Estudio de la contribución de los aminoácidos no excitadores en el edema citotóxico durante la hipoxia

Abstract

The role of the excitatory amino acids glutamate and aspartate in cerebral ischemia and excitotoxicity has been extensively studied. However, little is known about non excitatory amino acids’ contribution to brain injury, especially in combination with the hypoxia produced when cerebral blood flow is reduced after stroke or brain trauma. After a cerebral hemorrhage, plasmatic content, including amino acids, is released to the brain parenchyma. The generation of new hemorrhages or the expansion of previous ones propagates cell damage hours and days after traumatic brain injury onset, in a phenomenon called hemorrhagic progression of contusions. Furthermore, in focal stroke, amino acids released in the ischemic core from necrotic cells could contribute to penumbra expansion. In this work, we have used acute hippocampal murine slices to study the effect of several mixtures of amino acids at their plasmatic concentrations on field evoked potentials, basic neuronal membrane properties and cell volume, estimated by measuring changes in electrical resistance and light transmittance of the slices. Two-photon imaging of neurons and astrocytes is used to quantify changes at the cellular level, while electron microscopy is employed to reveal ultrastructural changes. Slice amino acid content is measured with HPLC techniques. We have found that in normoxic conditions, a mixture of seven amino acids (AGHQSTU: L-alanine, glycine, L-histidine, L-glutamine, L-serine, L-threonine, and taurine at their plasmatic concentrations) causes an increase in field potentials’ amplitude, through a mechanism independent of both NMDA receptor activation and amino acid transport system A activity. A concurrent rise in the applied amino acid slice content and electrical tissue resistance indicates a reduction of the extracellular space volume without severely affecting input resistance and neuronal membrane potential. The reversible loss of field potentials induced by a 40-minutes hypoxia period becomes irreversible in the presence of AGHQSTU, coinciding with an intense neuronal depolarization and a strong increase in the fiber volley amplitude. Moreover, the presence of the amino acids during hypoxia causes a rise in the electrical tissue resistance, indicating a reduction of the extracellular volume. Nevertheless, only specific amino acids cause these irreversible effects during hypoxia, suggesting that it cannot be entirely explained by the sole increase in the number of particles accumulated in the tissue, and that the identity of the amino acids determines their effect. A mixture of only four amino acids at their plasmatic concentrations (AGQS: L-alanine, glycine, L-glutamine, and L serine) mimics the AGHQSTU-induced deleterious effects on extracellular evoked potentials during hypoxia, without involving the activation of AMPA receptors. During hypoxia, AGQS causes neuronal and astroglial soma swelling, alongside irreversible dendritic damage. NMDA receptor blockade prevents hypoxic neuronal damage without affecting the increase in astroglial volume. ASCT2 (alanine-serine-cysteine transporter 2) exchanger and system N amino acid transporters could be involved in the amino acids-induced hypoxic damage, as inferred by the use of pharmacological inhibitors and specific substrates. Moreover, VRAC (volume-regulated anion channel) and Asc-1 (alanine-serine-cysteine transporter 1)-dependent release of glutamate, aspartate, and D-serine, an NMDA receptor co-agonist, could also be related to the irreversible loss of synaptic potentials mediated by amino acids in hypoxia. Results obtained in this Ph.D. Thesis imply that non-excitatory amino acids contribute to development and expansion of brain damage in neurological emergencies such as stroke and traumatic brain injury. Mechanisms mediating adverse effects of non-excitatory amino acids during hypoxia could point to novel therapeutic targets to ameliorate brain ischemia.