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

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

Diffuse brain injury is a leading cause of mortality in infants and children under 4 years of age and results in cognitive deficits in survivors. The anatomic basis for these behavioral deficits may be traumatic axonal injury (TAI), which manifests as impaired axonal transport (IAT) and neurofilament compaction (NFC), and may occur as a result of glutamate receptor activation. The extent of IAT and NFC was evaluated at 6, 24 and 72 h following non-contusive brain trauma in the 17 day-old rat to examine the causal relationship between these two pathologic entities; in addition, the effect of antagonists to the ionotropic glutamate receptors on TAI was evaluated. At 6 h post-injury, NFC was observed primarily in the cingulum, and appeared as swollen axons and terminal bulbs. By 24 h, swollen axons were additionally present in the corpus callosum and lateral white matter tracts, and appeared to increase in diameter. At 72 h, the extent of axonal swellings exhibiting compacted neurofilaments appeared to decrease, and was accompanied by punctate immunoreactivity within axon tracts suggestive of axonal degeneration. Although NFC was present in the same anatomical locations where axonal accumulation of amyloid precursor protein (APP) has been observed, double-label immunohistochemistry revealed no evidence of colocalization of compacted neurofilament and APP. Pre-injury treatment with either the NMDA receptor antagonist, ifenprodil, or the AMPA receptor antagonist, NBQX, had no significant effect on the extent of TAI, suggesting that excitotoxicity may not be a primary mechanism underlying TAI. Importantly, these data are indicative of the heterogeneity of mechanisms underlying TAI in the traumatically-injured immature brain.

Topics: Amyloid beta-Protein Precursor; Animals; Axonal Transport; Axons; Brain; Brain Injuries; Excitatory Amino Acid Antagonists; Female; Immunohistochemistry; Intermediate Filaments; Male; Neurons; Photomicrography; Piperidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Time Factors

To examine the possible role of inflammatory cytokines in mediating neonatal brain injury, we investigated effects of intracerebral injection of IL-1beta (IL-1beta) or tumor necrosis factor-alpha (TNFalpha) on brain injury in the neonatal rat. A stereotaxic intracerebral injection of IL-1beta or TNFalpha (10 ng per pup) was performed in postnatal day 5 (P5) SD rats. Although no necrosis of neurons was found, increased astrogliosis, as indicated by GFAP positive staining was observed 24 and 72 h following the injection of IL-1beta or TNFalpha. IL-1beta induced apoptotic cell death in the rat brain 24 h after the injection, as indicated by increases in positive TUNEL staining and caspase-3 activity, and apoptotic cell death was partially blocked by systemic administration of NBQX, an antagonist of the AMPA glutamate receptor. IL-1beta also significantly reduced the number of developing oligodendrocytes (OLs) 24 h after the injection and this impairment was not prevented by NBQX. On the contrary, TNFalpha induced a much smaller increase in the number of TUNEL positive cells and did not reduce the number of developing OLs. By P8, myelin basic protein (MBP) was clearly detected in the control rat brain, while MBP positive staining was very weak, if any, in the IL-1beta treated rat brain. MBP expression in the TNFalpha treated rat brain was less affected. The overall results indicate that IL-1beta may directly cause injuries to developing OLs and impair myelination in the neonatal rat brain and TNFalpha may have different roles in mediating brain injury.

Topics: Animals; Animals, Newborn; Apoptosis; Brain Injuries; Caspase 3; Caspases; Cerebral Cortex; Excitatory Amino Acid Antagonists; Humans; In Situ Nick-End Labeling; Infant; Inflammation; Interleukin-1; Myelin Basic Protein; Oligodendroglia; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Tumor Necrosis Factor-alpha

Tolerance to normally neurotoxic insults can be induced by prior a preconditioning exposure to a sublethal insult. Kainate toxicity can be attenuated by prior exposure to very low concentrations of kainate both in vivo and in vitro. Using organotypic hippocampal slice cultures from rats we have shown that 5 microM kainate induces a selective lesion in the CA3 region and this can be significantly attenuated by 1 microM kainate administered 1-5 days earlier. The time window for this effect was affected by the length of time in culture, and preconditioning was blocked by NBQX but not the selective AMPA receptor antagonist GYKI53655. These data demonstrate a role for kainate receptors in preconditioning for the first time and show that organotypic cultures can be used as a model to investigate long-term preconditioning mechanisms.

