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White matter damage is a significant problem in the human pre-term baby. Damage to white matter is usually associated with injury or insults to babies born prematurely, typically before 32 weeks' gestation, however there is increasing evidence of both grey and white matter damage occurring after 32 weeks' gestation. Astrocytes play a vital role in white matter, regulating molecules such as glutamate in the extracellular space and preventing excitotoxic damage to neighbouring oligodendrocytes and axons. We have previously described dramatic changes in grey matter astrocytes in response to a hypoxic/ischemic (H/I) insult around the time of birth. In this study, we have used GFAP immunohistochemistry and Golgi-Kopsch staining to examine the morphology of white matter astrocytes in control neonatal pig brains, and in the brains of animals exposed to the same (perinatal) H/I insult. We demonstrate that the areal percentage of the section occupied by GFAP-immunoreactive processes and cell bodies is significantly decreased (by 46%, P < 0.0001) in subcortical white matter from H/I brains. This loss of GFAP was accompanied by alterations in astrocyte morphology and an overall decrease in the size (field of section occupied by an individual astrocyte) of white matter astrocytes from 649 mu m(2) to 426 mu m(2), as revealed by Golgi-Kopsch staining and image analysis. These data suggest that astrocytes may contribute to the pathology of white matter damage following an H/I insult around the time of birth, and suggest that astrocytes may offer a novel target for therapies to improve outcomes after H/I. (C) 2010 Elsevier B.V. All rights reserved.

Uteroplacental vascular insufficiency in humans is a common cause of intrauterine growth restriction (IUGR) and is associated with an increased incidence of perinatal asphyxia and neurodevelopmental disorders compared to normal weight newborns. Experimental models that provide an opportunity to analyze the pathogenesis of these relationships are limited. Here, we used neonatal pigs from large litters in which there were piglets of normal birth weight (for controls) and of low birth weight (for uteroplacental vascular insufficiency). Hypoxia was induced in paired littermates by reducing the fraction of inspired oxygen to 4% for 25 min. Brain tissue was collected 4 h post-hypoxia. Cerebral levels of apoptosis were quantified morphologically and verified with caspase-3 activity and TUNEL. Expression of Bcl-2, Bcl-XL and Bax proteins was investigated using immunohistochemistry. Cellular positivity for Bcl-2 was consistently higher in the non-apoptotic white matter of the hypoxic IUGR animals compared with their littermates and reached significance at P < 0.05 in several pairs of littermates. Alterations in Bax showed a trend towards higher expression in the hypoxic IUGR littermates but rarely reached significance. The IUGR piglets showed a significantly greater amount of apoptosis in response to the hypoxia than the normal weight piglets, suggesting an increased vulnerability to apoptosis in the IUGR piglets.

The purpose of this study was to develop a newborn piglet model of hypoxia/ischaemia which would better emulate the clinical situation in the asphyxiated human neonate and produce a consistent degree of histopathological injury following the insult. One-day-old piglets (n=18) were anaesthetised with a mixture of propofol (10 mg/kg/h) and alfentinal (55.5 microg/kg/h) i.v. The piglets were intubated and ventilated. Physiological variables were monitored continuously. Hypoxia was induced by decreasing the inspired oxygen (FiO(2)) to 3-4% and adjusting FiO(2) to maintain the cerebral function monitor peak amplitude at < or =5 microV. The duration of the mild insult was 20 min while the severe insult was 30 min which included 10 min where the blood pressure was allowed to fall below 70% of baseline. Control piglets (n=4 of 18) were subjected to the same protocol except for the hypoxic/ischaemic insult. The piglets were allowed to recover from anaesthesia then euthanased 72 h after the insult. The brains were perfusion-fixed, removed and embedded in paraffin. Coronal sections were stained by haematoxylin/eosin. A blinded observer examined the frontal and parietal cortex, hippocampus, basal ganglia, thalamus and cerebellum for the degree of damage. The total mean histology score for the five areas of the brain for the severe insult was 15.6+/-4.4 (mean +/-S.D., n=7), whereas no damage was seen in either the mild insult (n=4) or control groups. This 'severe damage' model produces a consistent level of damage and will prove useful for examining potential neuroprotective therapies in the neonatal brain.

Damage to major white matter tracts is a hallmark mark feature of hypoxic-ischemic (HI) brain injury in the preterm neonate. There is, however, no therapeutic intervention to treat this injury. Neuroinflammation is thought to play a prominent role in the pathogenesis of the HI-induced white matter damage but identification of the key mediators that constitute the inflammatory response remain to be fully elucidated. Cyclooxygenase enzymes (COX-1 and COX-2) are candidate neuroinflammatory mediators that may contribute to the HI-induced demise of early oligodendrocyte progenitors and myelination. We investigated whether ibuprofen, a non-steroidal anti-inflammatory drug that inhibits COX enzymes, can attenuate neuroinflammation and associated white matter damage incurred in a rodent model of preterm HI. On postnatal day 3 (P3), HI was produced (right carotid artery ligation and 30 min 6% O(2)). An initial dose of ibuprofen (100mg/kg, s.c.) was administered 2h after HI followed by a maintenance dose (50mg/kg, s.c.) every 24h for 6 days. Post-HI ibuprofen treatment significantly attenuated the P3 HI-induced increases in COX-2 protein expression as well as interleukin-1beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) levels in the brain. Ibuprofen treatment also prevented the HI-induced loss O4- and O1-positive oligodendrocyte progenitor cells and myelin basic protein (MBP)-positive myelin content one week after P3 HI. These findings suggest that a repeated, daily, ibuprofen treatment regimen administered after an HI insult may be a potential therapeutic intervention to prevent HI-induced damage to white matter progenitors and early myelination in the preterm neonate.

