Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15213, USA.
Free Radic Biol Med. 2012;52(11-12):2312-9. doi: 10.1016/j.freeradbiomed.2012.04.011. Epub 2012 Apr 21.
Assessment of tissue free radical production is routinely accomplished by measuring secondary by-products of redox reactions and/or diminution of key antioxidants such as reduced thiols. However, immuno-spin trapping, a newly developed immunohistochemical technique for detection of free radical formation, is garnering considerable interest as it allows for the visualization of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-adducted molecules. Yet, to date, immuno-spin trapping reports have utilized in vivo models in which successful detection of free radical adducts required exposure to lethal levels of oxidative stress not reflective of chronic inflammatory disease. To study the extents and anatomic locations of more clinically relevant levels of radical formation, we examined tissues from high-fat (HF) diet-fed mice, a model of low-grade chronic inflammation known to demonstrate enhanced rates of reactive species production. Mice subjected to 20 weeks of HF diet displayed increased free radical formation (anti-DMPO mean fluorescence staining) in skeletal muscle (0.863±0.06 units vs 0.512±0.07 units), kidney (0.076±0.0036 vs 0.043±0.0025), and liver (0.275±0.012 vs 0.135±0.014) compared to control mice fed normal laboratory chow (NC). Western blot analysis of tissue homogenates confirmed these results showing enhanced DMPO immunoreactivity in HF mice compared to NC samples. The obesity-related results were confirmed in a rat model of pulmonary hypertension and right heart failure in which intense immunodetectable radical formation was observed in the lung and right ventricle of monocrotaline-treated rats compared to saline-treated controls. Combined, these data affirm the utility of immuno-spin trapping as a tool for in vivo assessment of altered extents of macromolecule oxidation to radical intermediates under chronic inflammatory conditions.
组织中自由基生成的评估通常通过测量氧化还原反应的次级副产物和/或关键抗氧化剂（如还原型硫醇）的减少来完成。然而，免疫自旋捕获，一种新开发的用于检测自由基形成的免疫组织化学技术，由于允许可视化 5,5-二甲基-1-吡咯啉 N-氧化物（DMPO）加合物分子，因此引起了相当大的兴趣。然而，迄今为止，免疫自旋捕获报告已经利用了体内模型，在这些模型中，成功检测自由基加合物需要暴露于不反映慢性炎症性疾病的致死水平的氧化应激下。为了研究更具临床相关性的自由基形成程度和解剖位置，我们检查了高脂肪（HF）饮食喂养的小鼠组织，该模型是一种已知具有增强活性物种产生率的低水平慢性炎症模型。接受 20 周 HF 饮食的小鼠在骨骼肌（抗 DMPO 平均荧光染色：0.863±0.06 单位比 0.512±0.07 单位）、肾脏（0.076±0.0036 比 0.043±0.0025）和肝脏（0.275±0.012 比 0.135±0.014）中显示出自由基形成增加（与对照相比），而对照小鼠则喂食正常实验室饲料（NC）。组织匀浆的 Western blot 分析证实了这些结果，表明 HF 小鼠中的 DMPO 免疫反应性增强。在肺动脉高压和右心衰竭的大鼠模型中，肥胖相关结果得到了证实，其中在给予单硝酸异山梨酯的大鼠的肺和右心室中观察到强烈的免疫可检测自由基形成，而在给予生理盐水的对照中则没有。综上所述，这些数据证实了免疫自旋捕获作为一种工具，可用于在慢性炎症条件下评估大分子氧化为自由基中间体的改变程度。