Topics: Animals; Animals, Newborn; Brain Injuries; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Kainic Acid; Organ Culture Techniques; Quinoxalines; Rats; Rats, Wistar; Time Factors

To understand the pathogenesis of diffuse axonal injury, we investigated the temporal and spatial profiles of neuronal degeneration in impact-acceleration injury in rats using Fluoro-Jade (FJ) staining. Impact-acceleration injury was produced in Wistar rats by the method described by Marmarou et al. with some modifications. Animals were sacrificed 1, 2, 7, 14, or 28 days after injury. Paraffin-embedded coronal sections were stained with HE or FJ, or analyzed immunohistochemically for GFAP or amyloid precursor protein (APP). FJ-positive degenerative neurons were found primarily in the dorsal brainstem and thalamus from 1 to 2 days following injury and these were associated with GFAP expression. However, FJ-positive cells were rarely found after 7 days. In all rats, significant expression of APP was observed primarily in the cingulum, cerebral peduncle and pontomedullary junction. FJ also stained these injured axons. Intrathecal administration of both NMDA and AMPA/kinate glutamate receptor antagonists MK-801 and NBQX, respectively, reduced the neuronal injury. NBQX showed more significant effects on axonal injury than MK-801. These observations indicate that not only axonal damage, but also primary neuronal damage occurs in this impact-acceleration injury model. It is also suggested that NBQX can act both directly on neuronal cells and white matter and that NMDA could have a significant protective effect against not only neuronal, but also axonal injury.

Topics: Amyloid beta-Protein Precursor; Animals; Brain Injuries; Brain Stem; Dizocilpine Maleate; Glial Fibrillary Acidic Protein; Male; Neurons; Quinoxalines; Rats; Rats, Wistar; Thalamus

The effects of three glutamate receptor antagonists, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate (MK-801) for the N-methyl-D-aspartate receptor, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide (NBQX) for the alpha-amino-3-hydroxy-5methyl-4-isoxazole propionate /kinate receptor and (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) for the metabotropic receptor, on c-fos and c-jun mRNA expression were investigated in cultured cortical glial cells following traumatic scratch injury. Expression of the two genes along the edges of wounds detected by in situ hybridization was not affected by MK-801 and NBQX. However, 100 and 500 microM of MCPG remarkably reduced the hybridization signals for both c-fos and c-jun mRNAs. The present results suggest that group I metabotropic glutamate receptors might have some association with immediate early gene induction after in vitro traumatic injury in glial cells.

Topics: Animals; Benzoates; Brain Injuries; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Fetus; Gene Expression Regulation; Genes, Immediate-Early; Gliosis; Glycine; Nerve Regeneration; Neuroglia; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Transcriptional Activation

Glutamate promotes neuronal survival during brain development and destroys neurons after injuries in the mature brain. Glutamate antagonists are in human clinical trials aiming to demonstrate limitation of neuronal injury after head trauma, which consists of both rapid and slowly progressing neurodegeneration. Furthermore, glutamate antagonists are considered for neuroprotection in chronic neurodegenerative disorders with slowly progressing cell death only. Therefore, humans suffering from Huntington's disease, characterized by slowly progressing neurodegeneration of the basal ganglia, are subjected to trials with glutamate antagonists. Here we demonstrate that progressive neurodegeneration in the basal ganglia induced by the mitochondrial toxin 3-nitropropionate or in the hippocampus by traumatic brain injury is enhanced by N-methyl-d-aspartate antagonists but ameliorated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonists. These observations reveal that N-methyl-d-aspartate antagonists may increase neurodestruction in mature brain undergoing slowly progressing neurodegeneration, whereas blockade of the action of glutamate at alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors may be neuroprotective.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain Injuries; Cell Death; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Memantine; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Nitro Compounds; Piperazines; Propionates; Quinoxalines; Rats; Rats, Wistar; Wounds and Injuries

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.

Topics: Animals; Brain Injuries; Bridged Bicyclo Compounds; Calcium; Cells, Cultured; Chlorides; Coculture Techniques; Cytosol; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Kainic Acid; Mice; Microscopy, Fluorescence; N-Methylaspartate; Quinoxalines; Receptors, Glutamate; Sodium; Veratridine

Topics: Acoustic Stimulation; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anticonvulsants; Brain Injuries; Brain Ischemia; Cerebral Cortex; Convulsants; Excitatory Amino Acid Antagonists; Exploratory Behavior; Kindling, Neurologic; Male; Mice; Mice, Inbred Strains; Neuroprotective Agents; Quinoxalines; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Rats, Wistar; Seizures

