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 #1 a 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 #1 b 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Cole, Alison E
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University of California San Francisco
Schools of Medicine
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Liu, Jia; Segal, Mark R; Kelly, Mark J S et al. (2013) 13C NMR metabolomic evaluation of immediate and delayed mild hypothermia in cerebrocortical slices after oxygen-glucose deprivation. Anesthesiology 119:1120-36
Liu, Jia; Litt, Lawrence; Segal, Mark R et al. (2011) Outcome-related metabolomic patterns from 1H/31P NMR after mild hypothermia treatments of oxygen-glucose deprivation in a neonatal brain slice model of asphyxia. J Cereb Blood Flow Metab 31:547-59
Liu, Jia; Hirai, Kiyoshi; Litt, Lawrence (2008) Fructose-1,6-bisphosphate does not preserve ATP in hypoxic-ischemic neonatal cerebrocortical slices. Brain Res 1238:230-8
Zeng, Jianying; Yang, Guo-Yuan; Ying, Weihai et al. (2007) Pyruvate improves recovery after PARP-1-associated energy failure induced by oxidative stress in neonatal rat cerebrocortical slices. J Cereb Blood Flow Metab 27:304-15
Zeng, Jianying; Hirai, Kiyoshi; Yang, Guo-Yuan et al. (2004) Using 31P NMR spectroscopy at 14.1 Tesla to investigate PARP-1 associated energy failure and metabolic rescue in cerebrocortical slices. J Bioenerg Biomembr 36:415-9
Hirai, K; Hayashi, T; Chan, P H et al. (2003) Akt phosphorylation and cell survival after hypoxia-induced cytochrome c release in superfused respiring neonatal rat cerebrocortical slices. Acta Neurochir Suppl 86:227-30
Litt, L; Hirai, K; Basus, V J et al. (2003) NTP and PCr responses to hypoxia by hypothermic and normothermic respiring, superfused, neonatal rat cerebrocortical slices: an NMR spectroscopy study at 14.1 Tesla. Acta Neurochir Suppl 86:71-4
Litt, Lawrence; Hirai, Kiyoshi; Basus, Vladimir J et al. (2003) Temperature control of respiring rat brain slices during high field NMR spectroscopy. Brain Res Brain Res Protoc 10:191-8
Hirai, Kiyoshi; Sugawara, Taku; Chan, Pak H et al. (2002) Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices. J Neurochem 83:309-19
Hasegawa, K; Litt, L; Espanol, M T et al. (1998) Expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices during NMDA-induced necrosis and apoptosis. Brain Res 785:262-78

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