Serotonergic fibres have a pervasive innervation of hypoxic-ischemic (HI)-affected areas in the neonatal brain and serotonin (5-HT) is pivotal in numerous neurobehaviours that match many HI-induced deficits. However, little is known about how neonatal HI affects the serotonergic system. We therefore examined whether neonatal HI can alter numbers of serotonergic raphe neurons in specific sub-divisions of the midbrain and brainstem since these nuclei are the primary sources of serotonin throughout the central nervous system (CNS). We utilised an established neonatal HI model in the postnatal day 3 (P3) rat pup (right common carotid artery ligation+30min 6% O2) and determined the effects of P3 HI on 5-HT counts in 5 raphe sub-divisions in the midbrain and brainstem one and six weeks later. After P3 HI, numbers of 5-HT-positive neurons were significantly decreased in the dorsal raphe dorsal, dorsal raphe ventrolateral and dorsal raphe caudal nuclei on P10 but only in the dorsal raphe dorsal and dorsal raphe ventrolateral nuclei on P45. In contrast, P3 HI did not alter counts in the dorsal raphe interfascicular and raphe magnus nuclei. We also discovered that P3 HI significantly reduces brainstem SERT protein expression; the key regulator of 5-HT in the CNS. In conclusion, neonatal HI injury caused significant disruption of the brainstem serotonergic system that can persist for up to six weeks after the insult. The different vulnerabilities of serotonergic populations in specific raphe nuclei suggest that certain raphe nuclei may underpin neurological deficits in HI-affected neonates through to adulthood.

Intrauterine growth restriction (IUGR) can increase susceptibility to perinatal hypoxic brain injury for reasons that are unknown. Previous studies of the neonatal IUGR brain have suggested that the cerebral mitochondrial capacity is reduced but the glycolytic capacity increased relative to normal weight (NW) neonates. In view of these two factors, we hypothesized that the generation of brain lactate during a mild hypoxic insult would be greater in neonatal IUGR piglets compared to NW piglets. Brain lactate/N-acetylaspartate (NAA) ratios and apparent diffusion coefficients (ADCs) were determined by proton magnetic resonance spectroscopy and imaging of the brain before, during and after hypoxia in seven neonatal piglets with asymmetric IUGR and six NW piglets. During hypoxia, IUGR piglets had significantly higher brain lactate/NAA ratios than NW piglets (P = 0.046). The lactate response in the IUGR piglets correlated inversely with apoptosis in the thalamus and frontal cortex of the brain measured 4 h post hypoxia (Pearson's r = 0.86, P < 0.05). Apoptosis in IUGR piglets with high brain lactate was similar to that in the NW piglets whereas IUGR piglets with low brain lactate had significantly higher apoptosis than NW piglets (P = 0.019). ADCs in the high lactate IUGR piglets were significantly lower during hypoxia than in all the other piglets. This signifies increased diffusion of water into brain cells during hypoxia, possibly in response to increased intracellular osmolality caused by high intracellular lactate concentrations. These findings support previous studies showing increased susceptibility to hypoxic brain injury in IUGR neonates but suggest that increased glycolysis during hypoxia confers neuroprotection in some IUGR piglets.

A comparison of a constant (continuous delivery of 4% FiO2) and a variable (initial 5% FiO2 with adjustments to induce low amplitude EEG (LAEEG) and hypotension) hypoxic/ischemic insult was performed to determine which insult was more effective in producing a consistent degree of survivable neuropathological damage in a newborn piglet model of perinatal asphyxia. We also examined which physiological responses contributed to this outcome. Thirty-nine 1-day-old piglets were subjected to either a constant hypoxic/ischemic insult of 30- to 37-min duration or a variable hypoxic/ischemic insult of 30-min low peak amplitude EEG (LAEEG

Multi-frequency bio-impedance has the potential to identify infants at risk of poor neurodevelopmental outcome following hypoxia by detecting cerebral edema. This study investigated the relationship between the severity of an hypoxic/ischemic episode, neurological outcome following the hypoxia and non-invasively measured cerebral bioelectrical impedance in piglets. One-day-old piglets were anaesthetised and ventilated. Hypoxia was induced by reducing the inspired oxygen concentration to 3-5%. Severe hypoxia was defined as hypoxia resulting in at least 30 min of low amplitude EEG (

The association of sustained cerebral edema with poor neurological outcome following hypoxia-ischaemia in the neonate suggests that measurement of cerebral edema may allow early prediction of outcome in these infants. Direct measurements of cerebral impedance have been widely used in animal studies to monitor cerebral edema, but such invasive measurements are not possible in the human neonate. This study investigated the ability of noninvasive cerebral impedance measurements to detect cerebral edema following hypoxia-ischaemia. One-day-old piglets were anaesthetized, intubated and ventilated. Hypoxia was induced by reducing the inspired oxygen concentration to 4-6% O-2. Noninvasive cerebral bioimpedance was measured using gel electrodes attached to the scalp. Cerebral bioimpedance was also measured directly by insertion of two silver-silver chloride electrodes subdurally. Noninvasive and invasive measurements were made before, during and after hypoxia. Whole body impedance was measured to assess overall fluid movements. Intracranial pressure was measured continuously via a catheter inserted subdurally, as an index of cerebral edema. There was good agreement between noninvasive and invasive measurements of cerebral impedance although externally obtained responses were attenuated. Noninvasive measurements were also well correlated with intracranial pressure. Whole body impedance changes did not account for increases in noninvasively measured cerebral impedance. Results suggest that noninvasive cerebral impedance measurements do reflect intracranial events, and are able to detect cerebral edema following hypoxia-ischaemia in the neonate. (C) 2002 Elsevier Science B.V. All rights reserved.