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预后。关键的、悬而未决的问题与最佳冷却温度、持续时间和复温方案有关。我们将使用超融合新生儿(P7)啮齿动物脑片结合缺氧-葡萄糖剥夺(OGD)来建立窒息模型，从而制定出一种代谢学方法来解决这些问题。治疗时间尺度(小时)将比临床实践中发生的时间(天)短得多。具体目标#1a是利用多变量分析来量化和比较在OGD之前和之后以及在低温抢救方案之前和之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。常温OGD对照组也将用于比较。目标#1b是对与目标#1a中切除的切片同时采集的单独切片进行组织学和免疫组织学评估。每个核磁共振代谢体将由20个1H TCA循环代谢物和3个31P代谢物(ATP、ADP和PCR)组成，并通过14.1Tesla 1H/31P/13C核磁共振谱进行定量。结果指标将根据高能磷酸盐保存和免疫组织学评分确定，将在缓慢复温后进行。特定的目标#2是用13C-核磁共振分析那些目标#1的实验，这些实验的超融合液富含13C标记的底物。获得的13C-核磁共振数据将用于确定以下方面的通量和浓度的变化：TCA循环；谷氨酸和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；以及丙酮酸循环。具体目标#3将评估组织损伤、乙锭荧光显微镜定量超氧化物和代谢组学之间的关系，在超氧化物产生被或不被三种外源抗氧化剂中的任何一种减弱的情况下：二聚体载脂蛋白(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，因此氧化应激可以发生在不同的细胞内位置。治疗和措施将针对缺氧后立即复氧，停止低温后立即复温。与公共卫生相关：最近发现，根据规程(持续时间、体温)进行的治疗性低温(降低体温)可以改善窒息(大脑缺氧剥夺)时期新生儿的神经结局，尽管反应各不相同。这项研究建议在啮齿动物模型中询问，在治疗性低温期间，是否可以使用对大脑代谢物整体的核磁共振波谱测量来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.