Stamenković Stefan, Pavićević Aleksandra, Mojović Miloš, Popović-Bijelić Ana, Selaković Vesna, Andjus Pavle, Bačić Goran
University of Belgrade - Faculty of Biology, Center for Laser Microscopy, Studentski trg 3, 11158 Belgrade, Serbia.
University of Belgrade - Faculty of Physical Chemistry, EPR Laboratory, Studentski trg 12-16, 11158 Belgrade, Serbia.
Free Radic Biol Med. 2017 Jul;108:258-269. doi: 10.1016/j.freeradbiomed.2017.03.034. Epub 2017 Mar 31.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting the motor pathways of the central nervous system. Although a number of pathophysiological mechanisms have been described in the disease, post mortem and animal model studies indicate blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. In this study, the BBB permeability and the brain tissue redox status of the SOD1 ALS rat model in the presymptomatic (preALS) and symptomatic (ALS) stages of the disease were investigated by in vivo EPR spectroscopy using three aminoxyl radicals with different cell membrane and BBB permeabilities, Tempol, 3-carbamoyl proxyl (3CP), and 3-carboxy proxyl (3CxP). Additionally, the redox status of the two brain regions previously implicated in disease pathology, brainstem and hippocampus, was investigated by spectrophotometric biochemical assays. The EPR results indicated that among the three spin probes, 3CP is the most suitable for reporting the intracellular redox status changes, as Tempol was reduced in vivo within minutes (t =2.0±0.5min), thus preventing reliable kinetic modeling, whereas 3CxP reduction kinetics gave divergent conclusions, most probably due to its membrane impermeability. It was observed that the reduction kinetics of 3CP in vivo, in the head of preALS and ALS SOD1 rats was altered compared to the controls. Pharmacokinetic modeling of 3CP reduction in vivo, revealed elevated tissue distribution and tissue reduction rate constants indicating an altered brain tissue redox status, and possibly BBB disruption in these animals. The preALS and ALS brain tissue homogenates also showed increased nitrilation, superoxide production, lipid peroxidation and manganese superoxide dismutase activity, and a decreased copper-zinc superoxide dismutase activity. The present study highlights in vivo EPR spectroscopy as a reliable tool for the investigation of changes in BBB permeability and for the unprecedented in vivo monitoring of the brain tissue redox status, as early markers of ALS.
肌萎缩侧索硬化症（ALS）是一种影响中枢神经系统运动通路的致命性神经退行性疾病。尽管该疾病已被描述了多种病理生理机制，但尸检和动物模型研究表明，血脑屏障（BBB）破坏和活性氧生成增加是疾病病理的主要促成因素。在本研究中，使用三种具有不同细胞膜和血脑屏障通透性的氨氧自由基（Tempol、3-氨甲酰基丙酰自由基（3CP）和3-羧基丙酰自由基（3CxP）），通过体内电子顺磁共振光谱法研究了SOD1 ALS大鼠模型在疾病症状前（preALS）和症状期（ALS）血脑屏障的通透性以及脑组织的氧化还原状态。此外，通过分光光度生化测定法研究了先前与疾病病理相关的两个脑区（脑干和海马体）的氧化还原状态。电子顺磁共振结果表明，在这三种自旋探针中，3CP最适合用于报告细胞内氧化还原状态的变化，因为Tempol在体内几分钟内（t =2.0±0.5分钟）就被还原，从而无法进行可靠的动力学建模，而3CxP的还原动力学得出了不同的结论，很可能是由于其膜不透性。观察到，与对照组相比，preALS和ALS SOD1大鼠头部3CP在体内的还原动力学发生了改变。3CP在体内还原的药代动力学建模显示，组织分布和组织还原速率常数升高，表明这些动物的脑组织氧化还原状态发生了改变，并且可能存在血脑屏障破坏。preALS和ALS脑组织匀浆还显示出腈化增加、超氧化物生成增加、脂质过氧化增加和锰超氧化物歧化酶活性增加，而铜锌超氧化物歧化酶活性降低。本研究强调了体内电子顺磁共振光谱法是一种可靠的工具，可用于研究血脑屏障通透性的变化以及前所未有的脑组织氧化还原状态的体内监测，作为ALS的早期标志物。
