Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
J Neurosci Methods. 2010 Nov 30;193(2):232-8. doi: 10.1016/j.jneumeth.2010.08.020. Epub 2010 Sep 15.
We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.
我们评估了使用荧光探针 JC-1（5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物）监测线粒体膜电位（ΔΨ）变化的有效性，用于定量评估新生儿缺氧缺血性脑损伤。7 日龄大鼠在单侧颈总动脉结扎后接受 2 小时 8%氧气。缺氧缺血（HI）后 24 小时获取脑组织进行 JC-1 染色，并将结果与其他同时测量的结果（如荧光标记的膜联蛋白 V/碘化丙啶（PI）的流式细胞术、末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色、氯化三苯基四氮唑（TTC）梗死面积和细胞溶质细胞色素 c 的 Western blot）进行比较。HI 后 24 小时，JC-1 的流式细胞图显示出具有不同ΔΨ的两个不同亚群，红色具有高ΔΨ，绿色具有低ΔΨ。这种 JC-1 荧光从红色到绿色的转移表明ΔΨ的崩溃。JC-1 低ΔΨ的百分比增加与同时增加的膜联蛋白 V(+)/PI(+)坏死细胞、TUNEL 阳性细胞、TTC 梗死面积和细胞溶质细胞色素 c 的 Western blot 呈显著正相关，与膜联蛋白 V(-)/PI(-)活细胞呈负相关。总之，JC-1 测量的低ΔΨ与用于评估 HI 后脑损伤程度的其他方法的结果显著相关。因此，JC-1 荧光细胞术分析ΔΨ可能是定量评估新生儿脑损伤的一种敏感可靠的技术。
