The development of plasmatic glutamate grabbers for the treatment of ischemic stroke

Abstract

Glutamate is subjected to strong homeostasis, which is regulated by excitatory amino acid transporters (EAATs), being the EAAT2 responsible for 90% of the uptake of extracellular glutamate. The balance disturbance of glutamate, as in ischemic stroke occurs, provokes that glutamate increases in the extracellular space which induces a complex process of pathogenic mechanisms. In order to mitigate these effects, neuroprotective drugs were developed, which showed no efficacy in human clinical practice. Because of this, studies describe the existence of glutamate concentration gradient between the endothelial cells that form the blood-brain barrier and the bloodstream. When the glutamate concentration of the brain endothelial cells is greater than the concentration of blood glutamate, the glutamate is carried from the brain to the bloodstream. On account of, the hypothesis that reducing blood levels of glutamate would increase the concentration gradient between brain and blood endothelium arises, thus favoring the removal of cerebral glutamate. This potential neuroprotective mechanism is called blood glutamate grabbering. For all the above, the combination of mechanisms of neuroprotection (by blood glutamate grabbing) and neurorepair (mediated by mesenchymal stem cells) constitute a promising therapeutic strategy in cerebral ischemia. Therefore, the objective of this work was the development of alternative blood glutamate grabbers for use in ischemic stroke. So we have used two strategies: enzymatic and cellular. The aim of enzymatic strategy was to analyze the protective effect of the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments (6.44 µg/100g, 12.88 µg/100g and 25.76 µg/100g) tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit; being the dose of 12.88 µg/100 g the most effective. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment. To perform cell strategy, electroporation technique was used to express the EAAT2 in mesenchymal stem cells and calcium phosphate transfection in HEK cells. Through immunofluorescence techniques, EAAT2 expression was demonstrated in transfected cells. Glutamate uptake experiments proved that EAAT2 was functional in transfected cells. Once demonstrated the ability of transfected cells to uptake glutamate in vitro, it was necessary to determine their effect in vivo. For that, healthy rats were used by stablish the adequate cell dose to induce a reduction of blood glutamate levels. The results showed that administration of transfected cells with EAAT2 provoked a decrease of blood glutamate levels in vivo. The therapeutic effect of the cells transfected with the EAAT2 in rats subjected to MCAO was studied. To determine the beneficial effects of this therapy, forty eight rats were employed and infarct volumes were assessed by MRI before treatment administration, and 24 h and 7 days after MCAO. Serum glutamate concentration was analyzed before MCAO, during the reperfussion period and 1, 3, 4 and 24 h after reperfussion period. Somatosensory test was performed at baseline and 7, 14 days after MCAO. The results showed that administration of transfected cells induced a decrease in serum levels of glutamate in ischemic animals; however the transfected cells did not cause a reduction of the infarct volume. As for the somatosensory test, only the transfected HEK cells showed an improvement at 14 days after ischemia. In conclusion, our results mean that the blood cell glutamate grabbing do not present better benefit than other glutamate grabbers already well described, as their protective effect is not higher than oxaloacetate.