2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and Hypoxia-Ischemia--Brain

The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis.

Topics: Animals; Animals, Newborn; Antigens; Brain Injuries; Carotid Artery Diseases; Disease Models, Animal; Excitatory Amino Acid Antagonists; Functional Laterality; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Leukoencephalopathies; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Minocycline; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Polysaccharides; Proteoglycans; Quinoxalines; Receptors, AMPA; Synapses; Vesicular Glutamate Transport Protein 1; Vesicular Glutamate Transport Protein 2

Periventricular leukomalacia is a form of hypoxic-ischemic cerebral white matter injury seen most commonly in premature infants and is the major antecedent of cerebral palsy. Glutamate receptor-mediated excitotoxicity is a predominant mechanism of hypoxic-ischemic injury to developing cerebral white matter. We have demonstrated previously the protective effect of AMPA-kainate-type glutamate receptor blockade in a rodent model of periventricular leukomalacia. The present study explores the therapeutic potential of glutamate receptor blockade for hypoxic-ischemic white matter injury. We demonstrate that AMPA receptors are expressed on developing human oligodendrocytes that populate fetal white matter at 23-32 weeks gestation, the period of highest risk for periventricular leukomalacia. We show that the clinically available anticonvulsant topiramate, when administered post-insult in vivo, is protective against selective hypoxic-ischemic white matter injury and decreases the subsequent neuromotor deficits. We further demonstrate that topiramate attenuates AMPA-kainate receptor-mediated cell death and calcium influx, as well as kainate-evoked currents in developing oligodendrocytes, similar to the AMPA-kainate receptor antagonist 6-nitro-7-sulfamoylbenzo-(f)quinoxaline-2,3-dione (NBQX). Notably, protective doses of NBQX and topiramate do not affect normal maturation and proliferation of oligodendrocytes either in vivo or in vitro. Taken together, these results suggest that AMPA-kainate receptor blockade may have potential for translation as a therapeutic strategy for periventricular leukomalacia and that the mechanism of protective efficacy of topiramate is caused at least in part by attenuation of excitotoxic injury to premyelinating oligodendrocytes in developing white matter.

Topics: Animals; Calcium; Cell Death; Cell Differentiation; Cell Division; Disease Models, Animal; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fructose; Gestational Age; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Kainic Acid; Leukomalacia, Periventricular; Movement Disorders; Neuroprotective Agents; Oligodendroglia; Quinoxalines; Rats; Receptors, AMPA; Receptors, Glutamate; Topiramate; Treatment Outcome

Glutamate release in ischaemia triggers neuronal death. The major glial glutamate transporter, GLT-1, might protect against glutamate-evoked death by removing extracellular glutamate, or contribute to death by reversing and releasing glutamate. Previous studies of the role of GLT-1 in ischaemia have often used the GLT-1 blocker dihydrokainate at concentrations that affect transporters other than GLT-1 and which affect kainate, N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In hippocampal slices from postnatal day 14 mice lacking GLT-1, the current response of area CA1 pyramidal cells to superfused AMPA and NMDA (which are not taken up) was unaffected, whereas the response to 100 microm glutamate was more than doubled relative to that in wild-type littermates, a finding consistent with a decrease in glutamate uptake. In response to a few minutes of simulated ischaemia, pyramidal cells in wild-type mice showed a large and sudden inward glutamate-evoked current [the anoxic depolarization (AD) current], which declined to a less inward plateau. In mice lacking GLT-1, the time to the occurrence of the AD current, its amplitude, the size of the subsequent plateau current and the block of the plateau current by glutamate receptor blockers were all indistinguishable from those in wild-type mice. We conclude that GLT-1 does not contribute significantly to glutamate release or glutamate removal from the extracellular space in early simulated ischaemia. These data are consistent with glutamate release being by reversal of neuronal transporters, and with uptake into glia being compromised by the ischaemia-evoked fall in the level of ATP needed to convert glutamate into glutamine.

