Cerebral Magnetic Resonance Spectroscopy Biomarkers and Outcome in Perinatal Asphyxia

Background: Neonatal encephalopathy is a common clinical condition affecting 2-3 per 1,000 neonates in the developed world. 10-15% of cases will die in the neonatal period. Therapeutic hypothermia has become the standard of care in the developed world for infants with moderate to severe neonatal encephalopathy only recently, after 2 decades of experimental and clinical studies. Approximately half the infants who receive therapeutic hypothermia still have an abnormal outcome. Research is now being focused on pre-clinical and Phase II clinical studies of drugs, which act synergistically or additively with therapeutic hypothermia. Magnetic resonance spectroscopy (MRS) techniques provide translational biomarkers, which may be used to speed up the development of safe and effective neuroprotective interventions. The precise relation between MRS and brain histology is unknown and it is unclear whether cooling itself alters the prognostic efficacy of MRS. Aim: (i) To explore the relation between MRS biomarkers and the degree of brain pathology observed in our pre-clinical piglet model of perinatal asphyxia; (ii) To assess the predictive value of MRS biomarkers in infants with neonatal encephalopathy Methods: In our piglet study, data from 2 large piglet asphyxia studies investigating neuroprotective agents were analysed. 1. The first was a study of Xenon-augmented hypothermia. Following transient hypoxia-ischemia, 36 piglets were randomised into 4 groups (each n=9), with intervention from 2-26 h: Group (i) normothermia (38.5oC); Group (ii) normothermia (38.5oC) + 24 h 50% inhaled xenon; Group (iii) 24 h therapeutic hypothermia (rectal temperature (Trectal) 33.5oC) or Group (iv) 24 h 50% inhaled xenon +24 h therapeutic hypothermia (Trectal 33.5oC). 2. The second was a study investigating Amiloride as a neuroprotective agent. Following transient hypoxia-ischemia, 18 male piglets (<24 h of age) were randomized to 2 groups (each n=9) (1) normothermia; or (2) 2.5mg/kg of methyl isobutyl amiloride (MIA) at 10 minutes after resuscitation and 8 hourly thereafter. Both studies were performed on a Bruker 4.7 Tesla MR system. Following resuscitation after hypoxia-ischemia, proton (1H) magnetic resonance (MR) spectra were acquired with repetition time (TR) 5 sec, 128 summed transients, and echo times (TE) 25 ms, 144 ms, and 288 ms. (we measured the following peak area ratios: Lactate/N acetyl aspartate (Lac/NAA) and Lactate/Creatine (Lac/Cr) in both white matter and thalamus). Phosphorus (31P) MR spectra were acquired from whole brain using single-pulse acquire with TR 10 s (we measured inorganic phosphate (Pi)/exchangeable phosphate pool (EPP = Pi + PCr + (2γ +β)-NTP), NTP/EPP). Immunohistochemistry was performed on brain sections for activated Caspase 3, TUNEL positive cells and Iba I (activated microglia) to quantify cell death and microglial activation. For the clinical study, we assessed 45 infants (median gestational age – 40 weeks) with moderate to severe neonatal encephalopathy admitted over a 3 year period to the neonatal unit at UCH. Their neurodevelopmental outcome was assessed at 18 months. Results: (i) Experimental Study - Early Biomarkers Early biomarkers (acquired between 2 and 4 hours after hypoxia-ischemia) in particular thalamic Lac/NAA, predicted the 1H MRS area under the curve values from 0-48h and quantitative immunohistochemistry at 48 hours. Late Biomarkers: WM Lac/Cr at 40-48h after HI demonstrated the highest positive correlation with Tunel positive cell death (R20.98, p = 0.01) and NTP/EPP with microglial ramification (R20.68, p = 0.006); this correlation was present in both treated and untreated piglets. (ii) In the clinical study, deep gray matter Lac/NAA was the most accurate predictor of long term adverse neurological outcome following NE; importantly the predictive accuracy of Lac/NAA was unaltered by preceding therapeutic hypothermia. Conclusions: (i) Early thalamic Lac/NAA predicted the subsequent trajectory of energy disruption; this has importance in understanding the relevance of very early MRS studies in babies. White matter lactate/Cr at 40-48h correlated best with quantitative immunohistochemistry (especially TUNEL positive cells) and this relationship was present with and without preceding therapeutic intervention. (ii) In babies with neonatal encephalopathy, the predictive accuracy of Lac/NAA was unaltered by therapeutic hypothermia.