Proton MR spectroscopy in neonates with perinatal cerebral hypoxic-ischemic injury: metabolite peak-area ratios, relaxation times, and absolute concentrations.

BACKGROUND: Results from cerebral proton (1)H-MR spectroscopy studies of neonates with perinatal hypoxic-ischemic injury have generally been presented as metabolite peak-area ratios, which are T1- and T2-weighted, rather than absolute metabolite concentrations. We hypothesized that compared with (1)H-MR spectroscopy peak-area ratios, calculation of absolute metabolite concentrations and relaxation times measured within the first 4 days after birth (1) would improve prognostic accuracy and (2) enhance the understanding of underlying neurochemical changes in neonates with neonatal encephalopathy. METHODS: Seventeen term infants with neonatal encephalopathy and 10 healthy controls were studied at 2.4T at 1 (1-3) and 2 (2-4) (median [interquartile range]) days after birth, respectively. Infants with neonatal encephalopathy were classified into 2 outcome groups (normal/mild and severe/fatal), according to neurodevelopmental assessments at 1 year. The MR spectroscopy peak-area ratios, relaxation times, absolute concentrations, and concentration ratios of lactate (Lac), creatine plus phosphocreatine (Cr), N-acetylaspartate (NAA), and choline-containing compounds (Cho) from a voxel centered on the thalami were analyzed according to outcome group. RESULTS: Comparing the severe/fatal group with the controls (significance assumed with P < 0.05), we found that Lac/NAA, Lac/Cho, and Lac/Cr peak-area ratios increased and NAA/Cr and NAA/Cho decreased; Lac, NAA, and Cr T2s were increased; [Lac] was increased and [Cho], [Cr], and [NAA] decreased; and among the concentration ratios, only [Lac]/[NAA] was increased. Comparison of the normal/mild group with controls revealed no differences in peak-area ratios, relaxation times, or concentration ratios but decreased [NAA], [Cho], and [Cr] were observed in the infants with normal/mild outcome. Comparison of the normal/mild and severe/fatal groups showed increased Lac/NAA and Lac/Cho and decreased NAA/Cr and NAA/Cho peak-area ratios, reduced [NAA], and increased Lac T2 in the infants with the worse outcome. CONCLUSIONS: Metabolite concentrations, in particular [NAA], enhance the prognostic accuracy of cerebral (1)H-MR spectroscopy-[NAA] was the only measurable to discriminate among all (control, normal/mild, and severe/fatal outcome) groups. However, peak-area ratios are more useful prognostic indicators than concentration ratios because they depend on metabolite concentrations and T2s, both of which are pathologically modulated. Concentration ratios depend only on the concentrations of the constituent metabolites. Increased Cr T2 may provide an indirect marker of impaired cellular energetics, and similarly, NAA T2 may constitute an index of exclusively neuronal energy status. Our recommendation is to collect data that enable calculation of brain metabolite concentrations. However, if time constraints make this impossible, metabolite peak-area ratios provide the next best method of assigning early prognosis in neonatal encephalopathy.

Follow up of a randomised trial of two different courses of dexamethasone for preterm babies at risk of chronic lung disease.

OBJECTIVES: To report 18 month outcome of a randomised trial of two courses of dexamethasone to prevent chronic lung disease of prematurity. STUDY DESIGN: Babies of birth weight 1250 g or less ventilated at 7 days of age were randomised to a 42 day reducing course (long) or a 3 day pulsed (pulse) course of dexamethasone. Growth, cardiovascular status, and respiratory and neurodevelopmental outcomes were assessed at 18 months. RESULTS: Seventy six babies were enrolled. Nine died and three were lost to follow up. Babies receiving the long course were weaned off oxygen more quickly than those receiving the pulse course (47% v 69% on oxygen at 28 days; p = 0.01), but there were no differences in 18 month outcomes. However, children averaged -1 SD for growth parameters, half had moderate or severe disability, and 35% and 19% respectively required oxygen at 36 weeks and discharge. CONCLUSIONS: The dexamethasone course used did not influence long term outcome. However, entry criteria for this study selected a group of babies at high risk of poor long term outcome.

