Characterization of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by multiparametric quantitative cardiac magnetic resonance imaging

Figures

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  Fig. 1.. TLR-7 agonist Resiquimod induces myocarditis, thrombocytopenia and cardiac hemorrhage. Cardiac involvement in Resiquimod-treated mice was assessed macroscopically and by microscopic histopathology. (A,B) Example and quantification of macroscopic hemorrhagic lesions on hearts of Resiquimod-treated mice. (C) Representative micrographs of H&E-stained heart sections of Resiquimod-treated mice showing immune cell infiltration, edema and red blood cells in the myocardial interstitial space. Degree of RBC extravasation was scored on a scale from 0 to 3, where 0 indicates none and 3 is the most severe. (D) Full platelet count, percentage drop in platelet number from baseline, and changes in hemoglobin content in response to Resiquimod-treatment. Mann–Whitney test was used for semiquantitative scores, n=3 (ctrl), n=10 (treated); unpaired two-tailed Student's t-test for hematological analysis, n=3. Symbols represent individual animals. All data presented as mean±s.e.m. *P<0.05, ****P<0.001.

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  Fig. 2.. Native T1 and T2 tissue mapping does not conclusively detect inflammatory cardiac damage in Resiquimod-treated mice. Heart tissue damage in Resiquimod-treated mice was assessed by histology and MRI. (A) Example of H&E and Picrosirius Red-stained heart sections showing immune cell infiltration and fibrosis. (B) Example of corresponding T1w and T2w images to demonstrate segmentation (A, anterior; AL, anterior lateral; AS, anterior septum; I, inferior; IL, inferior lateral; IS, inferior septum; T1w, T1 weighted; T2w, T2 weighted). (C) Global semiquantitative score for infiltration, fibrosis and RBC extravasation on a scale from 0 to 3, where 0 indicates none and 3 is the most severe. (D) Global values for T1 and T2 values of Resiquimod-treated mice compared with healthy controls. The Mann–Whitney test was used for semiquantitative histopathology scores, n=3 (ctrl), n=10 (treated); paired Student's two-tailed t-test for longitudinal MRI values, n=7; *P<0.05. (E,F) Regional analysis depicted as pie charts performed on 12 segments at mid level per mouse, showing infiltration, fibrosis and RBC accumulation (E), and T1 and T2 (F). Shading indicates significance compared with controls;…

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  Fig. 3.. Regional T1 and T2 mapping is strongly influenced by iron deposition in the tissue. Heart tissue damage was assessed by histology and MRI. (A) T1 and T2 maps illustrating heterogeneity of T1 and T2 relaxation times with regions of low T1, T2 (A1) and high T2 (A2). (B) Corresponding areas of interest (A1 and A2) in paraffin-embedded heart sections stained with H&E, Picrosirius Red and Perls Prussian Blue. (C) Histograms of signal distribution of T1 and T2 indices from regions of interest A1 and A2.

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  Fig. 4.. T2* reveals severe cardiac iron deposition impacting on T1 and T2 measurements. Heart tissue damage was assessed by histology and MRI. (A) Global histopathological scores of RBC extravasation, iron and T2* values of Resiquimod-treated mice compared with control mice. The Mann–Whitney test was used for semiquantitative histopathology scores, n=3 (ctrl), n=10 (treated); unpaired two-tailed Student's t-test for MRI values, n=5 (ctrl), n=3 (treated); *P<0.05, **P<0.005. (B) Examples of T1, T2 and T2* maps of mid-section LV with corresponding Perls Prussian Blue-stained paraffin-embedded heart sections of Resiquimod-treated and control mice showing significant iron deposition. (C) High-magnification images of tissue areas with iron deposition (Perls Prussian Blue), immune cell infiltration (H&E) and mild fibrosis (Picrosirius Red). (D) Histograms of T1, T2 and T2* signal distribution in the anterior septum showing the impact of iron on the behavior of each relaxation time index. (E) Correlation between T2* values and individual histopathology scores. Pearson's correlation was used, pooling data from three independent experiments, n=3-5 (ctrl), n=3-10 (treated); *correlation…

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  Fig. 5.. Comparison of the CMR protocol presented in this study and the established diagnostic approach for myocarditis in human patients based on the Lake Louise criteria (LLC). LLC-based measurements in human patients using clinical MRI field strengths. Edema leads to increased T2 weighted signal and increased T2 relaxation times, suggestive of acute inflammation and/or pericardial effusion. Hyperemia leads to increased early gadolinium enhancement (EGE) caused by leakage of gadolinium from capillaries. Necrosis or fibrosis can be identified by late gadolinium enhancement (LGE) because gadolinium crosses the damaged myocyte membrane (necrosis) or accumulates in the extended extracellular space (fibrosis). Example CMR images illustrate an example of increased edema (increased T2), capillary leakage (increased EGE) and nonischemic scar (positive LGE) at the inferolateral wall of a human myocarditis patient. *Fibrosis can also nonspecifically affect T2 values; however, T2 alone is not sufficient to diagnose fibrosis but needs to be combined with T1 or LGE measurements. **LGE can be enhanced in the presence of edema, but should not be used as a marker to identify myocardial edema, which…