Topics: 2-Amino-5-phosphonovalerate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Amino Acid Transporter 2; Glutamic Acid; Hippocampus; Hypoxia-Ischemia, Brain; Kainic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; Organ Culture Techniques; Patch-Clamp Techniques; Pyramidal Cells; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

This study investigated the influence of temperature or glutamate antagonism on the immediate outcome of perinatal asphyxia. Perinatal asphyxia was produced by water immersion of fetus-containing uterus horns removed by cesarean section from ready to deliver rats. The uterus horns were kept in a water bath for different time periods, before the pups were delivered and stimulated to breathe. After delivery, the pups were assessed for behavior and for systemic glutamate, aspartate, lactate and pyruvate levels measured with in vivo microdialysis, or ex vivo for energy-rich phosphates, including adenosine triphosphate (ATP), in brain, heart and kidney. In a series of experiments, asphyxia was initiated in a water bath at 37 degrees C, before the pup-containing uterus horns were moved for different time intervals to a 15 degrees C bath. In another series of experiments, the mothers were treated with N-methyl-D-aspartate (NMDA) antagonist, dizocilpine (MK-801), or alpha-amino-3-hydroxy-methylisoxazole-4-propionic acid (AMPA) antagonist,2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f) quinoxalin NBQX) 1 h before hysterectomy and asphyxia at 37 degrees C. The rate of survival rapidly decreased following exposure to more than 16 min of asphyxia, and no survival could be observed after 22 min of asphyxia. An LD50 was estimated to occur at approximately 19 min of asphyxia. The outcome was paralleled by a decrease in ATP in kidney, followed by a decrease in heart and brain. A maximal decrease in ATP was observed after 20 min of asphyxia in all tissues. Systemic microdialysis revealed that glutamate, aspartate and pyruvate levels were increased with a peak after 5 min of asphyxia. In contrast, lactate levels increased along with the length of the insult. Survival was increased when the pup-containing uterus horns were moved from a 37 degrees C to a 15 degrees C bath, at 15 min of asphyxia (the LD50 was thus increased to 30 min). If the shift occurred at 10 or 5 min of asphyxia, the LD50 increased to 80 or 110 min, respectively. The effect of glutamate antagonism was minor compared to hypothermia; the best effect (an increase in the LD50 to approximately 22 min) was observed after combining AMPA and NMDA antagonists.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Aspartic Acid; Asphyxia Neonatorum; Behavior, Animal; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Heart; Humans; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Infant, Newborn; Kidney; Lactic Acid; Maternal Behavior; Microdialysis; Pyruvic Acid; Quinoxalines; Rats; Receptors, Glutamate; Survival Rate; Treatment Outcome

The excitatory neurotransmitter glutamate is released from axons and glia under hypoxic/ischemic conditions. In vitro, oligodendrocytes (OLs) express non-NMDA glutamate receptors (GluRs) and are susceptible to GluR-mediated excitotoxicity. We evaluated the role of GluR-mediated OL excitotoxicity in hypoxic/ischemic white matter injury in the developing brain. Hypoxic/ischemic white matter injury is thought to mediate periventricular leukomalacia, an age-dependent white matter lesion seen in preterm infants and a common antecedent to cerebral palsy. Hypoxia/ischemia in rat pups at postnatal day 7 (P7) produced selective white matter lesions and OL death. Furthermore, OLs in pericallosal white matter express non-NMDA GluRs at P7. Unilateral carotid ligation in combination with hypoxia (6% O(2) for 1 hr) resulted in selective, subcortical white matter injury with a marked ipsilateral decrease in immature and myelin basic protein-expressing OLs that was also significantly attenuated by 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). Intracerebral AMPA demonstrated greater susceptibility to OL injury at P7 than in younger or older pups, and this was attenuated by systemic pretreatment with the AMPA antagonist NBQX. These results indicate a parallel, maturation-dependent susceptibility of immature OLs to AMPA and hypoxia/ischemia. The protective efficacy of NBQX suggests a role for glutamate receptor-mediated excitotoxic OL injury in immature white matter in vivo.

Topics: Aging; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypoxia-Ischemia, Brain; Injections, Intraperitoneal; Male; Microinjections; Nerve Fibers, Myelinated; Neuroprotective Agents; Oligodendroglia; Quinoxalines; Rats; Rats, Long-Evans; Receptors, AMPA; Receptors, Glutamate