Twenty-four hours of mild hypothermia in unsedated newborn pigs starting after a severe global hypoxic-ischemic insult is not neuroprotective.

Three to 12 h of mild hypothermia (HT) starting after hypoxia-ischemia is neuroprotective in piglets that are anesthetized during HT. Newborn infants suffering from neonatal encephalopathy often ventilate spontaneously and are not necessarily sedated. We aimed to test whether mild posthypoxic HT lasting 24 h was neuroprotective if the animals were not sedated. Thirty-nine piglets (median weight 1.6 kg, range 0.8-2.2 kg; median age 24 h, range 7-48 h) were anesthetized and ventilated and subjected to a 45-min hypoxic (FiO(2) approximately 6%) global insult (n = 36) or sham hypoxia (n = 3). On reoxygenation, 18 were maintained normothermic (NT, 39.0 degrees C) for 72 h, and 21 were cooled from 39 (NT) to 35 degrees C (HT) for the first 24 h before NT was resumed (18 experimental, three sham hypoxia). Cardiovascular parameters and intermittent EEG were documented throughout. The brain was perfusion fixed for neuropathology and five main areas examined using light microscopy. The insult severity (duration in minutes of EEG amplitude < 7 microV) was similar in the NT and HT groups, mean +/- SD (28 +/- 7.2 versus 27 +/- 8.6 min), as was the mean FiO(2) (5.9 +/- 0.7 versus 5.8 +/- 0.8%) during the insult. Six NT and seven HT piglets developed posthypoxic seizures that lasted 29 and 30% of the time, respectively. The distribution and degree of injury (0.0-4.0, normal-maximal damage) within the brain (hippocampus, cortex/white matter, cerebellum, basal ganglia, thalamus) were similar in the NT and HT groups (overall score, mean +/- SD, 2.3 +/- 1.5 versus 2.4 +/- 1.3) as was the EEG background amplitude at 3 h (13 +/- 3.5 versus 10 +/- 3.3 microV). The HT animals shivered and were more active. The sham control group (n = 3) shivered but had normal physiology and neuropathology. Plasma cortisol was significantly higher in the HT group during the HT period, 766 +/- 277 versus 244 +/- 144 microM at 24 h. Mild postinsult HT for 24 h was not neuroprotective in unsedated piglets and did not reduce the number of animals that developed posthypoxic seizures. Cortisol reached 3 times the NT value at the end of HT. We speculate that the stress of shivering and feeling cold interfered with the previously shown neuroprotective effect of HT. Research on the appropriateness of sedation during clinical HT is urgent.

Magnesium sulfate treatment after transient hypoxia-ischemia in the newborn piglet does not protect against cerebral damage.

Transient perinatal hypoxia-ischemia (HI) can lead to delayed cerebral damage beginning 8-24 h after resuscitation. Cerebroprotective therapies applied soon after HI may thus reduce the severity of brain injury. We have previously shown that MgSO4 administration to newborn piglets after HI fails to prevent the delayed global impairment in cerebral energy metabolism characteristic of severe brain damage. However, high extracellular concentrations of magnesium ions have been found to prevent specific excitotoxic neural cell death in vivo and in vitro. This study therefore examined the hypothesis that MgSO4 administration after HI reduces damage in some regions of the brain even though global energy metabolism is unaffected. Twelve newborn piglets were subjected to global cerebral HI by transient occlusion of both common carotid arteries and reduction of the inspired oxygen fraction to 0.12 until cerebral high-energy phosphates, measured by magnetic resonance spectroscopy, were significantly depleted. Subjects were randomly assigned to two groups of six: the first received MgSO4 (three doses, 400 mg/kg 1 h after resuscitation and 200 mg/kg at 12 and 24 h), and the second received placebo infusions. At 48 h after the start of the experiment, the piglets were killed and their brains were perfused, fixed, and embedded in paraffin wax. Five-micrometer sections were stained with hematoxylin and eosin to allow semiquantitative analysis of the severity and extent of injury to the hippocampus, cerebellum, cerebral cortex, caudate nucleus, thalamus, and striatum and the white matter tracts. There was no difference in the severity of tissue damage between the MgSO4-treated group and the placebo-treated animals in any brain region.

