S. T. Bjorkman

Seizures are associated with brain injury severity in a neonatal model of hypoxia-ischemia.

Hypoxia-ischemia is a significant cause of brain damage in the human newborn and can result in long-term neurodevelopmental disability. The loss of oxygen and glucose supply to the developing brain leads to excitotoxic neuronal cell damage and death; such over-excitation of nerve cells can also manifest as seizures. The newborn brain is highly susceptible to seizures although it is unclear what role they have in hypoxic-ischemic (H/I) injury. The aim of this study was to determine an association between seizures and severity of brain injury in a piglet model of perinatal H/I and, whether injury severity was related to type of seizure, i.e. sub-clinical (electrographic seizures only) or clinical (electrographic seizures+physical signs). Hypoxia (4% O(2)) was induced in anaesthetised newborn piglets for 30 min with a final 10 min period of hypotension; animals were recovered and survived to 72 h. Animals were monitored daily for seizures both visually and with electroencephalogram (EEG) recordings. Brain injury was assessed with magnetic resonance imaging (MRI), (1)H-MR spectroscopy ((1)H-MRS), EEG and by histology (haematoxylin and eosin). EEG seizures were observed in 75% of all H/I animals, 46% displayed clinical seizures and 29% sub-clinical seizures. Seizure animals showed significantly lower background amplitude EEG across all post-insult days. Presence of seizures was associated with lower cortical apparent diffusion coefficient (ADC) scores and changes in (1)H-MRS metabolite ratios at both 24 and 72 h post-insult. On post-mortem examination animals with seizures showed the greatest degree of neuropathological injury compared to animals without seizures. Furthermore, clinical seizure animals had significantly greater histological injury compared with sub-clinical seizure animals; this difference was not apparent on MRI or (1)H-MRS measures. In conclusion we report that both sub-clinical and clinical seizures are associated with increased severity of H/I injury in a term model of neonatal H/I.

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

Standard loading controls are not reliable for western blot quantification across brain development or in pathological conditions

A frequently utilized method of data quantification in Western blot analysis is comparison of the protein of interest with a house keeping gene or control protein. Commonly used proteins include β‐actin, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), and α‐tubulin. Various reliability issues have been raised when using this technique for data analysis—particularly when investigating protein expression changes during development and in disease states. In this study, we have demonstrated that β‐actin, GAPDH, and α‐tubulin are not appropriate controls in the study of development and hypoxic‐ischemic induced damage in the piglet brain. We have also shown that using an in‐house pooled standard, loaded on all blots is a reliable method for controlling interassay variability and data normalization in protein expression analysis.

Developmental Expression and Distribution of GABAA Receptor α1-, α3- and β2-Subunits in Pig Brain

The principal function of the γ-aminobutyric acid (GABA) system in the adult brain is inhibition; however, in the neonatal brain, GABA provides much of the excitatory drive. As the brain develops, transmembrane chloride gradients change and the inhibitory role of GABA is initiated and continues throughout juvenile and adult life. Previous studies have shown that GABA_A receptor subunit expression is developmentally regulated, and it is thought that the change in GABA function from excitation to inhibition corresponds to the changeover in expression of ‘immature’ to ‘mature’ subunit isoforms. We examined the protein expression pattern and distribution of GABA type A (GABA_A) receptor α₁-, α₃- and β₂-subunits in the parietal cortex and hippocampus of the developing piglet brain. Four perinatal ages were studied; 14 days preterm (P–14), 10 days preterm (P–10), day of birth (P0) and at postnatal day 7 (P7). Animals were obtained by either caesarean section or spontaneous birth. Protein expression levels and subunit localization were analysed by Western blotting and immunohistochemistry, respectively. In the cortex and hippocampus, GABA_A receptor α₁-subunit showed greatest expression at P7 when compared to all other age groups (p < 0.05). In contrast, α₃ expression in the cortex was elevated in preterm brain, peaking at P0, followed by a significant reduction by P7 (p < 0.05); a similar trend was observed in the hippocampus. GABA_A receptor β₂-subunit protein expression appeared relatively constant across all time points studied in both the cortex and hippocampus. Immunolabelling of the α₁-, α₃- and β₂-subunits was observed throughout all cortical layers at every age. GABA_A receptor α₃-subunit appeared to show specific localization to layers V and VI whilst labelling for the β₂-subunit was observed in layer IV. In the hippocampus of all animals, the α₁- and β₂-subunits were shown to immunolabel various cells and processes in the dentate gyrus (DG), CA1 and CA3; the α₃-subunit was barely observed except at the stratum moleculare of the DG. We report for the first time the ontogenesis of GABA_A receptor subunits α₁, α₃ and β₂ in the perinatal pig brain.

