S. Tracey Bjorkman

Cytoskeletal Anchoring of GLAST Determines Susceptibility to Brain Damage AN IDENTIFIED ROLE FOR GFAP

Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of d-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.

Hypoxic/Ischemic models in newborn piglet: Comparison of constant FiO2 versus variable FiO2 delivery

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 <5 microV) including 10 min of low mean arterial blood pressure (MABP <70% of baseline). Control animals (n = 6) received 21% FiO2 for the duration of the experiment. At 72 h, the piglets were euthanased, their brains removed and fixed in 4% paraformaldehyde and assessed for hypoxic/ischemic injury by histological analysis. Based on neuropathology scores, piglets were grouped as undamaged or damaged; piglets that did not survive to 72 h were grouped separately as dead. The variable insult resulted in a greater number of piglets with neuropathological damage (undamaged = 12.5%, damaged = 68.75%, dead = 18.75%) while the constant insult resulted in a large proportion of undamaged piglets (undamaged = 50%, damaged = 22.2%, dead = 27.8%). A hypoxic insult varied to maintain peak amplitude EEG <5 microV results in a greater number of survivors with a consistent degree of neuropathological damage than a constant hypoxic insult. Physiological variables MABP, LAEEG, pH and arterial base excess were found to be significantly associated with neuropathological outcome.

Rapid loss of glutamine synthetase from astrocytes in response to hypoxia: Implications for excitotoxicity

We have examined brains of neonatal pigs that were rendered hypoxic. Glutamine synthetase (GS), a key enzyme in the detoxification of glutamate and ammonia, was rapidly lost from astrocytes in regions susceptible to damage, including the CA1 of hippocampus and various cortical regions. Conversely, resilient areas such as the dentate gyrus exhibited little or no loss of GS. Onset of loss was rapid, patches of loss being evident by 1h post-insult, and loss was extensive by 24h and did not recover by 72 h. Examination of counterstained sections revealed that GS losses preceded any overt neuronal damage. Loss of GS from astrocytes would plausibly lead to a rise in intracellular glutamate, and could explain why reversal of astrocytic glutamate transport during hypoxia/ischaemia is conceptually possible.

Structural remodeling of gray matter astrocytes in the neonatal pig brain after hypoxia/Ischemia

Astrocytes play a vital role in the brain; their structural integrity and sustained function are essential for neuronal viability, especially after injury or insult. In this study, we have examined the response of astrocytes to hypoxia/ischemia (H/I), employing multiple methods (immunohistochemistry, iontophoretic cell injection, Golgi‐Kopsch staining, and D‐aspartate uptake) in a neonatal pig model of H/I. We have identified morphological changes in cortical gray matter astrocytes in response to H/I. Initial astrocytic changes were evident as early as 8 h post‐insult, before histological evidence for neuronal damage. By 72 h post‐insult, astrocytes exhibited significantly fewer processes that were shorter, thicker, and had abnormal terminal swellings, compared with astrocytes from control brains that exhibited a complex structure with multiple fine branching processes. Quantification and image analysis of astrocytes at 72 h post‐insult revealed significant decreases in the average astrocyte size, from 686 μm2 in controls to 401 μm2 in H/I brains. Sholl analysis revealed a significant decrease (>60%) in the complexity of astrocyte branching between 5 and 20 μm from the cell body. D‐Aspartate uptake studies revealed that the H/I insult resulted in impaired astrocyte function, with significantly reduced clearance of the glutamate analog, D‐aspartate. These results suggest that astrocytes may be involved in the pathophysiological events of H/I brain damage at a far earlier time point than first thought. Developing therapies that prevent or reverse these astrocytic changes may potentially improve neuronal survival and thus might be a useful strategy to minimize brain damage after an H/I insult.

Review: Neuroinflammation in intrauterine growth restriction

Disruption to the maternal environment during pregnancy from events such as hypoxia, stress, toxins, inflammation, and reduced placental blood flow can affect fetal development. Intrauterine growth restriction (IUGR) is commonly caused by chronic placental insufficiency, interrupting supply of oxygen and nutrients to the fetus resulting in abnormal fetal growth. IUGR is a major cause of perinatal morbidity and mortality, occurring in approximately 5-10% of pregnancies. The fetal brain is particularly vulnerable in IUGR and there is an increased risk of long-term neurological disorders including cerebral palsy, epilepsy, learning difficulties, behavioural difficulties and psychiatric diagnoses. Few studies have focused on how growth restriction interferes with normal brain development in the IUGR neonate but recent studies in growth restricted animal models demonstrate increased neuroinflammation. This review describes the role of neuroinflammation in the progression of brain injury in growth restricted neonates. Identifying the mediators responsible for alterations in brain development in the IUGR infant is key to prevention and treatment of brain injury in these infants.

