A period of secondary energy failure consisting of a decline in phosphocreatine/inorganic phosphate (PCr/Pi), a rise in brain lactate, and alkaline intracellular pH (pH i ) has been described in infants with neonatal encephalopathy. Strategies that ameliorate this energy failure may be neuroprotective. We hypothesized that a neonatal rat brain slice model undergoes a progressive decline in energetics, which can be ameliorated with hypothermia or amiloride. Interleaved phosphorus ( 31 P) and proton ( 1 H) magnetic resonance (MR) spectra were obtained from 350 m neonatal rat brain slices over 8 h in a bicarbonate buffer at 37°C and at 32°C in 7-and 14-d models.31 P MR spectra were obtained with amiloride in a bicarbonate-free buffer at 37°C in the 14-d model. Findings were similar in 7-and 14-d models. In the 14-d model, there was a Pi doublet structure corresponding to alkaline pH i values of 7.50 Ϯ 0.02 and 7.21 Ϯ 0.04. Compared with the stabilized baseline of 100, at 5 h PCr/Pi was 65 Ϯ 6.3 and lactate/NAA was 187 Ϯ 3 at 37°C, but PCr/Pi and lactate/NAA were not significantly different from baseline at 32°C. Nucleotide triphosphate (NTP)/phosphomonoester (PME) was 0.93 Ϯ 0.23 at 37°C and 1.81 Ϯ 0.21 at 32°C at 5 h. With amiloride exposure in the 14-d model, baseline pH i values were 7.25 Ϯ 0.09 and 6.98 Ϯ 0.02 and NTP/PME was 1.81 Ϯ 0.05; these parameters were not significantly different at 5 h. Our interpretation of these findings is that the brain slice model underwent secondary energy failure, which was delayed with hypothermia or amiloride. Studies in term infants with neonatal encephalopathy (NE) suggest that, although antenatal or genetic factors might predispose some infants to perinatal brain injury (1,2), events in the immediate perinatal period are important in neonatal brain injury (3). A consistent pattern of energy derangement has been observed by several groups in infants with NE due to intrapartum asphyxia using phosphorus ( 31 P) and proton ( 1 H) magnetic resonance (MRS), spectroscopy (4 -7). 31 P and 1 H MR spectra were often normal within the first few hours after resuscitation, but after 8 -24 h there was a progressive decline in PCr/Pi and rise in brain lactate despite adequate oxygenation and circulation in the infant. The nadir of the energetic disturbance was seen after 12-24 h and the magnitude of the fall in PCr/Pi and rise in brain lactate correlated with the subsequent neurodevelopmental abnormality (8,9). This sequence of events was termed "secondary energy failure" to distinguish it from the "primary" decline in PCr and ATP and rise in lactate seen during the insult in experimental animal models of hypoxia-ischemia (HI) (10,11). The biphasic pattern of energy failure during and after HI has been the cornerstone behind the realization that neuroprotective strategies might interrupt the cascade of irreversible injury if administered within hours of a perinatal insult (12), and the concept has been an important catalyst in the development of neuroprotection trials with moderate hypoth...