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经历窒息的新生儿的神经系统预后。关键的，悬而未决的问题涉及到最佳冷却温度，持续时间和复温方案。我们将制定代谢组学方法来解决这些问题，使用过量的新生儿（P7）啮齿动物大脑切片和氧葡萄糖剥夺（OGD）来模拟窒息。治疗时间（小时）将大大少于临床实践（天）。具体目的#1a是量化和比较，使用多变量分析，在OGD前后以及低温救援方案前后去除的切片的高氯酸（PCA）提取物中发现的1H和31P代谢物的集合。正常OGD对照组也将用于比较。Aim #1b是对单独的切片进行组织学和免疫组织学评估，这些切片与Aim #1a中取出的切片同时取下。每个NMR代谢组将由20个1H TCA Cycle代谢物和3个31P代谢物（ATP， ADP和PCr）组成，并通过14.1 Tesla 1H/31P/13C NMR谱进行定量。在缓慢复温后，将根据高能磷酸盐保存和免疫组织学评分来确定结局指标。特定的目标2是用13C-NMR分析目标1中含有13c标记底物的过浓液的实验。获得的13c核磁共振数据将用于确定三羧酸循环中通量和浓度的变化；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途径；戊糖磷酸途径利用；还有丙酮酸回收。特异性目标#3将评估组织损伤、用乙硫铵荧光显微镜定量超氧化物和代谢组学之间的关系，在三种外源性抗氧化剂：二聚罗泼宁（抑制nadpd氧化酶）、S-PBN和丙酮酸乙酯中的任何一种都能减弱或不减弱超氧化物产生的情况下。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，氧化应激可以发生在细胞内不同的位置。治疗和措施将针对缺氧后立即再充氧，并在停止低温后立即重新加热。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）可改善经历过一段时间窒息（脑氧剥夺）的新生儿的神经系统预后，尽管反应各不相同。这项研究计划在啮齿动物模型中询问，在治疗性低温期间，脑代谢物的核磁共振光谱测量是否可以用于评估神经系统结果和损伤，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.