Ann Lorek

Delayed ("secondary") cerebral energy failure after acute hypoxia-ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy

ABSTRACT: Phosphorus (31P) spectra from the brains of severely birth-asphyxiated human infants are commonly normal on the first day of life. Later, cerebral energy failure develops, which carries a serious prognosis. The main purpose of this study was to test the hypothesis that this delayed (“secondary”) energy failure could be reproduced in the newborn piglet after a severe acute reversed cerebral hypoxicischemic insult. Twelve piglets were subjected to temporary occlusion of the common carotid arteries and hypoxemia [mean arterial Po2 3.1 (SD 0.6) kPa]. Mean cerebral phosphocreatine concentration [PCr]/inorganic orthophosphate concentration [Pi] decreased from 1.40 (SD 0.29) to 0.01 (SD 0.02), and nucleotide triphosphate concentration [NTP]/exchangeable phosphate pool concentration [EPP] decreased from 0.19 (SD 0.02) to 0.06 (SD 0.04) (p<0.001 for each decrease). On reperfusion and reoxygenation of the brain, mean [PCr]/[Pi] and [NTP]/[EPP] returned to baseline. Observations continuing for the next 48 h showed that [PCr]/[Pi] again decreased, in spite of normal arterial Po2, mean arterial blood pressure, and blood glucose, to 0.62 (SD 0.61) at 24 h (p<0.01) and 0.49 (SD 0.37) at 48 h (p<0.001). [NTP]/[EPP] also decreased, but to a lesser degree. Intracellular pH remained unchanged. These findings appeared identical with those seen in birth-asphyxiated human infants. No changes in cerebral metabolite concentrations took place in six control piglets. The severity of secondary energy failure, as judged by the lowest [PCr]/[Pi] recorded at 24-48 h, was directly related to the extent of acute energy depletion, obtained as the time integral of reduction in [NTP]/[EPP] (p<0.0001). This animal model of secondary energy failure may prove useful for testing cerebroprotective strategies.

Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet

ABSTRACT: Severely birth-asphyxiated human infants develop delayed (“secondary”) cerebral energy failure, which carries a poor prognosis, during the first few days of life. This study tested the hypothesis that mild hypothermia after severe transient cerebral hypoxia-ischemia decreases the severity of delayed energy failure in the newborn piglet. Six piglets underwent temporary occlusion of the common carotid arteries and hypoxemia. Resuscitation was started when cerebral [phosphocreatine (PCr)]/ [inorganic phosphate (Pi)] as determined by phosphorus magnetic resonance spectroscopy had fallen almost to zero and [nucleotide triphosphate (NTP)]/[exchangeable phosphate pool (EPP)] had fallen below about 30% of baseline. Rectal and tympanic temperatures were then reduced to 35°C for 12 h after which normothermia (38.5°C) was resumed. Spectroscopy results over the next 64 h were compared with previously established data from 12 piglets similarly subjected to transient cerebral hypoxia-ischemia, but maintained normothermic, and six sham-operated controls.The mean severity of the primary insult (judged by the time integral of depletion of [NTP]/[EPP]) was similar in the hypothermic and normothermic groups. In the normothermic group, [PCr]/[Pi] and [NTP]/[EPP] recovered after the acute insult and then fell again. Minimum values for these variables observed between 24 and 48 h were significantly higher in the hypothermic group and not significantly different from the control values (p < 0.05, analysis of variance). A large reduction in secondary energy failure relative to the extent of the primary insult was shown and no further fall in either [PCr]/[Pi] or [NTP]/[EPP] took place up to 64 h in the hypothermic piglets. We conclude that mild hypothermia after a severe acute cerebral hypoxicischemic insult ameliorated delayed energy failure.

Proton Magnetic Resonance Spectroscopy of the Brain during Acute Hypoxia-Ischemia and Delayed Cerebral Energy Failure in the Newborn Piglet

Studies of the brains of severely birth-asphyxiated infants using proton(1H) magnetic resonance spectroscopy (MRS) have shown changes indicating a rise in cerebral lactate (Lac) and a fall in N- acetylaspartate (Naa). The aim of this study was to test two hypotheses: 1) that these changes can be reproduced in the newborn piglet after transient reversed cerebral hypoxia-ischemia, and their time course determined; and 2) that changes in Lac peak-area ratios are related to changes in phosphorylation potential as determined by phosphorus(31P) MRS. Eighteen piglets aged <24 h were anesthetized and ventilated. Twelve underwent temporary occlusion of the carotid arteries and hypoxemia, and six served as sham-operated controls. 1H and 31P spectra were acquired alternately, both during the insult and for the next 48 h, using a 7-tesla spectrometer. During hypoxia-ischemia, the median Lac/total creatine (Cr) peak-area ratio rose from a baseline of 0.14 (interquartile range 0.07-0.27), to a maximum of 4.34 (3.33-7.45). After resuscitation, Lac/Cr fell to 0.75 (0.45-1.64) by 2 h, and then increased again to 2.43(1.13-3.08) by 48 h. At all stages after resuscitation Lac/Cr remained significantly above baseline and control values. Naa/Cr was significantly reduced below baseline and control values by 48 h after resuscitation. The increases in the Lac peak-area ratios were concomitant with the falls in the[phosphocreatine (PCr)*]/[inorganic phosphate (Pi)] ratio, during both acute hypoxia-ischemia and delayed energy failure. The maximum Lac/Naa during delayed energy failure correlated strongly with the minimum[nucleotide triphosphate (NTP)]/[exchangeable phosphate pool (EPP)](r = -0.94, p < 0.0001). We conclude that both hypotheses have been confirmed.

Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult

An increase in the number of cells undergoing apoptosis was observed in the cingulate sulcus of newborn piglets 48 h after a global hypoxic-ischaemic insult. Apoptotic death was identified morphologically (by light and electron microscopy) and by DNA fragmentation, detected by in situ end labelling. The number of apoptotic cells was directly related to the degree of high-energy phosphate depletion during hypoxia-ischaemia, measured using continuous 31P magnetic resonance spectroscopy. These results may have implications for the understanding and treatment of perinatal hypoxic-ischaemic brain injury.

Early brain proton magnetic resonance spectroscopy and neonatal neurology related to neurodevelopmental outcome at 1 year in term infants after presumed hypoxic-ischaemic brain injury

This study investigated the accuracy of prediction of neurodevelopmental outcome at 1 year using cerebral proton magnetic resonance spectroscopy (MRS) and structured neonatal neurological assessment in term infants after presumed hypoxic–ischaemic brain injury. Eighteen control infants and 28 infants with presumed hypoxic–ischaemic brain injury underwent proton MRS investigation. Studies were carried out as soon as possible after the cerebral insult, most within 48 hours. Infants had an early structured neurological assessment at a median of 19 hours (range 0 hours to 9 days) from the presumed hypoxic–ischaemic insult and a late assessment at a median of 7 days (range 3 to 25 days) during recovery. The maximum cerebral peak–area ratio lactate:N‐acetylaspartate measured by proton MRS accurately predicted adverse outcome at 1 year with a specificity of 93% and positive predictive value of 92%. Neurological assessment had a tendency for false‐positive predictions. However, both early and late neurological examination can be used as a reliable indicator for a favourable outcome at 1 year having negative predictive values of 100% and 91% respectively.

Mild Hypothermia after Severe Transient Hypoxia-Ischemia Reduces the Delayed Rise in Cerebral Lactate in the Newborn Piglet

This study tested the hypothesis that mild hypothermia after severe transient hypoxia-ischemia reduces the subsequent delayed rise in cerebral lactate peak-area ratios as determined by proton (1H) magnetic resonance spectroscopy (MRS) in the newborn piglet. Nine piglets aged <24 h underwent temporary occlusion of the common carotid arteries and hypoxemia. Resuscitation was started when cerebral [phosphocreatine]/[inorganic phosphate] had fallen close to zero and [nucleotide triphosphate(NTP)]/[exchangeable phosphate pool (EPP)] was below about a third of baseline. On resuscitation rectal and tympanic temperatures were lowered to 35°C for 12 h after which normothermia (38.5 °C) was resumed. 1H MRS data collected over 48 or 64 h after resuscitation were compared with concurrently established data from 12 piglets similarly subjected to transient cerebral hypoxia-ischemia, but maintained normothermic, and six shamoperated controls. The severity of the primary insult (judged from the time integral of depletion of [NTP]/[EPP]) was similar in the hypothermic and normothermic groups. The maximum lactate/N-acetylaspartate ratio observed between 24 and 48 h after resuscitation in the hypothermic group was 0.10 (0.05-0.97), median (interquartile range), which was significantly lower than that observed in the normothermic group, 1.28 (0.97-2.14), and not significantly different from that observed in the control group, 0.08 (0.06-0.11). Similar results were obtained for lactate/choline and lactate/total creatine. We conclude that mild hypothermia after a severe acute cerebral hypoxic-ischemic insult reduces the delayed elevation in lactate peak-area ratios, thus reflecting reduced lactate accumulation.

Brain-metabolite transverse relaxation times in magnetic resonance spectroscopy increase as adenosine triphosphate depletes during secondary energy failure following acute hypoxia-ischaemia in the newborn piglet.

The adenosine triphosphate (ATP)-dependent sodium/potassium pump extrudes intracellular sodium in exchange for extracellular potassium. Low ATP causes pump dysfunction increasing both intracellular sodium and water thereby enhancing metabolite mobility. This should be detectable by proton magnetic resonance spectroscopy (MRS) as increased metabolite transverse relaxation times (T2s). During secondary cerebral energy failure in the newborn piglet, proton and phosphorus MRS showed large increases in the T2s of choline, creatine, N-acetylaspartate, and lactate that correlated with ATP depletion. These results provide insight into factors affecting metabolite T2s and show that T2s may be useful for studying cellular oedema.

