Department of Surgery, Washington University in Saint Louis, Saint Louis, MO, USA.
Surgery. 2012 Aug;152(2):247-53. doi: 10.1016/j.surg.2012.02.012. Epub 2012 Jun 13.
Although inflow occlusion techniques have given surgeons the ability to carry out increasingly complex liver resections, ischemia-reperfusion (IR) injury continues to be a source of morbidity. Efforts to ameliorate IR injury have been hindered in absence of adequate preclinical models. The goal of the present study was to develop a simple, efficient, and cost-effective means of studying hepatic IR injury.
Liver cubes were procured from normal (C57BL/6) mice. After hepatectomy, 4-mm punch biopsies were taken for individual placement in culture wells containing hepatocyte media. Experimental cubes underwent hypoxia for 60 minutes, whereas controls remained normoxic. Supernatants were collected from individual wells after 0, 6, and 12 hours of rediffusion for transaminase and cytokine measurement. Histologic examination was performed on individual cubes.
Extensive histologic injury was seen in the experimental cubes compared with controls with greater staining for activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling at 6 and 24 hours, respectively. Changes consistent with ischemic injury occurred more centrally in liver cubes, whereas markers for rediffusion injury were appreciated along the periphery. Transaminases were significantly higher at 6 hours after rediffusion in experimental cubes compared with controls (P = .02). tumor necrosis factor-α and interleukin-1β were significantly higher in the media of experimental cubes compared with controls at 12 hours rediffusion (P = .05 and .03 respectively).
In vitro IR of cubes produces a significant injury with a pattern reflective of hepatic lobular architecture. This novel technique may open new avenues for uncoupling the mechanisms of IR while facilitating rapid screening of potential therapies.
尽管流入阻断技术使外科医生能够进行越来越复杂的肝切除术，但缺血再灌注（IR）损伤仍然是发病率的一个来源。由于缺乏充分的临床前模型，改善 IR 损伤的努力受到阻碍。本研究的目的是开发一种简单、高效且经济有效的方法来研究肝 IR 损伤。
从正常（C57BL/6）小鼠中获取肝块。肝切除术后，取 4mm 活检用于单独放置在含有肝细胞培养基的培养孔中。实验性肝块经历 60 分钟的缺氧，而对照物保持正常氧合。在重新扩散 0、6 和 12 小时后，从各个孔中收集上清液用于转氨酶和细胞因子测量。对各个肝块进行组织学检查。
与对照物相比，实验组的肝块广泛出现组织学损伤，在 6 和 24 小时时，分别显示出更多的激活 caspase-3 和末端脱氧核苷酸转移酶 dUTP 缺口末端标记染色。在肝块中，缺血性损伤的变化更集中在中央，而再扩散损伤的标志物则沿周边出现。与对照组相比，实验组在再扩散后 6 小时的转氨酶明显升高（P =.02）。与对照组相比，实验组在再扩散 12 小时时培养基中的肿瘤坏死因子-α和白细胞介素-1β明显升高（分别为 P =.05 和.03）。
在体外对肝块进行 IR 会导致明显的损伤，其模式反映了肝小叶的结构。这种新的技术可能为分离 IR 机制开辟新途径，同时促进潜在治疗方法的快速筛选。