Cerebral tissue water spin-spin relaxation times in human neonates at 2.4 tesla: methodology and the effects of maturation.

Using a 4-echo spin-echo sequence, cerebral T2 was measured in specific anatomic regions in eleven healthy newborn infants, whose gestational plus postnatal ages (GPAs) lay between 37 and 42 weeks. For a region in the pons, T2 was 141+/-9 ms (mean +/- standard deviation), and no significant dependence upon GPA was seen. In the thalamus mean T2 was 136+/-13 ms, and T2 demonstrated a significant negative linear dependence upon age (r = 0.690; p < 0.02). In periventricular and frontal regions, mean T2 were 217+/-33, and 228+/-32 ms respectively, and more marked negative linear correlations with age were observed (r = 0.833; p < 0.001 and r = 0.722; p < 0.02). For these regions, the rate of T2 decrease with age appeared to be related to known patterns of myelination. For the parietal region studied, mean T2 was 204+/-34 ms, no significant dependence upon GPA being seen. T2 shows promise as an objective measure of cerebral development in the perinatal period.

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.

Use of mitochondrial inhibitors to demonstrate that cytochrome oxidase near-infrared spectroscopy can measure mitochondrial dysfunction noninvasively in the brain.

The use of near-infrared spectroscopy to measure noninvasively changes in the redox state of cerebral cytochrome oxidase in vivo is controversial. We therefore tested these measurements using a multiwavelength detector in the neonatal pig brain. Exchange transfusion with perfluorocarbons revealed that the spectrum of cytochrome oxidase in the near-infrared was identical in the neonatal pig, the adult rat, and in the purified enzyme. Under normoxic conditions, the neonatal pig brain contained 15 micromol/L deoxyhemoglobin, 29 micromol/L oxyhemoglobin, and 1.2 micromol/L oxidized cytochrome oxidase. The mitochondrial inhibitor cyanide was used to determine whether redox changes in cytochrome oxidase could be detected in the presence of the larger cerebral hemoglobin concentration. Addition of cyanide induced full reduction of cytochrome oxidase in both blooded and bloodless animals. In the blooded animals, subsequent anoxia caused large changes in hemoglobin oxygenation and concentration but did not affect the cytochrome oxidase near-infrared signal. Simultaneous blood oxygenation level-dependent magnetic resonance imaging measurements showed a good correlation with near-infrared measurements of deoxyhemoglobin concentration. Possible interference in the near-infrared measurements from light scattering changes was discounted by simultaneous measurements of the optical pathlength using the cerebral water absorbance as a standard chromophore. We conclude that, under these conditions, near-infrared spectroscopy can accurately measure changes in the cerebral cytochrome oxidase redox state.

Temporal and anatomical variations of brain water apparent diffusion coefficient in perinatal cerebral hypoxic-ischemic injury: relationships to cerebral energy metabolism.

