Levine Glenn K, Deutschman Clifford S, Helfaer Mark A, Margulies Susan S
Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, and Department of Anesthesia, University of Pennsylvania School of Medicine, Philadelphia 19104-6392, USA.
Crit Care Med. 2006 Jun;34(6):1746-51. doi: 10.1097/01.CCM.0000218813.77367.E2.
Previous in vitro models have shown that cellular deformation causes dose-dependent injury and death in healthy rat alveolar epithelial cells (AECs). We compared the viability of AECs from septic rats with those from nonseptic rats after 1 hr of cyclic equibiaxial stretch. We hypothesized that sepsis would increase stretch-induced cell death.
Laboratory investigation.
University research laboratory.
Thirty-seven male Sprague-Dawley rats weighing 240-260 g.
Anesthetized rats were subjected to cecal ligation and double puncture (2CLP) or sham laparotomy without cecal ligation or puncture (sham). After 24 or 48 hrs, AECs were isolated, seeded in custom wells, and maintained in culture for 48 hrs before study. AECs were stretched cyclically (15/min) to a 0%, 12%, 25%, or 37% change in surface area (DeltaSA) for 1 hr. Cell viability, phenotypic markers, and nuclear factor-kappaB intracellular localization were assessed using fluorescent immunocytochemistry.
Phase and fluorescent images were evaluated for all studies. Response to stretch was the same at 24 and 48 hrs after 2CLP. Relative to sham, 2CLP significantly increased cell death at 25 and 37% DeltaSA (p<.003, analysis of variance). Relative to sham, 2CLP did not alter expression of type I or type II phenotypic markers. Nuclear factor-kappaB within the nuclear compartment was observed after 2CLP in unstretched cells and after 1 hr of cyclic stretch at 37% DeltaSA. In sham, nuclear factor-kappaB within the nuclear compartment was seen only after stretch.
AECs isolated from septic rats are more vulnerable to mechanical deformation injury than AECs from nonseptic animals.
先前的体外模型表明，细胞变形会导致健康大鼠肺泡上皮细胞（AECs）出现剂量依赖性损伤和死亡。我们比较了循环等双轴拉伸1小时后，脓毒症大鼠与非脓毒症大鼠的AECs活力。我们假设脓毒症会增加拉伸诱导的细胞死亡。
实验室研究。
大学研究实验室。
37只体重240 - 260克的雄性Sprague-Dawley大鼠。
将麻醉后的大鼠进行盲肠结扎和双穿刺（2CLP），或进行不进行盲肠结扎或穿刺的假剖腹手术（假手术）。24或48小时后，分离AECs，接种于定制孔中，并在研究前在培养中维持48小时。将AECs以15次/分钟的频率循环拉伸，使表面积变化（ΔSA）为0%、12%、25%或37%，持续1小时。使用荧光免疫细胞化学评估细胞活力、表型标志物和核因子-κB细胞内定位。
对所有研究的相位和荧光图像进行评估。2CLP后24和48小时对拉伸的反应相同。相对于假手术组，2CLP在ΔSA为25%和37%时显著增加细胞死亡（方差分析，p<0.003）。相对于假手术组，2CLP未改变I型或II型表型标志物的表达。在2CLP组中，未拉伸细胞以及在ΔSA为37%的情况下循环拉伸1小时后，观察到核内有核因子-κB。在假手术组中，仅在拉伸后观察到核内有核因子-κB。
从脓毒症大鼠分离的AECs比非脓毒症动物的AECs更容易受到机械变形损伤。
