Stroke is a major cause of morbidity, mortality, and disability. During ischemic stroke, a marked and prolonged rise of glutamate concentration in the brain causes neuronal cell death. This study explores the protective effect of a bioconjugate form of glutamate oxaloacetate transaminase (hrGOT), which catalyzes the depletion of blood glutamate in the bloodstream for ~6 days following a single administration. When treated with this bioconjugate, a significant reduction of the infarct volume and a better retention of sensorimotor function was observed for ischemic rats compared to those treated with saline. Moreover, the equivalent dose of native hrGOT yielded similar results to the saline treated group for some tests. Targeting the bioconjugate to the blood-brain-barrier did not improve its performance. The data suggest that the bioconjugates draw glutamate out of the brain by displacing homeostasis between the different glutamate pools of the body.

RNA-binding motif protein 3 (RBM3) is a molecular marker of hypothermia that has proved neuroprotective in neurodegenerative disease models. However, its relationship to the well-recognized therapeutic effect of hypothermia in ischemic stroke had not been studied. In this work, the expression of RBM3 was investigated in ischemic animal models subjected to systemic and focal brain hypothermia, specifically the effects of RBM3 silencing and overexpression on ischemic lesions. Moreover, the association of RBM3 levels with body temperature and clinical outcome was evaluated in two independent cohorts of acute ischemic stroke patients (n = 215); these levels were also determined in a third cohort of 31 patients derived from the phase III EuroHYP-1 trial of therapeutic cooling in ischemic stroke. The preclinical data confirmed the increase of brain RBM3 levels in ischemic animals subjected to systemic and focal hypothermia; this increase was selectively higher in the cooled hemisphere of animals undergoing focal brain hypothermia, thus confirming the direct effect of hypothermia on RBM3 expression, while RBM3 up-regulation in ischemic brain regions led to functional recovery. Clinically, patients with body temperature <37.5 °C in the first two cohorts had higher RBM3 values at 24 hours and good outcome at 3 months post-ischemic stroke, while RBM3 levels in the cooled third cohort tended to exceed those in placebo-treated patients. These results make RBM3 a molecular marker associated with the effect of hypothermia in ischemic stroke and suggest its potential application as a promising protective target.

BackgroundExcitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection.MethodsTo address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 106 and 9 × 106 cells/animal.FindingsThe expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2–HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2–MSCs by another mechanism independent of the glutamate-grabbing capacity.InterpretationAlthough the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.

Proper occlusion of the medial cerebral artery, as determined by laser Doppler monitoring, during cerebral ischaemia in rat models is an important inclusion criterion in experimental studies. However, successful occlusion of the artery does not always guarantee a reproducible infarct volume, which is crucial for validating the efficacy of new protective drugs. In a rat intraluminal ischaemic model, laser Doppler monitoring alone was compared with laser Doppler monitoring in combination with magnetic resonance angiography (MRA) and diffusion-weighted imaging (DWI). Twenty-eight animals showed successful occlusion and reperfusion determined with Doppler monitoring, with an infarct size at 24 h of 16.7±11.5% (determined as ischaemic damage with respect to the ipsilateral hemisphere volume). However, when arterial occlusion and infarct damage were analysed in these animals using MRA and DWI, respectively, 15 animals were excluded and only 13 animals were included, with an infarct size at 24 h of 21.6±6.1%, showing a variability in the infarct size significantly lower (P<0.05, F-test) than that obtained with Doppler monitoring alone. We also observed that blocking of the pterygopalatine artery (a maxillary artery that is usually occluded in the intraluminal ischaemic model) was not relevant for this model, at least in terms of infarct variability. These results show that laser Doppler monitoring is a necessary procedure, but not sufficient to guarantee a reproducible infarct volume, in a rat ischaemic model. Therefore, laser Doppler monitoring in combination with DWI and MRA represents a reliable inclusion protocol during ischaemic surgery for the analysis of new protective drugs.

No abstract