Cerebral apparent diffusion coefficients (ADCs) were determined in nine newborn piglets before and for 48 h after transient hypoxia-ischemia. Phosphorus MRS revealed severely reduced cerebral energy metabolism during the insult and an apparently complete recovery 2 h after resuscitation commenced. At this time, mean ADC over the imaging slice (ADCglobal) was 0.88 (0.04) x 10(-9) m2 x s(-1) (mean (SD)), which was close to the baseline value of 0.92 (0.4) x 10(-9) m2 x s(-1). In seven of the animals, a "secondary" failure of energy metabolism then evolved, accompanied by a decline in ADCglobal to 0.64 (0.17) x 10(-9) m2 x s(-1) at 46 h postresuscitation (P < 0.001 versus baseline). For these seven animals, ADCglobal correlated linearly with the concentration ratio [phosphocreatine (PCr)]/[inorganic phosphate (Pi)] (0.94 < r < 0.99; P < 0.001). A nonlinear relationship was demonstrated between ADCglobal and the concentration ratio [nucleotide triphosphate (NTP)]/[Pi + PCr + 3 NTP]. The ADC reduction commenced in the parasagittal cortex before spreading in a characteristic pattern throughout the brain. ADC seems to be closely related to cerebral energy status and shows considerable potential for the assessment of hypoxic-ischemic injury in the newborn brain.

Organ pathology following mild hypothermia used as neural rescue therapy in newborn piglets.

The aim of this study was to assess the possible adverse effects of hypothermia, used as neural rescue therapy in a newborn piglet model. Sixteen newborn piglets were subjected to transient cerebral hypoxia-ischaemia by temporary occlusion of the carotid arteries and reduction of the fractional inspired oxygen to 0.12. On resuscitation 11 piglets were maintained normothermic (38.5-39.0 degrees C) and, in order to assess the cerebroprotective effect of hypothermia, 5 piglets were cooled to 35 degrees C for 12 h before normothermia was resumed. At 48 or 64 h following resuscitation the animals were sacrificed and the heart, left kidney, specimens of distal small bowel, lung and liver were removed and histologically sectioned. No microscopic abnormalities of the heart, bowel or lung were observed in hypothermic or normothermic animals. All kidney specimens were normal except one from the normothermic group. Abnormal liver pathology suggestive of hypoperfusion injury was found in 5 normothermic and 3 hypothermic piglets. There was no significant difference in the proportion of piglets with liver abnormality between the two groups. Mild hypothermia following cerebral hypoxia-ischaemia in the newborn piglet was not associated with an increased incidence of non-cerebral organ damage. The hepatic injury observed may be related to umbilical venous catheterisation and has potential relevance to neonatal intensive care.

Relation of impaired energy metabolism to apoptosis and necrosis following transient cerebral hypoxia-ischaemia.

This study investigated whether both mild and severe hypoxia-ischaemia (HI) caused significant numbers of cells to die by apoptosis in the developing brain in vivo. Newborn piglets were subjected to transient global HI and the fraction of all cells in the cingulate gyrus that were apoptotic or necrotic counted 48 h after resuscitation. The mean (S.D.) proportion of apoptotic cells was 11.9% (6.7%) (sham operated controls 4.1% (2.7%)), while 11.4% (8.4%) were necrotic (controls 0.7% (1.3%)) (P<0.05). Apoptotic and necrotic cell counts were both linearly related to the severity of impaired cerebral energy metabolism measured by magnetic resonance spectroscopy (P<0.05), as shown by: (1) the decline in the ratio of nucleotide triphosphates to the exchangeable phosphate pool during HI; (2) the fall in the ratio of phosphocreatine to inorganic phosphate 8 - 48 h after HI; and (3) an increased ratio of lactate to total creatine at both these times. Thus both apoptosis and necrosis occurred in the cingulate gyrus after both severe and mild HI in vivo in proportion to the severity of the insult.

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 hypoxiaischemia, 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 hypoxiaischemia, 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 hypoxiaischemia 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.

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 degrees C for 12 h after which normothermia (38.5 degrees 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 sham-operated 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.

Magnesium sulfate after transient hypoxia-ischemia fails to prevent delayed cerebral energy failure in the newborn piglet.