Short-Term Dose–Response Characteristics of 2-Iminobiotin Immediately Postinsult in the Neonatal Piglet After Hypoxia-Ischemia

Background and Purpose— To determine the optimal dose of 2-iminobiotin (2-IB) for the treatment of moderate to severe asphyxia in a neonatal piglet model of hypoxia-ischemia. Methods— Newborn piglets were subjected to a 30-minute hypoxia-ischemia insult and randomly treated with vehicle or 2-IB (0.1 mg/kg, 0.2 mg/kg, or 1.0 mg/kg). aEEG background and seizure activity were scored after hypoxia-ischemia every 4 h until 24 h and at 48 h and neurobehavioral scores were obtained. Brain tissue was collected and processed for analysis of caspase-3 activity, histology, and tyrosine nitration. Results— A dose range of 0.1 to 1.0 mg/kg/dose of 2-IB improved short-term outcome as demonstrated by an increased survival with a normal aEEG and decreased nitrotyrosine staining in the 2-IB–treated animals, indicating decreased cellular damage. Neurobehavior, caspase-3 activity in thalamus, and histology scores were not significantly different. Conclusions— Based on survival with a normal aEEG, 0.2 mg/kg 2-IB is likely to be the most appropriate dose for use in future clinical trials in neonates with perinatal hypoxia-ischemia.

Neonatal seizures are associated with redistribution and loss of GABAA α‐subunits in the hypoxic‐ischaemic pig

Seizures are a common manifestation of hypoxic‐ischaemic brain injury in the neonate. In status epilepticus models alterations to GABAAR subunit expression have been suggested to contribute to (i) abnormal development of the GABAergic system, (ii) why seizures become self‐sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABAAR subunit protein expression after neonatal hypoxia‐ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α‐subunits of the GABAAR; α1, α2 and α3. Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude‐integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post‐mortem and GABAAR α‐subunit protein expression was analysed using western blot and immunohistochemistry. GABAAR α1 and α3 protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α1 and α3 expression, and α1 redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABAAR α3 reduction. This is the first study to show loss and redistribution of GABAAR α‐subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.

Origin and Detection of Neonatal Seizures: Animal and Clinical Studies

Neonatal seizures remain a major clinical problem worldwide and are harmful to the developing brain. Seizures are associated with poor neurodevelopmental outcomes and significant risk of death requiring urgent diagnosis and intervention. Current antiepileptic drugs however have limited efficacy and are potentially harmful to the developing newborn brain. Despite this, standard clinical practice for the treatment of neonatal seizures remains unchanged. This chapter describes a clinically relevant neonatal animal model of HI-induced seizures.

Therapeutic potential to reduce brain injury in growth restricted newborns

Brain injury in intrauterine growth restricted (IUGR) infants is a major contributing factor to morbidity and mortality worldwide. Adverse outcomes range from mild learning difficulties, to attention difficulties, neurobehavioral issues, cerebral palsy, epilepsy, and other cognitive and psychiatric disorders. While the use of medication to ameliorate neurological deficits in IUGR neonates has been identified as warranting urgent research for several years, few trials have been reported. This review summarises clinical trials focusing on brain protection in the IUGR newborn as well as therapeutic interventions trialled in animal models of IUGR. Therapeutically targeting mechanisms of brain injury in the IUGR neonate is fundamental to improving long‐term neurodevelopmental outcomes. Inflammation is a key mechanism in neonatal brain injury; and therefore an appealing target. Ibuprofen, an anti‐inflammatory drug currently used in the preterm neonate, may be a potential therapeutic candidate to treat brain injury in the IUGR neonate. To better understand the potential of ibuprofen and other therapeutic agents to be neuroprotective in the IUGR neonate, long‐term follow‐up information of neurodevelopmental outcomes must be studied. Where agents such as ibuprofen are shown to be effective, have a good safety profile and are relatively inexpensive, they can be widely adopted and lead to improved outcomes.

Supporting Preterm Cardiovascular Function

Preterm infants are at higher risk of adverse neurodevelopmental outcomes. Inadequate cerebral oxygen delivery resulting from poor cardiovascular function is likely to be a significant contributor to preterm brain injury. In this context, improved support of cardiovascular function is integral to improving preterm outcomes. Many of the treatments used to support preterm cardiovascular function are based on adult physiology and may not be appropriate for the unique physiology of the preterm infant. The preterm heart is structurally immature with reduced contractility and low cardiac output. However, there is limited evidence that inotropic support with dopamine and/or dobutamine is effective in preterm babies. Hypovolemia may also contribute to poor preterm cardiovascular function; there is evidence that capillary leakage results in considerable loss of plasma from the circulation of newborn preterm babies. In addition, the vasoconstrictor response to acute stimuli does not develop until quite late in gestation and is limited in the preterm infant. This may lead to inappropriate vasodilatation adding to functional hypovolemia. The first line treatment for hypotension in preterm infants is volume expansion with crystalloid solutions, but this has limited efficacy in the preterm infant. More effective methods of volume expansion are required. Effective support of preterm cardiovascular function requires better understanding of preterm cardiovascular physiology so that treatments can target mechanisms that are sufficiently mature to respond.