tetrodotoxin 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

Neurofilaments are key structural components of white matter axons. The effect of in vitro anoxia or oxygen-glucose deprivation (OGD) on the integrity of the 160 and 200 kDa neurofilament isoforms was studied by immunoblot, and correlated with physiological function. Adult rat optic nerves were exposed to 60 min of either anoxia or OGD. Compound action potential area recovered to 22+/-6% of control after 60 min of anoxia, and to 4+/-1% after 60 min of OGD. Ca(2+)-free (+EGTA) perfusate allowed complete recovery after OGD (108+/-42%). Tetrodotoxin (TTX, 1 microM) was less protective (45+/-6%). Both anoxia and OGD induced breakdown of neurofilament 160 (NF160) and NF200 revealed by the appearance of multiple lower molecular weight bands mainly in the 75-100 kDa range. Zero-Ca(2+)/EGTA completely prevented NF breakdown. TTX only partially reduced NF160 degradation. Non-phosphorylated NF200 appeared after reperfusion post-anoxia or OGD, and was also greatly reduced by zero-Ca(2+) or TTX. Calpain inhibitors (10 microM calpain inhibitor I or 50 microM MDL 28,170) significantly reduced NF160 and NF200 breakdown/dephosphorylation, but did not improve electrophysiological recovery. Significant calpain-mediated breakdown of NF160 and NF200 indicates structural damage to the axonal cytoskeleton, which was completely Ca(2+)-dependent. While pharmacological inhibition of calpain alone greatly reduced NF proteolysis, there was no concomitant improvement in function. These results imply that calpain inhibition is necessary but not sufficient for white matter protection, and emphasize the existence of multiple Ca(2+)-dependent degradative pathways activated in injured white matter.

Topics: Action Potentials; Animals; Axons; Calcium; Calcium Signaling; Calpain; Central Nervous System; Chelating Agents; Enzyme Inhibitors; Hypoxia-Ischemia, Brain; Male; Nerve Degeneration; Nerve Fibers, Myelinated; Neurofilament Proteins; Optic Nerve; Organ Culture Techniques; Rats; Rats, Long-Evans; Recovery of Function; Tetrodotoxin