Cold ischemia time alters cell-type specific senescence leading to loss of cellular integrity in mouse lungs

<b>Purpose:</b> Ischemia-reperfusion injury (IRI) is a major challenge in lung transplantation often causing graft dysfunction and chronic airway illnesses in recipients. To prevent potential transplant related complications, strict guidelines were put in place to choose viable donor lungs with minimal risk of IRI. These regulations deem most of the donor organs unfit for transplant which then are donated for research to understand the mechanisms of health and diseases in human. However, resected organs that are being transported undergo cold ischemia that can negatively affect the tissue architecture and other cellular functions under study. Thus, it is important to assess how cold ischemia time (CIT) affects the physiological mechanism. In this respect, we are interested in studying how CIT affects cellular senescence in normal aging and various pulmonary pathologies. We thus hypothesized that prolonged CIT exhibits cell-type specific changes in lung cellular senescence in mice. <b>Methods:</b> Lung lobes from C57BL/6J (<i>n</i> = 5–8) mice were harvested and stored in UW Belzer cold storage solution for 0, 4-, 9-, 12-, 24-, and 48-h CIT. Lung cellular senescence was determined using fluorescence (C₁₂FdG) assay and co-immunolabelling was performed to identify changes in individual cell types. <b>Results:</b> We found a rapid decline in the overall lung cellular senescence after 4-h of CIT in our study. Co-immunolabelling revealed the endothelial cells to be most affected by cold ischemia, demonstrating significant decrease in the endothelial cell senescence immediately after harvest. Annexin V-PI staining further revealed a prominent increase in the number of necrotic cells at 4-h CIT, thus suggesting that most of the cells undergo cell death within a few hours of cold ischemic injury. <b>Conclusions:</b> We thus concluded that CIT significantly lowers the cellular senescence in lung tissues and must be considered as a confounding factor for mechanistic studies in the future.

研究背景与目的：缺血再灌注损伤（Ischemia-reperfusion injury, IRI）是肺移植领域的重大挑战，常导致受者出现移植肺功能障碍与慢性气道疾病。为防范移植相关并发症，临床已建立严格指南，以筛选缺血再灌注损伤风险最低的合格供肺。此类判定标准导致多数供体器官被认定不适用于移植，转而用于医学研究，以探究人类健康与疾病的发生机制。然而，术中切除并处于运输过程中的供肺会经历冷缺血过程，这可能对后续研究中的组织结构与细胞功能产生负面影响。因此，评估冷缺血时间（cold ischemia time, CIT）对生理机制的影响至关重要。本研究旨在探讨冷缺血时间在正常衰老及多种肺部病理状态下对细胞衰老的调控作用，并提出假说：延长冷缺血时间会在小鼠肺组织中引发细胞类型特异性的细胞衰老变化。
实验方法：选取C57BL/6J品系小鼠（n=5~8），摘取其肺叶，置于UW Belzer冷保存液中，分别进行0、4、9、12、24及48小时的冷缺血处理。采用荧光（C₁₂FdG）检测法测定肺组织细胞衰老水平，并通过免疫荧光双标技术鉴定不同细胞类型的衰老变化情况。
实验结果：本研究发现，冷缺血处理4小时后，肺组织整体细胞衰老水平迅速下降。免疫荧光双标结果显示，内皮细胞受冷缺血影响最为显著，取材后即刻即可观察到内皮细胞衰老水平显著降低。Annexin V-PI染色进一步证实，冷缺血4小时后坏死细胞数量显著增加，提示多数细胞在冷缺血损伤后的数小时内即发生细胞死亡。
研究结论：综上，冷缺血时间可显著降低肺组织细胞衰老水平，未来的机制研究需将其作为潜在混杂因素纳入考量。