GABAA receptor expression and white matter disruption in intrauterine growth restricted piglets

Intrauterine growth restriction (IUGR) is one of the most common causes of perinatal mortality and morbidity. White matter and neuronal injury are major pathophysiological features of the IUGR neonatal brain. GABAA (γ‐aminobutyric acid type A) receptors have been shown to play a role in oligodendrocyte differentiation and proliferation in the neonatal brain and may be a key factor in white matter injury and myelination in IUGR neonates. Whether there are impairments to the GABAergic system and neuronal cytoskeleton in IUGR brain has yet to be elucidated. This study aims to examine GABAA receptor α1 and α3 subunit protein expression and distribution in parietal cortex and hippocampus of the IUGR piglet at four different ages (term = 115 d – days gestational age), 100 d, 104 d, birth (postnatal day 0–P0) and P7 and to examine neuronal and myelination patterns. Significant alterations to GABAA receptor α1 and α3 protein expression levels were observed in the IUGR piglet brain of P7 IUGR piglets with significantly greater α3 expression compared to α1 expression in the hippocampus while there was virtually no difference between the two subunits in the parietal cortex. However a significantly lower α1/α3 ratio was evident in P7 IUGR cortex when compared with P7 NG cortex. Neuronal somatodendrites studied using MAP2 immunohistochemistry showed reduced and disrupted somatodendrites while MBP immunolabelling showed loss of axonal fibres from gestational day 104 d through to P7. These findings provide insights into the effects of IUGR on the development of the GABA system, altered developmental maturation of GABAA receptor subunit expression in the IUGR brain may influence myelination and may partly explain the cognitive disabilities observed in IUGR. Understanding the mechanisms behind grey and white matter injury in the IUGR infant is essential to identifying targets for treatments to improve long‐term outcomes for IUGR infants.

A Pig Model of the Preterm Neonate: Anthropometric and Physiological Characteristics

Background and Aims Large animal models are an essential research tool to investigate the physiology of the preterm infant, which remains poorly understood. We aim to describe the pig model of the preterm neonate in terms of growth, maturation and requirement for intensive care over a range of gestational ages and determine the effects of maternal glucocorticoid exposure and sex. Methods Twenty-nine litters of piglets (N=305) were delivered by C-section at 91d, 94d, 97d, 100d, 104d and 113d (term 115d). Some litters received maternal betamethasone treatment (0.19mg/kg body wt; IM) at 48h and 24h prior to delivery. At 97d piglets were resuscitated, surfactant administered, and piglets were ventilated, sedated and monitored for 6–8h post-birth using standard NICU techniques. Results At 91d, piglets were half the weight of term animals, had fused eyelids, very thin skin, no hair, and survived a maximum of 3h due to difficulties with ventilation. At 97d piglets were able to be maintained for at least 6–8h but physiology was unstable for 1–2h. Piglets 100d and older breathed spontaneously. Only near term piglets were able to maintain body temperatures. Males were heavier than females at 113d gestation (p=0.021). Exposure to maternal glucocorticoids resulted in larger females and influenced brain:body wt. Conclusions The piglet provides a useful model of preterm neonatal physiology as very preterm piglets can be survived under standard intensive care conditions. The large litters allow for parallel experiments or the use of littermates as controls.

302 Seizure Burden and Neurobehavioral Scores after Therapeutic Hypothermia in the Newborn Piglet

Background Therapeutic hypothermia (TH) is standard of care in newborns with hypoxic-ischemic encephalopathy (HIE). Although the predictive value of amplitude-integrated EEG (aEEG) after HIE has been studied, the predictive value of aEEG during TH remains to be established. Aim To study aEEG characteristics and timing of recovery of neurobehavior in a newborn piglet model of HIE following TH. Methods Newborn piglets (N=14) were subjected to 30 min hypoxia-ischemia and survived to 72h. Animals were randomly assigned to hypothermia (N=8) or normothermia for 24h after hypoxia-ischemia (N=6). aEEG was continuously recorded until ~40h post-insult and at 48 and 72h post-insult. Background pattern aEEG and presence of seizures were analysed. Neurobehavior was scored from 40 until 72h post-insult. Results In hypothermic piglets aEEG background pattern recovered to continuous low voltage (CLV) within 2h post-insult until 36h post-insult. Normothermic piglets recovered within 2h post-insult to continuous normal voltage (CNV) until 36h, where there was a decrease in background pattern to CLV. aEEG recovered to CNV in both groups by 72h post-insult. Seizures were recorded in 50% of hypothermic piglets c.f. 83% in normothermic piglets. At 48h post insult both groups showed a maximum of epileptic activity. The neurobehavioral score in the normothermic piglets showed an earlier return to baseline compared to the hypothermic piglets. Conclusions Electrographic seizure burden was decreased following TH. aEEG background pattern and neurobehaviour score recovered earlier in normothermic piglets, suggesting that in the clinical situation conclusions based on aEEG and neurological examination should not be performed too early.

Hypercapnic acidosis following hypoxia in newborn piglet, a potential neuroprotectant?

catenin, known to serve as a linker between the cadherin and actin cytoskeleton, results in destabilization of synaptic contacts. On the contrary, overexpression of this catenin causes excess spine formation and reduced spine turnover. Pharmacological suppression of neural activities in hippocampal cultures induces a release of a N-catenin from synapses, whereas elevation of neural activities have opposite effects, i.e., enrichment of this molecule in synapses, suggesting the existence of an activity-dependent mechanism to control the association of a Ncatenin with synapses. In addition, it is known that a number of different cadherin subtypes with distinct adhesive specificities, generated due to the sequence diversity of their extracellular domain, are expressed in the nervous system, and each neuron has a unique set of these cadherins. These observations suggest that the cadherin–catenin complex regulates synapse dynamics from the cytoplasmic side, and possibly synaptic specificity from the extracellular side, although the latter idea is awaiting experimental tests. PL1 CONTROL OF SYNAPTIC JUNCTION DYNAMICS: ROLES OF THE CADHERIN–CATENIN COMPLEX Takeichi, M. RIKEN Center for Developmental Biology, Kobe, Japan