Effects of N-omega-nitro-L-arginine methyl ester on the cerebral circulation of newborn piglets quantified in vivo by near-infrared spectroscopy

ABSTRACT: The effects of N--nitro-L-arginine methyl ester (L-NAME) on basal cerebral vascular tone, the vasodilatory effects of acetylcholine (ACh), and the cerebrovascular response to alterations in arterial carbon dioxide tension (CBVR) were investigated using near-infrared spectroscopy. Seven newborn piglets were anesthetized and mechanically ventilated; mean arterial blood pressure (MAP) was monitored and near-infrared spectroscopy used to measure changes in total cerebral Hb concentration. At the beginning of the experiment, CBVR was measured and then 10, 20, 30, and 100 mg.kg−1 L-NAME were administered sequentially; ACh (1, 2, 3, and 5 μg) was given before and after each injection of L-NAME. At the end of this sequence, CBVR was measured again and finally sodium nitroprusside (1.5 mg.kg−1) was administered. Ten and 20 mg.kg−1 L-NAME caused a significant decrease in total cerebral Hb concentration of −0.59 (-3.21 to −0.02) and −1.46 (-6.50 to −0.15) μmol.L−1 (median and range), respectively (Wilcoxon p < 0.05), but subsequent injections did not. Ten, 20, and 100 mg.kg−1 L-NAME caused an increase in MAP (VVilcoxon p < 0.05). ACh caused an increase in total cerebral Hb concentration and a decrease in MAP that was impaired but not abolished by L-NAME (ANOVA p < 0.05). CBVR was not affected by L-NAME. Sodium nitroprusside caused a reduction in mean (SD) MAP of 4.7 (1.6) kPa, and a slower rise in [tHb] of 13.44 (2.03) μmol.L−1. Postmortem examination of three animals revealed NADPH-diaphorase staining in neurons, cerebral blood vessels, carotid artery, and jugular vein. These results demonstrate that nitric oxide modulates basal vasodilation and part of the vasodilatory action of ACh in the newborn piglet brain, but that these effects are quantitatively small. Nitric oxide was not shown to be an important participant in CBVR. This is consistent with the suggestion that the mechanism mediated by nitric oxide may coordinate perfusion rather than control bulk flow in the developing brain.

Quantitation of phosphorus metabolites in newborn human brain using internal water as reference standard

A new method for noninvasive, in vivo quantitation of cerebral phosphorus (31P) metabolites is described. The technique employs point-resolved spectroscopy (PRESS) to obtain both 31P-metabolite and proton (1H) water spectra: brain water is used as an internal concentration reference. Spin-spin relaxation times (T2s) of cerebral 31P metabolites are much longer than the minimum echo time (TE) usable on a spectrometer equipped with actively shielded gradient coils. With short-TE (approximately 10 ms) 31P PRESS, T2 relaxation is minimal and phase modulation of the nucleotide triphosphate (NTP) multiplets can be accounted for 1H water spectra were acquired using several TEs so that extra- and intracellular water signals could be separated from that due to cerebrospinal fluid. Prior calibration of the 31P and 1H spectrometer channels and an assumed brain-water concentration enabled estimations of metabolite concentrations. Using this method, mean 31P metabolite concentrations in the brains of eight normal infants of gestational plus postnatal age 34 to 39 wk were: phosphomonoester (PME) 5.6 (SD 0.9); inorganic phosphate 1.4 (0.4); mobile phosphodiester 2.3 (0.6); phosphocreatine 2.9 (0.3); nucleotide triphosphate 2.8 (0.6); and total mobile phosphate 21.4 (2.8) mmol/kg wet.

107 MOTOR TESTS OF INTERHEMISPHERIC CONTROL AND COGNITIVE FUNCTION IN VERY PRETERM INFANTS AT EIGHT YEARS

We have previously reported that damage to the posterior corpus callosum might explain some of the subtle cognitive processing deficits found later in many preterm infants. To test this hypothesis we carried out motor tests of interhemispheric control in 197 consecutively examined very preterm infants(<33 weeks gestation) at eight years. In addition to the Kaufman Assessment Battery for Children (K-ABC) and other studies, two tests of mirrorwise(Tm) and alternate(Ta) forearm diadochokinetic (pronation-supination) movements were performed. The alternate movement represents the most complicated form of interhemispheric control and reciprocal inhibition. The time difference between the two movements (Ta-Tm) is considered to give an indication of the efficiency of transfer of motor information between the hemispheres (particularly the posterior corpus callosum). There was a significant and linear relation between this time difference and the Kaufman Mental Processing Composite (K-MPC) score (p<0.002, DF 2). We conclude that the corpus callosum has an important role in cognitive development and posterior corpus callosum efficiency is related to cognitive outcome.