The application of neutrophil extracellular traps to thrombus age Estimation in rat deep vein thrombosis model

In experimental models of deep vein thrombosis (DVT), neutrophil extracellular traps (NETs) have been established as pivotal mediators in the initial phases of thrombus formation. Through the establishment of a chromatin-based scaffold, NETs provide a structural foundation that enhances platelet adhesion. Furthermore, they significantly contribute to the perpetuation of a self-amplifying cycle of venous endothelial cell injury, thereby exacerbating thrombogenesis. While extant research has predominantly concentrated on the role of NETs in the initiation of DVT, there remains a paucity of investigation into the temporal dynamics of NETs content across the sequential stages of thrombus development, including formation, elongation, organization, and recanalization. The present study elucidates the forensic application of NETs for temporal assessment of thrombus age in cases of sudden death resulting from pulmonary embolism secondary to deep vein thrombosis. This was accomplished through the establishment of a time-gradient DVT model in a rat model system. Utilizing myeloperoxidase (MPO) antibody, a well-established marker of neutrophil activation, in conjunction with CitH3 histone antibody, a specific marker for neutrophil extracellular traps, we performed dual immunofluorescence co-localization studies. These investigations confirmed the integral involvement of NETs in the thrombotic process. Complementary Western blot analyses demonstrated temporal variations in NETs content within the thrombotic mass. The CitH3 levels exhibited no significant elevation during the initial 3-6 h post-ligation period, followed by a gradual increase after 24 h, reaching maximal expression on day 3. Subsequently, a progressive decline was observed, culminating in complete resolution by day 21. Quantitative analysis of positive cell counts (×1000, across five representative fields) at sequential time points revealed distinct temporal patterns. During the initial 3-hour to 1-day post-ligation period, MPO-positive cells demonstrated a significantly more rapid increase compared to CitH3-positive cells. By day 3, MPO levels commenced a decline, ultimately falling below CitH3 levels. Subsequent analysis of the CitH3/MPO ratio yielded significant forensic implications. Our findings demonstrate that a CitH3/MPO ratio approximating 1.0 corresponds to a thrombus formation time within 5 days. The degree of ratio variation among multiple samples serves as a temporal indicator: minimal variation (approaching 1.0) suggests a shorter thrombus formation interval, while ratios exceeding 2.0 or demonstrating substantial variation are indicative of thrombus formation times surpassing 7 days.