Ion-selective microelectrodes were used to study acute effects of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy- 5-methyl-4-isoxazole (AMPA) receptor blockade on posttraumatic calcium disturbances. An autoradiographic technique with 45 Ca2+ was used to study calcium disturbances at 8, 24, and 72 h. Compression contusion trauma of the cerebral cortex was produced by a 21-g weight dropped from a height of 35 cm onto a piston that compressed the brain 2 mm. Pre- and posttrauma interstitial [Ca2+] ([Ca2+]e) concentrations were measured in the perimeter, i.e., the shear stress zone (SSZ) and in the central region (CR) of the trauma site. For the [Ca2+]e studies the animals were divided into controls and groups pretreated with dizocilipine maleate (MK-801) or with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[F]quinoxaline (NBQX). In all groups, [Ca2+]e decreased from pretrauma values of approximately 1 mM to posttraumatic values of 0.1 mM in both the CR and the SSZ. This was followed by a slow restitution toward pretraumatic levels during the 2-h observation period. There was no significant difference in recovery pattern between controls and pretreated animals. Accumulation of 45Ca2+ and serum proteins was seen in the entire SSZ, while neuronal necrosis was confined to a narrow band within the SSZ. The CR was unaffected apart from occasional eosinophilic neurons and showed no accumulation of 45Ca2+. Posttraumatic treatment with MK-801 or NBQX had no obvious effect on neuronal injury in the SSZ. We conclude that (a) acute [Ca2+]e disturbances in compression contusion brain trauma are not affected by blockade of NMDA or AMPA receptors, (b) 45Ca2+ accumulation in the SSZ reflects mainly protein accumulation due to blood-brain barrier breakdown rather than cell death, and (c) acute cellular Ca2+ over-load per se does not seem to be a major determinant of cell death after cerebral trauma in our model.

Topics: Animals; Autoradiography; Brain Injuries; Calcium; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Ion Channels; Male; Necrosis; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

The mechanisms of neuronal degeneration following traumatic head injury are not well understood and no adequate treatment is currently available for the prevention of traumatic brain damage in humans. Traumatic head injury leads to primary (at impact) and secondary (distant) damage to the brain. Mechanical percussion of the rat cortex mimics primary damage seen after traumatic head injury in humans; no animal model mimicking the secondary damage following traumatic head injury has yet been established. Rats subjected to percussion trauma of the cortex showed primary damage in the cortex and secondary damage in the hippocampus. Morphometric analysis demonstrated that both cortical and hippocampal damage was mitigated by pretreatment with either the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)- 2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX). Neither treatment prevented primary damage in the cortex when therapy was started after trauma. Surprisingly, delayed treatment of rats with NBQX, but not with CPP, beginning between 1 and 7 hr after trauma prevented hippocampal damage. No protection was seen when therapy with NBQX was started 10 hr after trauma. These data indicate that both NMDA- and non-NMDA-dependent mechanisms contribute to the development of primary damage in the cortex, whereas non-NMDA mechanisms are involved in the evolution of secondary damage in the hippocampus in rats subjected to traumatic head injury. The wide therapeutic time-window documented for NBQX suggests that antagonism at non-NMDA receptors may offer a novel therapeutic approach for preventing deterioration of the brain after head injury.

Topics: Animals; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Excitatory Amino Acid Antagonists; Hippocampus; Humans; Male; N-Methylaspartate; Nerve Degeneration; Neurons; Piperazines; Quinoxalines; Rats; Rats, Inbred F344; Time Factors; Wounds, Nonpenetrating

Recent evidence implicates the endogenous excitatory neurotransmitters, glutamate (Glu) and aspartate, in the pathophysiology of traumatic injury in the adult CNS, but it is not known whether similar excitotoxic mechanisms mediate traumatic injury in the immature CNS. Therefore, we developed a model of brain contusion injury in infant rats and used this model to study the nature and evolution of the acute cytopathologic changes and to evaluate the ability of Glu receptor antagonists to protect the immature brain against such changes. Seven-day-old rat pups were subjected to contusion injury and were killed 0, 0.5, 1, 2, 4, and 6 h later for histologic evaluation of the brain. Physical tearing of the dura and minor disruption of underlying brain tissue was noted at 0 h. At 30 min a discrete zone of neuronal necrosis began to appear at the border of the trauma site; this zone progressively expanded over a period of 4 h. The cytopathologic changes closely resembled the type of changes Glu is known to cause; these changes consisted of swollen dendrites, degenerating neurons with pyknotic nuclei and markedly swollen cytoplasm, and dark cells with vacuolated cytoplasm. The noncompetitive N-methyl-D-aspartate (NMDA) antagonist, dizocilpine maleate, when administered 30 min before or 1 h after trauma, significantly attenuated the lesion. The competitive NMDA antagonist, 3-((-2)-carboxypiperazine-4-yl)-propyl-1-phosphonate, was also neuroprotective. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline did not significantly suppress the lesion when given as three treatments (30 mg/kg each) 30 min before plus 15 and 75 min after the insult. These findings suggest that traumatic injury in the infant rat brain is mediated by endogenous excitotoxins (Glu and aspartate) acting at NMDA receptors and can be substantially mitigated by timely treatment with NMDA receptor antagonists.

Topics: Animals; Brain Injuries; Dendrites; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Piperazines; Quinoxalines; Rats; Rats, Sprague-Dawley; Time Factors; Wounds and Injuries