Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
Department of Cardiovascular Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.
Int J Legal Med. 2020 May;134(3):1061-1066. doi: 10.1007/s00414-019-02168-0. Epub 2019 Nov 12.
We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To perform the immunohistochemical analyses, we used anti-LC3 antibody and anti-p62 antibody for detecting the intrathrombotic autophagic functions. We estimated dynamics of the intrathrombotic autophagy as LC3 cell number (×1000, five fields) with the thrombus ages (each group n = 5). The number of LC3 cells was once decreased at 3 days, and then increased until 10 days. On the contrary, the number of p62 cells progressively increased until 10 days after the inferior vena cava (IVC) ligation, and then gradually decreased. Especially, in all of thrombus samples with the postligation intervals of 5-10 days, both numbers were larger than 10. Subsequently, we compared the number of LC3 cells to that of p62 cells. Although, at 1 day after the IVC ligation, LC3 cell number significantly exceeded p62 cell number, the former was significantly or relatively less than the latter at 3 days or more after the IVC ligation. Thus, positive cells of > 10 in both LC3 and p62 indicated the thrombus age of 5-10 days. Upon comparison of immunopositive cells in LC3 and p62, the p62/LC3 ratio was > 1.0 in 29 out of 30 thrombus samples aged 3-21 days, and all of 1-day-old thrombus had the p62/LC3 ratio of < 0.5. Thus, the ratio of > 1.0 and that of < 0.5 could indicate thrombus age of 3 days or more and that of 1 day, respectively. Collectively, our study implied that the detection of autophagy-related molecules such as LC3 and p62 would be useful for the determination of thrombus age.
我们使用小鼠深静脉血栓形成 (DVT) 模型，通过免疫组织化学方法检查血栓形成过程中的自噬的血栓内动态变化。为了进行免疫组织化学分析，我们使用抗 LC3 抗体和抗 p62 抗体来检测血栓内自噬功能。我们根据血栓形成的年龄来估计血栓内自噬的动态变化，将 LC3 细胞数（×1000，5 个视野）作为指标（每组 n = 5）。LC3 细胞数在 3 天内首次减少，然后增加到 10 天。相反，p62 细胞数在结扎下腔静脉 (IVC) 后逐渐增加，直到 10 天，然后逐渐减少。特别是在所有结扎后 5-10 天的血栓样本中，这两个数值都大于 10。随后，我们比较了 LC3 细胞数和 p62 细胞数。尽管在 IVC 结扎后 1 天，LC3 细胞数明显超过 p62 细胞数，但在 IVC 结扎后 3 天或更长时间，前者明显或相对小于后者。因此，LC3 和 p62 均为阳性细胞> 10 表示血栓形成年龄为 5-10 天。比较 LC3 和 p62 免疫阳性细胞，在年龄为 3-21 天的 30 个血栓样本中有 29 个的 p62/LC3 比值> 1.0，所有 1 天的血栓的 p62/LC3 比值均<0.5。因此，比值> 1.0 和<0.5 可分别指示血栓形成年龄为 3 天或以上和 1 天。总的来说，我们的研究表明，检测自噬相关分子如 LC3 和 p62 将有助于确定血栓形成的年龄。
