6-cyano-7-nitroquinoxaline-2-3-dione and Hypoxia-Ischemia--Brain

Brain ischemia triggers excessive release of neurotransmitters that mediate neuronal damage following ischemic injury. The striatum is one of the areas most sensitive to ischemia. Release of dopamine (DA) from ischemic neurons is neurotoxic and directly contributes to the cell death in affected areas. Astrocytes are known to be critically involved in the physiopathology of cerebrovascular disease. However, their response to ischemia and their role in neuroprotection in striatum are not completely understood. In this study, we used an in vitro model to evaluate the mechanisms of ischemia-induced DA release, and to study whether astrocytes modulate the release of DA in response to short-term ischemic conditions. Using slices of adult mouse brain exposed to oxygen and glucose deprivation (OGD), we measured the OGD-evoked DA efflux using fast cyclic voltammetry and also assessed metabolic impairment by 2,3,5-triphenyltetrazolium chloride (TTC) and tissue viability by propidium iodide (PI) staining. Our data indicate that ischemia induces massive release of DA by dual mechanisms: one which operates via vesicular exocytosis and is action potential dependent and another involving reverse transport by the dopamine transporter (DAT). Simultaneous blockade of astrocyte glutamate transporters and DAT prevented the massive release of dopamine and reduced the brain tissue damage. The present results provide the first experimental evidence that astrocytes function as a key cellular element of ischemia-induced DA release in striatum, constituting a novel and promising therapeutic target in ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Astrocytes; Calcium; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Exocytosis; Functional Laterality; Hypoxia-Ischemia, Brain; In Vitro Techniques; Mice; Mice, Inbred C57BL; Neurotoxicity Syndromes; Oxidopamine; Piperazines; Tetrodotoxin; Time Factors

Perinatal hypoxia-ischemia is one of the most common risk factors for neonatal mortality and permanent neurodevelopmental disability. Topiramate [2,3:4,5-bis-o-(1-methylethylidene) beta-D-fructo-pyranose sulfamate; TPM] is widely used as an antiepileptic agent with multiple targets. In the present study, we found that treatment with TPM reduced the neuronal damage induced by oxygen-glucose deprivation in vitro with strong inhibition of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor. Because perinatal hypoxia is mediated, at least in part, by aberrant glutamatergic excitation, we tested whether treatment with TPM was effective against perinatal brain hypoxia-ischemia. Intraperitoneal or oral pretreatment with TPM was found to reduce the brain damage and subsequent cognitive impairments induced by transient hypoxia-ischemia in perinatal rats. A potent neuroprotective effect of TPM was also observed in a post-treatment regime although post-treatment window appears to be relatively narrow (<2 h). These results suggest that TPM treatment may be beneficial for perinatal hypoxia-ischemia and related damage.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Administration, Oral; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; Behavior, Animal; Cell Hypoxia; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fructose; Hypoxia-Ischemia, Brain; Injections, Intraperitoneal; Maze Learning; N-Methylaspartate; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Topiramate

Decreased cerebral blood flow, hence decreased oxygen and glucose, leads to ischemic brain injury via complex pathophysiological events, including excitotoxicity, mitochondrial dysfunction, increased intracellular Ca2+, and reactive oxygen species (ROS) generation. Each of these could also contribute to cerebral edema, which is the primary cause of patient mortality after stroke. In vitro brain slices are widely used to study ischemia. Here we introduce a slice model to investigate ischemia-induced edema. Significant water gain was induced in coronal slices of rat brain by 5 min of oxygen and glucose deprivation (OGD) at 35 degrees C, with progressive edema formation after return to normoxic, normoglycemic medium. Edema increased with increasing injury severity, determined by OGD duration (5-30 min). Underlying factors were assessed using glutamate-receptor antagonists (AP5/CNQX), blockade of mitochondrial permeability transition [cyclosporin A (CsA) versus FK506], inhibition of Na+/Ca2+ exchange (KB-R7943), and ROS scavengers (ascorbate, Trolox, dimethylthiourea, Tempol). All agents except KB-R7943 and FK506 significantly attenuated edema when applied after OGD; KB-R7943 was effective when applied before OGD. Significantly, complete prevention of ischemia-induced edema was achieved with a cocktail of AP5/CNQX, CsA and Tempo applied after OGD, which demonstrates the involvement of multiple, additive mechanisms. The efficacy of this cocktail further shows the potential value of combination therapies for the treatment of cerebral ischemia.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Antioxidants; Brain Edema; Cell Hypoxia; Cyclic N-Oxides; Cyclosporine; Drug Therapy, Combination; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia-Ischemia, Brain; In Vitro Techniques; Male; Mitochondria; Neuroprotective Agents; Rats; Rats, Long-Evans; Reactive Oxygen Species; Receptors, Glutamate; Sodium-Calcium Exchanger; Spin Labels; Water