Molecular Magnetic Resonance Imaging of Fibrin Deposition in the Liver as an Indicator of Tissue Injury and Inflammation

Abstract

Rationale and objectives: Liver inflammation is associated with nonalcoholic steatohepatitis and other pathologies, but noninvasive methods to assess liver inflammation are limited. Inflammation causes endothelial disruption and leakage of plasma proteins into the interstitial space and can result in extravascular coagulation with fibrin deposition. Here we assess the feasibility of using the established fibrin-specific magnetic resonance probe EP-2104R for the noninvasive imaging of fibrin as a marker of liver inflammation.

Methods: Weekly 100 mg/kg diethylnitrosamine (DEN) dosing was used to generate liver fibrosis in male rats; control animals received vehicle. Magnetic resonance imaging at 1.5 T with EP-2104R, a matched non-fibrin-binding control linear peptide, or the collagen-specific probe EP-3533 was performed at 1 day or 7 days after the last DEN administration. Imaging data were compared with quantitative histological measures of fibrosis and inflammation.

Results: After 4 or 5 DEN administrations, the liver becomes moderately fibrotic, and fibrosis is the same if the animal is killed 1 day (Ishak score, 3.62 ± 0.31) or 7 days (Ishak score, 3.82 ± 0.25) after the last DEN dose, but inflammation is significantly higher at 1 day compared with 7 days after the last DEN dose (histological activity index from 0-4, 3.54 ± 0.14 vs 1.61 ± 0.16, respectively; P < 0.0001). Peak EP-2104R signal enhancement was significantly higher in animals imaged at 1 day post-DEN compared with 7 days post-DEN or control rats (29.0% ± 3.2% vs 22.4% ± 2.0% vs 17.0% ± 0.2%, respectively; P = 0.017). Signal enhancement with EP-2104R was significantly higher than control linear peptide at 1 day post-DEN but not at 7 days post-DEN indicating specific fibrin binding during the inflammatory phase. Collagen molecular magnetic resonance with EP-3533 showed equivalent T1 change when imaging rats 1 day or 7 days post-DEN, consistent with equivalent fibrosis.

Conclusions: EP-2104R can specifically detect fibrin associated with inflammation in a rat model of liver inflammation and fibrosis.

Figure 1.

Histological analysis of liver specimens from rats given 4 weekly i.p. doses of PBS (left) or 100 mg/kg of DEN and sacrificed at 1 day (middle) or 7 days (right) following the last DEN administration, scale bar = 250 μm. (A) Hematoxylin and eosin (H&E) staining of liver tissue. Inflammatory regions are labeled with pointed black arrows. (B) Sirius Red staining of liver tissue. Fibrosis is shown by intense red staining of collagen fibers. (C) Immunohistochemistry staining of fibrin in liver tissue. (D) Morphometric analysis of Sirius Red stained slides to give collagen proportional area showing moderate fibrosis in both DEN groups that is significantly higher than in PBS controls. (E) Ishak scoring of liver fibrosis showing moderate fibrosis in both DEN groups that is significantly higher than in PBS controls. (F) Histological activity index for inflammation (0 – 4) showing that inflammation is significantly higher at 1 day post DEN than 7 days post DEN. (G) Morphometric analysis of the area of the slide stained positive for fibrin by immunohistochemistry. ** P<0.01, *** P<0.0001, one way ANOVA with post hoc Tukey test.

Figure 2.

(A) Representative T1-weighted axial images of rats dosed with PBS (left), with DEN and imaged 7 days after 4^th DEN dose (middle), and imaged one day after 4^th DEN dose (right). False color overlay represents subtraction image of pre-contrast image from the one minute post EP-2104R image. Peak liver signal enhancement (%ΔSI) at 1 min post injection of EP-2104R (B) and liver signal enhancement area under the curve (C) are significantly higher in the DEN treated rats than in PBS controls. * P<0.05, one way ANOVA with post hoc Tukey test.

Figure 3.

Representative liver signal enhancement versus time post probe injection for EP-2014R (filled squares) and CLP (open circles) in rats imaged 7 days (A) or 1 day (D) post the last DEN dose. Peak (at 1 minute post injection) liver %ΔSI (B) and liver AUC (C) are not significantly different for EP-2104R and control probe CLP at 7 days post DEN. At 1 day post DEN, both peak liver (at 1 minute post injection) %ΔSI (E) and liver AUC (F) are significantly higher for EP-2104R indicating elevated fibrin levels during the inflammatory phase which results in increased relaxivity of EP-2104R and increased retention in liver tissue. * P<0.05, two tailed t-test.

Figure 4.

(A) ΔR1 of liver after administration of collagen-targeted EP-3533 measured 1 day or 7 days following the last DEN administration. EP-3533 reports on fibrosis and this data suggests no change in fibrotic burden after one week of withdrawal from DEN as expected. Peak EP-2104R liver signal enhancement (B) and EP-2104R liver signal enhancement AUC (C) are significantly decreased from 1 day post DEN to 7 days post DEN in these animals. * P<0.05, two tailed t-test.