S. M. Sullivan

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.

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.

MAP2 provides reliable early assessment of neural injury in the newborn piglet model of birth asphyxia

Reduction in microtubule-associated-protein-2 (MAP2) immunoreactivity is a sensitive and quantifiable early marker of neural injury in rats. This study assessed the reliability of MAP2 as an early marker of neural injury following hypoxia/ischaemia in neonatal piglets, and compared the effects of perfusion and immersion fixation on MAP2 immunoreactivity. Hypoxia was induced in newborn piglets (n=23) by reducing the FiO2 to 4% for 0, 25, 35 or 50 min. Six hours after the end of hypoxia piglets were killed, and the brain removed and immunolabelled for MAP2. Significant reductions in MAP2 immunoreactivity were seen in cortex, hippocampus, basal ganglia and thalamus. Reductions correlated with duration of hypoxia, pH at the end of hypoxia, cerebral function monitor amplitude and cerebral impedance 6h after hypoxia, and with early histological evidence of ischaemic changes. Regions with reduced immunoreactivity correlated with areas where damage is present in later histological examination in this model. Immersion fixation with postmortem delays up to 30 min did not affect MAP2 immunoreactivity compared to perfusion-fixed tissue. Results indicate that MAP2 immunoreactivity 6h after hypoxia/ischaemia is a reliable marker of neural injury in the neonatal piglet.

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.

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.