Hypothermia, anesthesia and NMR metabolomics in ischemic neonatal brain slices

描述（由申请人提供）：最近的大型临床试验发现，治疗性低温可以改善窒息新生儿的神经功能结局。关键的，未回答的问题涉及最佳冷却温度，持续时间和复温方案。我们将制定一个代谢组学的方法，这些问题使用灌流新生儿（P7）啮齿动物脑切片与氧-葡萄糖剥夺（OGD）模型窒息。治疗时间尺度（小时）将远小于临床实践中发生的时间尺度（天）。具体目标#1a是使用多变量分析定量和比较在OGD前后以及低温抢救方案前后取出的切片的高氯酸（PCA）浸提液中发现的1H和31 P代谢物的集合。常温OGD对照组也将用于比较。目的#1b是对与目的#1a中取出的切片同时采集的单独切片进行组织学和免疫组织学评价。每个NMR代谢物组将由20个1H TCA循环代谢物和3个31 P代谢物（ATP、ADP和PCr）组成，并通过14.1 T 1H/31 P/13 C NMR光谱定量获得。将在缓慢复温后进行结果测量，其将根据高能磷酸盐保存和免疫组织学评分来定义。具体目标#2是用13 C-NMR分析目标#1实验，这些实验的灌流液富含13 C标记的底物。获得的13 C-NMR NMR将用于确定以下方面的通量和浓度变化：TCA循环;谷氨酸能和GABA能途径;丙酮酸羧化酶途径相对于丙酮酸脱氢酶途径;戊糖磷酸途径利用;和丙酮酸再循环。具体目标#3将评估组织损伤、用乙锭荧光显微镜定量超氧化物和代谢组学之间的关联，在超氧化物产生被或不被三种外源性抗氧化剂中的任何一种减弱的情况下：二聚夹竹桃素（抑制NADPD氧化酶）、S-PBN和丙酮酸乙酯。由于超氧化物可以由质膜结合的NADPH氧化酶以及线粒体和黄嘌呤氧化酶产生，因此氧化应激可以在单独的细胞内位置发生。治疗和措施的目标是缺氧后立即复氧，停止低温后立即复温。公共卫生相关性：最近发现，根据方案（持续时间、温度）实施的治疗性低温（降低体温）可改善窒息（脑缺氧）一段时间的新生儿的神经功能结局，尽管反应是可变的。这项研究建议在啮齿动物模型中询问，在治疗性低温期间，脑代谢物集合的NMR光谱测量是否可用于评估神经学结果和损伤，并单独指导每个新生儿的医疗管理，而不是根据协议进行管理。

DESCRIPTION (provided by applicant): Recent large clinical trials have found that therapeutic hypothermia can improve neurological outcomes in neonates who experienced asphyxia. Crucial, unanswered questions relate to optimal cooling temperatures, durations, and rewarming regimens. We will formulate a metabolomic approach to such questions using superfused neonatal (P7) rodent brain slices together with oxygen-glucose deprivation (OGD) to model asphyxia. Treatment time scales (hours) will be much less that those occurring in clinical practice (days). Specific Aim #1a is to quantify and compare, using multivariate analyses, ensembles of 1H and 31P metabolites found in perchloric acid (PCA) extracts of slices removed before and after OGD, as well as before and after hypothermic rescue regimens. A normothermic OGD control group will also be used for comparisons. Aim #1b is to make histological and immunohistological evaluations of separate slices, taken simultaneously with those removed in Aim #1a. Each NMR metabolome will consist of twenty 1H TCA Cycle metabolites and three 31P metabolites (ATP, ADP, and PCr), and be obtained from quantifications with 14.1 Tesla 1H/31P/13C NMR spectroscopy. Outcome measures, which will be defined from high energy phosphate preservation and immunohistological scores, will be made after slow rewarming. Specific Aim #2 is to analyze with 13C-NMR those Aim #1 experiments that had the superfusate enriched with 13C-labeled substrates. The 13C-NMR dataso obtained will be used to determine changes in fluxes and concentrations in: the TCA cycle; glutamatergic and GABAergic pathways; the pyruvate carboxylase pathway relative to the pyruvate dehydrogenate pathway; pentose phosphate pathway utilization; and pyruvate recycling. Specific Aim #3 will assess associations between tissue injury, quantifications of superoxide with ethidium fluorescence microscopy, and metabolomics, in situations where superoxide production is or is not attenuated by any of three exogenous antioxidants: dimeric apocynin (inhibits NADPD-oxidase), S-PBN, and ethyl pyruvate. Because superoxide can be produced by plasma membrane bound NADPH oxidase as well as by mitochondria and xanthine oxidase, oxidative stress can occur at separate intracellular locations. Treatments and measures will target reoxygenation immediately after hypoxia, and rewarming immediately after stopping hypothermia. PUBLIC HEALTH RELEVANCE: Therapeutic hypothermia (lowering of body temperature), administered according to protocols (duration, temperature) has recently been found to improve neurological outcomes in neonates who have suffered a period of asphyxia (brain oxygen deprivation) although responses are variable. This research proposal asks in a rodent model if, during therapeutic hypothermia, NMR spectroscopy measurements of ensembles of brain metabolites can be used to assess neurologic outcome and injury, and individually guide each neonate's medical management, rather than manage according to a protocol.