Hypothermia and Amiloride Preserve Energetics in a Neonatal Brain Slice Model

Abstract

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...

N-Methyl-isobutyl-amiloride Ameliorates Brain Injury When Commenced Before Hypoxia Ischemia in Neonatal Mice

“…In a tissue culture model of cerebral ischemia, the prevention of rebound alkalosis using amiloride after in vitro HI delayed the onset of injury for as long as the cells remained acidotic (25). Recent studies in an ex vivo model of neonatal rat brain slices demonstrated a preservation of brain slice energetics over several hours in those brain slices exposed to amiloride; this preservation of brain slice energetics with amiloride was similar to that observed in brain slices main- tained under hypothermic conditions (26). Further in vivo studies in neonatal models are required to demonstrate whether amiloride retains its neuroprotective effect when treatment is commenced after HI and whether its neuroprotective effect is enhanced in combination with other strategies such as hypothermia.…”

S100B Secretion in Acute Brain Slices: Modulation by Extracellular Levels of Ca2+ and K+

“…Interestingly, ATP content in these preparations is apparently the same, either in HEPES-or in bicarbonate-buffered medium [38]. Based on extracellular lactate, it is possible to observe a significant energetic variation during the first hour and a recovery from 1 h on.…”

Na+/H+ Exchangers and Intracellular pH in Perinatal Brain Injury

“…During in vitro cerebral ischemia, NHE inhibitors (DMA, harmaline [46], SM-20220 [88], amiloride [89], and HOE-642 [85, 87, 90]) had a protective effect, in terms of delayed pH i normalization both in neuronal cells and in astrocytes [46, 89–92], inhibition of microglia [90], and less intracellular calcium accumulation [87, 88]. This anoxia-induced alkalization was also ameliorated in NHE1−/− CA1 neurons.…”