Mapping Lipid Oxidation in Traumatic Brain Injury by Mass Spectrometric Imaging

描述（由申请人提供）：脂质过氧化有助于创伤性脑损伤（TBI）的继发性损伤的演变；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) -是脑外伤诱导氧化的主要靶点。这些氧化反应由细胞色素c （cyt c）与CL和PS的复合物催化，分别与程序性细胞死亡的线粒体阶段和专业吞噬细胞对受损细胞的识别有关。实验性脑外伤研究表明，皮层、海马体和丘脑有选择性地易受损伤。然而，关于磷脂及其氧化产物在大脑各区域的空间分布信息缺乏。本应用程序的目标是通过开发和应用一种新技术-成像质谱法（IMS）来填补这一知识空白，该技术用于对磷脂及其氧化产物的不同分子种类进行时空映射，并将其叠加到受伤大脑的神经病理学上。这一信息对于设计和开发靶向抗氧化疗法以及评估其在创伤性脑损伤中的疗效至关重要。我们将利用傅立叶变换离子回旋共振（FT-ICR）质谱仪（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。我们将通过提高空间分辨率的新型IMS技术来补充这些研究：i)矩阵辅助激光解吸电离-定位-离子迁移-正交飞行时间质谱（MALDI-POST-IM-oTOFMS），采用纳米级矩阵；ii)过采样-激光步进MALDI-FTICR， iii)基质微沉积。我们将把这些信息与荧光显微成像相结合，以揭示大脑脆弱区域的结构和代谢功能。这将是首次对不同脑区CL和PS进行全面的脂质组学、氧化脂质组学和IMS分析。这项技术将解决大脑中脂质过氧化反应的空间限制问题，否则无法轻易检查。我们还将使用氧化脂组学和IMS检查从顽固性颅内高压TBI患者和脑库对照组织中切除的脑组织。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关于脂质生物化学的新信息应该引起广泛的根本兴趣。我们在新的线粒体靶向电子清除剂（gramicidin偶联氮氧化物）和cyt c/ CL过氧化物酶抑制剂（三苯膦偶联咪唑脂肪酸）方面的进展将有助于我们从药理学上描述细胞内氧化CL和PS在TBI中的作用。IMS在代谢组学模式下用于这些小分子，将揭示这些假定的神经保护剂对TBI的处置和功效的关键分子药理学信息。总的来说，IMS技术和线粒体CL和线粒体外PS失衡的潜在贡献可能对TBI研究很重要，对其他中枢神经系统疾病也有影响。

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.