Time-dependent organic changes of intravenous thrombi in stasis-induced deep vein thrombosis model and its application to thrombus age determination

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Abstract

Using histochemical and immunohistochemical techniques, we examined the intrathrombotic collagen contents and the appearance of hemosiderin-positive cells, neovessels, and myofibroblasts in a stasis-induced venous thrombosis model. The intrathrombotic collagen deposition area occupied about 20% at 5 days, and exceeded 80% at 21 days after ligation of the inferior vena cava (IVC). Hemosiderin-positive cells in the thrombus first appeared at 3 days in only one of the five samples, and positive cells were constantly detected in all thrombi at 5 days or later. CD31-positive neovessels in the thrombus first appeared at 5 days in one of five samples and were detected in all samples after 10 days. At 7 days, αSMA-positive myofibroblasts at the periphery of the thrombus first appeared in three of five samples, and were detected and enhanced time-dependently in all samples after 10 days. These observations demonstrated that these markers would be applicable for thrombus age determination.

Introduction

Recently, medical treatment for thrombosis has progressed markedly and much information regarding the formation mechanism of thrombosis has been accumulated [1], [2], [3], [4], [5], [6], [7]. In forensic practice, pulmonary thromboembolism is one of the major causes of sudden unexpected death; however, further forensic studies on deep vein thrombosis and pulmonary thromboembolism are necessary from the aspects of pathophysiology and etiology [8], [9].

Several lines of accumulating evidence have established a model of stasis-induced venous thrombi in rodents [10], [11], [12]. We and other groups have conducted several studies on the age determination of skin wounds [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]; similarly, it is important to estimate thrombus age. The immunohistochemical detection of neutrophils and macrophages in venous thrombi could be presumed to give significant information for age determination of venous thrombi [8]. In addition to neutrophils and macrophages, the dynamics of other types of cells and extracellular matrix is essentially involved in the formation and resolution of venous thrombi. Thus, in the present study, intrathrombotic collagen contents, and the appearance of hemosiderin-positive cells, myofibroblasts and neovessels were examined using histochemical and immunohistochemical techniques.

Section snippets

Antibodies (Abs)

The following monoclonal Abs (mAbs) were used in this study: mouse anti-human α-smooth muscle actin (αSMA) mAb (clone 1A4; Dako Cytomation, Kyoto, Japan), and rat anti-mouse CD31 mAb (Pharmingen, San Diego, CA).

Mice

Specific pathogen-free 8–10-week-old male mice were obtained from SLC (Shizuoka, Japan). All mice were housed individually in cages under specific pathogen-free conditions during experiments. All animal experiments were approved by the Committee on Animal Care and Use of Wakayama Medical

Intrathrombotic appearance of collagen deposition and hemosiderin-positive cells

As reported previously [8], IVC ligation caused intravenous thrombus formation posterior to the ligated point in a time-dependent manner. Until 3 days after IVC ligation, organic changes could not be detected. Thus, at 1 and 3 days after IVC ligation, the collagen-positive area was hardly detected in all thrombi; thereafter, the collagen-positive area started from the peripheral area of the thrombi (Fig. 1a–d). Five days after ligation, the collagen content area reached about 20%, and,

Discussion

There have been many forensic studies on the age estimation of skin wounds or brain contusions [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [26], [27], [28], [29], [30], [31], [32], [33] and those on postmortem diagnosis [34], [35], [36], [37] with the development of immunohistochemistry. However, to the best of our knowledge, there have been only two forensic studies on thrombus age estimation using immunostaining [8], [38]. Various cells, such as red blood cells,

Acknowledgements

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-Aids for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.

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