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 i.v. magnesium sulfate (MgSO4) after severe transient cerebral hypoxia-ischemia decreases the severity of delayed energy failure in the newborn piglet. Twelve 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 virtually to zero, and nucleotide triphosphate (NTP) had fallen below a third of baseline. The piglets were randomized to receive, blind, either: 1) three i.v. infusions of 12.5% MgSO4 heptahydrate solution: 400 mg.kg-1 MgSO4.7H2O starting 1 h after resuscitation, and 200 mg.kg-1 12 and 24 h later (n = 6); or 2) three infusions of placebo, 0.9% NaCl (n = 6). Phosphorus and proton spectroscopy were continued until 48 h after resuscitation, and values were compared between the two groups. Mean plasma magnesium levels, 1 h after each of the three doses of MgSO4, were 2.1, 2.0, and 1.9 mmol.L-1, respectively. The severity of the primary insult, determined by the time-integral of depletion of cerebral [NTP]/[exchangeable phosphate pool (EPP)], was similar in the MgSO4-treated and placebo groups. After resuscitation, there was no difference in the progression or severity of delayed energy failure between the two groups, as judged by cerebral [PCr]/[Pi], [NTP]/[EPP], or lactate/creatine and N-acetylaspartate/creatine peak-area ratios. We conclude that MgSO4 did not decrease the severity of delayed cerebral energy failure.

Apoptosis and necrosis in the newborn piglet brain following transient cerebral hypoxia-ischaemia.

We have used a porcine model of global hypoxia-ischaemia to examine the mode and extent of cell damage to the newborn brain. Apoptosis and necrosis were observed in neurons and glial cells following transient cerebral hypoxic-ischaemic injury (HII) by haematoxylin and eosin staining and by in situ end labelling (ISEL). Quantitative neuropathological analysis of the cingulate gyrus, the hippocampus and the cerebellum showed that the degree of both apoptosis and necrosis increased with the severity of injury in these brain areas. The hippocampus and cerebellar cortex were particularly sensitive to HII. Furthermore, some cell types were more susceptible to a particular mode of cell death. In the cerebellum. Purkinje cells died by necrosis but never by apoptosis. In contrast, cerebellar granule cells were frequently apoptotic, but never necrotic. In the hippocampus, apoptosis occurred in the inner layer neurons of the dentate fascia and necrosis in the more mature outer layer neurons. This suggests that immature neurons may be more prone to apoptotic death while terminally differentiated neurons die by necrosis. Apoptosis but not necrosis was seen in cerebral white matter. This model may help to elucidate the factors that determine cell fate following HII and aid the development of cerebroprotective strategies.

Anisotropic water diffusion in white and gray matter of the neonatal piglet brain before and after transient hypoxia-ischaemia.

Measurements of tissue water apparent diffusion coefficient (ADC) performed with diffusion sensitization applied separately along the x, y, and z axes revealed significant diffusion anisotropy in both cerebral white and gray matter in six newborn (< 24 h old) piglets. Mean baseline white matter ADC for a particular region of interest was 125.8% (SD 32.0%; p < .001) greater when the diffusion gradients were applied along the y axis as compared to along the x. For the cortical gray matter region considered, the situation was reversed, the mean ADC value measured along x exceeding that along y by 15.2% (SD 6.1%; p < .01). Forty-three hours subsequent to a transient cerebral hypoxic-ischaemic insult, phosphorous MRS measurements indicated that the animals had suffered severe secondary cerebral energy failure. This was accompanied by a significant (p < .01) decrease in the white matter anisotropy, such that the mean y direction ADC now exceeded that along the x by only 70.9% (SD 29.4%; p < .03). There was no change in the gray matter anisotropy. The average of the ADC values measured in the x, y, and z directions had decreased by 35.3% (SD 18.5%; p < .01) in white matter and 31.4% (SD 21.9%; p < .05) in cortical gray matter. Diffusion anisotropy measurements may provide additional information useful in the characterisation of hypoxic-ischaemic injury in the neonatal brain, and must be considered if tissue water ADC values are to be unambiguously interpreted in this context.