Leemreis Jan R, Versteilen Amanda M G, Sipkema Pieter, Groeneveld A B Johan, Musters René J P
Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Centre (VUMC), Amsterdam, The Netherlands.
Cytometry A. 2006 Sep 1;69(9):973-8. doi: 10.1002/cyto.a.20269.
Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function.
Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phalloïdin. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans.
Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phalloïdin fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine.
The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).
受损和/或功能失调的微血管内皮与缺血性急性肾衰竭（ARF）的发病机制有关。体外观察到的内皮F-肌动蛋白细胞骨架的快速变化可能是原因，但已证明难以在原位准确测量。因此，本研究的目的是研究几种数字图像分析方法，以量化肾缺血再灌注（I/R）后内皮F-肌动蛋白的变化，并将这些变化与肾功能恶化相关联。
将假手术组和I/R大鼠肾脏的冰冻切片用4%甲醛固定，并用罗丹明-鬼笔环肽染色。使用3i Marianastrade mark数字成像荧光显微镜工作站捕获微血管结构。使用3i SlideBooktrade mark软件，采用几种掩蔽技术和线扫描对图像进行分析。
数字图像分析显示，I/R后罗丹明-鬼笔环肽荧光的平均强度从1030±187任意单位降至735±121任意单位（n = 7）。每像素F-肌动蛋白片段的数量从（15.8±4.9）×10⁻⁵增加到（20.7±3.5）×10⁻⁵（n = 7），表明细胞骨架断裂。此外，线扫描分析显示I/R后F-肌动蛋白细胞骨架的空间取向紊乱。最后，F-肌动蛋白的丢失与血浆肌酐升高相关。
本研究中描述的数字图像分析方法表明，肾I/R可诱导微血管内皮细胞F-肌动蛋白细胞骨架结构发生深刻变化，提示微血管内皮受损且功能失调，这可能导致急性肾衰竭（ARF）。
