Mapping Lipid Oxidation in Traumatic Brain Injury by Mass Spectrometric Imaging

描述（由申请人提供）：脂质过氧化作用有助于创伤性脑损伤（TBI）继发性损伤的发展;然而，关于氧化分子靶点的基本信息在很大程度上是未知的。我们报道了两种阴离子磷脂-线粒体心磷脂（CL）和线粒体外磷脂酰丝氨酸（PS）-是TBI诱导的脑氧化的主要靶点。这些氧化反应，催化复合物的细胞色素c（cyt c）与CL和PS，与线粒体阶段的程序性细胞死亡和识别受损细胞的专业吞噬细胞，分别。对实验性TBI的研究表明，皮质、海马和丘脑选择性地易受损伤。然而，磷脂及其氧化产物在不同脑区的空间分布的信息是缺乏的。本申请的目标是通过开发和应用一种新技术-成像质谱（IMS）-来填补这一知识空白，该技术用于磷脂及其氧化产物的不同分子种类的空间和时间映射，并将其叠加到受损大脑的神经病理学上。这些信息对于靶向抗氧化治疗的设计和开发以及评估其在TBI中的疗效至关重要。我们将使用傅里叶变换离子回旋共振（FT-ICR）MS（Bruker Solarix）的高质量分辨率和测量精度，同时对数千个脂质信号进行全景快照，以获得大脑的脂质图谱。我们将通过具有改进的空间分辨率的新型IMS技术来补充这些研究：i）采用纳米尺度基质的基质辅助激光解吸电离-定位-离子迁移-正交飞行时间MS（MALDI-POST-IM-oTOFMS）; ii）过采样-激光步进MALDI-FTICR，以及iii）基质的微沉积。我们将把这些信息与荧光显微成像相结合，以揭示脆弱大脑区域的结构和代谢功能。这将是第一个全面的脂质组学，氧化脂质组学和IMS分析CL和PS在不同的大脑区域。这种使能技术将解决大脑中脂质过氧化反应的空间限制问题，否则无法容易地进行检查。我们还将使用氧化脂质组学和IMS检查从患有难治性颅内高压的TBI患者和脑库对照组织中取出的脑组织。由于脂质和氧化脂质是重要的信号分子，因此这种技术的发展和脂质生物化学的新信息应该具有广泛的根本利益。我们的进展与新的线粒体靶向电子清除剂（短杆菌肽共轭氮氧自由基）和抑制剂的细胞色素c/ CL过氧化物酶（三苯基磷共轭咪唑脂肪酸）将促进我们的能力，以重新划定的roe细胞内氧化的CL和PS在TBI。IMS用于代谢组学模式对这些小分子将揭示这些推定的神经保护剂对TBI的处置和功效的关键分子药理学信息。总体而言，IMS技术和线粒体CL和线粒体外PS的动态平衡障碍的潜在贡献可能对TBI研究很重要，并对其他CNS疾病有影响。 公共卫生相关性：严重创伤性脑损伤（TBI）是儿童和年轻人死亡的主要原因。脂质过氧化反应参与了脑外伤后继发性损伤的发展。本申请的目标是开发和应用一种新技术-成像质谱（IMS）-用于磷脂及其氧化产物的不同分子种类的空间和时间映射，并将其叠加到受损大脑的神经病理学上。这种使能技术将解决大脑中脂质过氧化反应的空间限制问题，否则无法容易地进行检查。这些信息对于靶向抗氧化疗法的设计和开发以及评估其在TBI中的疗效至关重要。将使用这些方法测试新型线粒体靶向疗法的神经保护功效。

DESCRIPTION (provided by applicant): Lipid peroxidation contributes to the evolution of secondary damage in traumatic brain injury (TBI) however; essential information on molecular targets of oxidation is largely unknown. We reported that two anionic phospholipids-mitochondrial cardiolipin (CL) and extramitochondrial phosphatidylserine (PS) - are major targets of TBI-induced oxidation in brain. These oxidation reactions, catalyzed by complexes of cytochrome c (cyt c) with CL and PS, are associated with mitochondrial stages of programmed cell death and recognition of damaged cells by professional phagocytes, respectively. Studies in experimental TBI have revealed that the cortex, hippocampus and thalamus are selectively vulnerable to injury. However, information on spatial distribution of phospholipids and their oxidation products in various brain regions is lacking. The goal of this application is to fill thi gap of knowledge by developing and applying a new technology - imaging mass spectrometry (IMS) - for spatial and temporal mapping of diverse molecular species of phospholipids and their oxidation products and superimposing them onto neuropathology of the injured brain. This information will be critical for the design and development of targeted antioxidant therapies and evaluating their efficacy in TBI. We will use the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS (Bruker Solarix) for a panoramic snap-shot of thousands of lipid signals simultaneously to obtain lipid maps of the brain. We will complement these studies by novel IMS technologies with improved spatial resolution: i) Matrix Assisted Laser Desorption Ionization-Postionization-Ion Mobility- orthogonal Time of Flight MS (MALDI-POST-IM-oTOFMS) with employment of nano-scale matrices; ii) oversampling-laser stepping MALDI-FTICR, and iii) micro-deposition of matrix. We will merge this information with fluorescent microscopic imaging to reveal structure and metabolic function of the vulnerable brain regions. This will be the first comprehensive lipidomics, oxidative lipidomics and IMS analysis of CL and PS in different brain regions. This enabling technology will resolve issues of spatial confinements of peroxidation reactions in lipids in the brain that cannot otherwise be readily examined. We will also examine brain tissue removed from TBI patients with refractory intracranial hypertension and brain-bank control tissue using oxidative lipidomics and IMS. As lipids and oxidized lipids are vital signaling molecules, the development of such technology and new information on the biochemistry of lipids should be of broad fundamental interest. Our progress with novel mitochondria targeted electron scavengers (gramicidin conjugated nitroxides) and inhibitors of cyt c/ CL peroxidase (triphenylphosphonium conjugated imidazole fatty acids) will facilitate our ability to pharmacologically delineate the roe of intracellular oxidized CL and PS in TBI. IMS used in a metabolomic mode towards these small molecules will reveal critical molecular pharmacologic information on the disposition and efficacy of these putative neuroprotectants against TBI. Overall, IMS technology and the underlying contribution of dyshomeostasis of mitochondrial CL and extramitochondrial PS are likely to be important for TBI studies and have implications for other CNS disorders. PUBLIC HEALTH RELEVANCE: Severe traumatic brain injury (TBI) is the leading cause of death in children and young adults. Lipid peroxidation contributes to evolution of secondary damage after TBI. The goal of this application is to develop and apply a new technology - imaging mass spectrometry (IMS) - for spatial and temporal mapping of diverse molecular species of phospholipids and their oxidation products and superimposing them onto neuropathology of the injured brain. This enabling technology will resolve issues of spatial confinements of peroxidation reactions in lipids in the brain that cannot otherwise be readily examined. This information is critical for the design and development of targeted antioxidant therapies and evaluating their efficacy in TBI. Novel mitochondria targeted therapies will be tested for their neuroprotective efficacy using these approaches.