P N Amess

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.

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.

MRI measurements of cerebral deoxyhaemoglobin concentration [dHb] - correlation with near infrared spectroscopy (NIRS)

Changes in physiological parameters such as cerebral blood flow, cerebral blood volume, oxygen extraction, and the size and distribution of cerebral blood vessels, result in changes in the local concentration of deoxyhaemoglobin ([dHb]). The purpose of this study was to quantitatively investigate the dependence of the R2* relaxation rate upon the [dHb] per voxel. Five neonatal piglets were studied in a 7 T/20 cm bore magnet. MRI was conducted using a 2.5 cm diameter surface coil placed over the parietal lobes. Four progressively T2*‐weighted images were acquired, allowing the absolute quantitation of R2*. Simultaneous near infrared spectroscopy (NIRS) measurements were made from an area encompassing the MR imaging slice, and allowed the absolute quantitation of [dHb]. The arterial oxygen saturation (SaO2) of the piglet was lowered stepwise by decreasing the fractional inspired oxygen concentration (FiO2), which precipitated a change in [dHb]. NIRS and MRI measurements were made at each FiO2 step. The results demonstrate an extremely strong, linear relationship between R2* as determined by MRI and [dHb], as measured by NIRS. Whereas NIRS can only give us a global measure of [dHb], the results suggest the future use of MRI in producing high resolution relaxation rate maps related to the [dHb] distribution of the brain.

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 μmol/L deoxyhemoglobin, 29 μmol/L oxyhemoglobin, and 1.2 μmol/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.

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.

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.

Measurement of cytochrome oxidase redox state by near infrared spectroscopy.

Although near infrared spectroscopy (NIRS) is primarily used to probe changes in oxyhaemoglobin (HbO2) and deoxyhaemoglobin (dHb) concentrations, it has long been realised that there is a significant oxygen-concentration dependent near infrared signal from the mitochondrial enzyme cytochrome c oxidase. In this paper we discuss the origins of this near infrared (NIR) signal, the possible factors affecting its intensity and its likely physiological and clinical significance. This paper complements our recent review on this subject1.

Early cerebral-metabolite quantification in perinatal hypoxic-ischaemic encephalopathy by proton and phosphorus magnetic resonance spectroscopy

Acute cerebral hypoxia-ischaemia, often due to birth asphyxia, is the major cause of perinatal brain injury. Phosphorus (31P) magnetic resonance spectroscopy (MRS) enables non-invasive monitoring of cerebral high-energy metabolites such as phosphocreatine (PCr) and nucleotide triphosphates (NTP; mainly adenosine triphosphate) and can evaluate hypoxic-ischaemic cerebral damage in the newbom.1,2 Following severe birth asphyxia, 31P spectra often appear normal for up to 24 h presumably due to apparent recovery of energy generation soon after the acute intrapartum insult. However, subsequent spectra often show reduced [ PCr] / [ inorganic phosphate (Pi)] : a measure of phosphorylation potential. In very severe cases, [NTP] eventually falls indicating impaired cerebral energy generation. This sequence of events has been termed “secondary energy failure” (SEF) .’ Proton (‘H) MRS has also been applied to studies of newborn brain enabling observation of choline-containing compounds (Cho) , total creatine (Cr) , N-acetylaspartate (Naa) and lactate (Lac) .3-5 Both 31P and ‘H spectra can be local&d to specific brain regions4s6 and metabolite concentrations can be quantitated.4~5~7*8 This report describes the early application of quantitative, local&d, ‘H and 31P MRS in severe perinatal hypoxic-ischaemic encephalopathy.

Correlation between Absolute Deoxyhaemoglobin [dHb] Measured by Near Infrared Spectroscopy (NIRS) and Absolute R2′ as Determined by Magnetic Resonance Imaging (MRI)

Magnetic Resonance Imaging (MRI) is a technique which provides high spatial resolution combined with a wide range of image contrast mechanisms. Standard MR image contrast relies upon tissue differences in MR visible hydrogen spin density, spin-lattice (T1) relaxation and spin-spin (T2) relaxation. In soft tissue, the variation of MR visible hydrogen spin density between tissues types is small (up to 30%) whereas T1 and T2 relaxation times can vary considerably (>100%). However, despite this sensitivity, these standard forms of contrast mainly reflect the longer term disruption of tissue structure. Since alterations in tissue function normally precede structural damage, MR image contrast which accurately reflects this tissue function holds the promise of earlier detection of pathophysiology.