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

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Abstract

We immunohistochemically examined the expression of IFNγ, TNFα, and TNF receptor p55 (TNF-Rp55) in a stasis-induced venous thrombus murine model. IFNγ+, TNFα+, 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, IFNγ/macrophage ratio (IFNγ/Mϕ), TNFα/macrophage ratio (TNFα/Mϕ), and TNF-Rp55/macrophage ratio (TNF-Rp55/Mϕ) were <0.2, <0.2, and <0.4, respectively. In contrast, IFNγ/Mϕ and TNFα/Mϕ were greater than 0.2 and 0.3 at ≥10 days after the IVC ligation, respectively. These observations suggested that IFNγ/Mϕ ratios of >0.2 and TNFα/Mϕ ratios of >0.3 indicated thrombus age of ≥10 days. Moreover, TNF-Rp55/Mϕ ratios of >0.7 strongly suggested thrombus age of >14 days. The present study demonstrated that the immunohistochemical detection of IFNγ, TNFα, and TNF-Rp55 was suitable to estimate the age of venous thrombi. However, the variation in TNF-Rp55/Mϕ ratios is wider than those of the other two markers. Thus, IFNγ and TNFα could be more practical markers.

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Acknowledgments

We thank Ms. Mariko Kawaguchi for her excellent assistance in the preparation of this manuscript. This study was financially supported in part by Grants-in-Aid for Scientific Research (A) and (C) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.

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Correspondence to Toshikazu Kondo.

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Nosaka, M., Ishida, Y., Kimura, A. et al. Immunohistochemical detection of intrathrombotic macrophage-derived cytokines and its application to thrombus age estimation in murine deep vein thrombosis model. Int J Legal Med 127, 937–942 (2013). https://doi.org/10.1007/s00414-013-0873-0

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  • DOI: https://doi.org/10.1007/s00414-013-0873-0

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