Immunohistochemical detection of intrathrombotic macrophage-derived cytokines and its application to thrombus age estimation in murine deep vein thrombosis model.

Abstract

We immunohistochemically examined the expression of IFN&#x3b3;, TNF&#x3b1;, and TNF receptor p55 (TNF-Rp55) in a stasis-induced venous thrombus murine model. IFN&#x3b3;(+), TNF&#x3b1;(+), and TNF-Rp55(+) cells could be first detected 7, 3, and 3 days after inferior vena cava (IVC) ligation. Thereafter, the numbers of these positive cells increased with time after IVC ligation. Double-color immunofluorescence analyses demonstrated that each molecule was expressed by intrathrombotic macrophages. In all samples with postligation intervals of 7 days or less, IFN&#x3b3;/macrophage ratio (IFN&#x3b3;/M&#x3d5;), TNF&#x3b1;/macrophage ratio (TNF&#x3b1;/M&#x3d5;), and TNF-Rp55/macrophage ratio (TNF-Rp55/M&#x3d5;) were <0.2, <0.2, and <0.4, respectively. In contrast, IFN&#x3b3;/M&#x3d5; and TNF&#x3b1;/M&#x3d5; were greater than 0.2 and 0.3 at &#x2265;10 days after the IVC ligation, respectively. These observations suggested that IFN&#x3b3;/M&#x3d5; ratios of >0.2 and TNF&#x3b1;/M&#x3d5; ratios of >0.3 indicated thrombus age of &#x2265;10 days. Moreover, TNF-Rp55/M&#x3d5; ratios of >0.7 strongly suggested thrombus age of >14 days. The present study demonstrated that the immunohistochemical detection of IFN&#x3b3;, TNF&#x3b1;, and TNF-Rp55 was suitable to estimate the age of venous thrombi. However, the variation in TNF-Rp55/M&#x3d5; ratios is wider than those of the other two markers. Thus, IFN&#x3b3; and TNF&#x3b1; could be more practical markers.