-plates of the 2-D Li-Cor scan showing T-peptide enhancement in the MAL (A). Measurement of the T-peptide collagen enhancement in the MAL (B). Measurement of the T-peptide specificity in the enhancement at the disease level (C). Measurement of the T-peptide specificity in the enhancement at the basal level (D). Representative well-plates of the 2-D Li-Cor scan showing cyclic peptide enhancement in the MAL (E). Measurement of the cyclic peptide collagen enhancement in the MAL (F). Mean ± SEM, *p < 0.05 vs. Tg by unpaired t-test; n = 5.