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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Nosaka M, Ishida Y, Kimura A, Kondo T (2009) Time-dependent appearance of intrathrombus neutrophils and macrophages in a stasis-induced deep vein thrombosis model and its application to thrombus age determination. Int J Legal Med 123:235–240
Nosaka M, Ishida Y, Kuninaka Y, Kimura A, Kondo T (2012) Immunohistochemical detection of uPA, tPA, and PAI-1 in a stasis-induced deep vein thrombosis model and its application to thrombus age estimation. Int J Legal Med 126:421–425
Nosaka M, Ishida Y, Kimura A, Kondo T (2010) Immunohistochemical detection of MMP-2 and MMP-9 in a stasis-induced deep vein thrombosis model and its application to thrombus age estimation. Int J Legal Med 124:439–444
Nosaka M, Ishida Y, Kimura A, Kondo T (2010) Time-dependent organic changes of intravenous thrombi in stasis-induced deep vein thrombosis model and its application to thrombus age determination. Forensic Sci Int 195:143–147
Nosaka M, Ishida Y, Kimura A, Kuninaka Y, Inui M, Mukaida N, Kondo T (2011) Absence of IFN-gamma accelerates thrombus resolution through enhanced MMP-9 and VEGF expression in mice. J Clin Invest 121:2911–2920
Ishida Y, Kimura A, Kuninaka Y, Inui M, Matsushima K, Mukaida N, Kondo T (2012) Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing. J Clin Invest 122:711–721
Ishida Y, Kondo T, Kimura A, Matsushima K, Mukaida N (2006) Absence of IL-1 receptor antagonist impaired wound healing along with aberrant NF-κB activation and a reciprocal suppression of TGF-beta signal pathway. J Immunol 176:5598–5606
Ishida Y, Kondo T, Takayasu T, Iwakura Y, Mukaida N (2004) The essential involvement of cross-talk between IFN-γ and TGF-β in the skin wound-healing process. J Immunol 172:1848–1855
Lin ZQ, Kondo T, Ishida Y, Takayasu T, Mukaida N (2003) Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. J Leukoc Biol 73:713–721
Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N (2002) Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 16:963–974
Cecchi R (2010) Estimating wound age: looking into the future. Int J Legal Med 124:523–536
Zheng JL, Yu TS, Li XN, Fan YY, Ma WX, Du Y, Zhao R, Guan DW (2012) Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice. Int J Legal Med 126:807–814
Ishida Y, Kimura A, Nosaka M, Kuninaka Y, Takayasu T, Eisenmenger W, Kondo T (2012) Immunohistochemical analysis on cyclooxygenase-2 for wound age determination. Int J Legal Med 126:435–440
Ma WX, Yu TS, Fan YY, Zhang ST, Ren P, Wang SB, Zhao R, Pi JB, Guan DW (2011) Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice. Int J Legal Med 125:549–558
Sun JH, Nan LH, Gao CR, Wang YY (2012) Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR. Int J Legal Med 126:113–120
Kondo T, Ishida Y (2010) Molecular pathology of wound healing. Forensic Sci Int 203:93–98
Kondo T, Ohshima T (1996) The dynamics of inflammatory cytokines in the healing process of mouse skin wound: a preliminary study for possible wound age determination. Int J Legal Med 108:231–236
Kondo T (2007) Timing of skin wounds. Leg Med (Tokyo) 9:109–114
Fineschi V, Turillazzi E, Neri M, Pomara C, Riezzo I (2009) Histological age determination of venous thrombosis: a neglected forensic task in fatal pulmonary thrombo-embolism. Forensic Sci Int 186:22–28
Farrar MA, Schreiber RD (1993) The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 11:571–611
Tracey KJ (1994) Tumor necrosis factor-alpha. In: Thomson A (ed) The cytokine handbook, 2nd edn. Academic, London, pp 289–304
Lewis M, Tartaglia LA, Lee A, Bennett GL, Rice GC, Wong GH, Chen EY, Goeddel DV (1991) Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc Natl Acad Sci U S A 88:2830–2834
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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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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