Stroke is the leading cause of death and adult disability in Portugal and the second cause of death worldwide. In the coming years, this health condition will worsen due to the increase in average life expectancy and the increase in the number of individuals with risk factors for stroke. Currently, the recommended treatment for stroke patients is the intravenous thrombolysis therapy (with alteplase or endovascular thrombectomy), although it has limited efficacy due to the narrow therapeutic window where it can be applied without causing hemorrhagic transformations, limiting the benefit to many patients and highlighting the need to develop alternative and safer therapies. The development of strategies aiming to potentiate endogenous neuroprotective mechanisms has emerged as a safe alternative to thrombolysis, however, these approaches have undervalued the importance of the cerebral vasculature in the post-stroke recovery. Previously, it was demonstrated that cerebral vascular remodeling is correlated with higher survival rates and improved long-term neurological function in stroke patients. Accordingly, in this research work, it was proposed the modulation of the Argonaute (Ago)-2 endogenous mechanism to enhance the brain vascular function compromised by cerebral ischemia and, consequently, contributing to full parenchymal recovery after a stroke. Besides being the major coordinator of microRNA traffic and activity, Ago2 protein is pivotal for cell survival and tube network formation in peripheral endothelial cells. However, these mechanisms and the role of Ago2 in the ischemic brain vasculature remain unknown. In this sense, the potential of this protein in the inflammatory process was first assessed. This pathological process is one of the most important post-stroke pathological events that can lead to short-term mortality or promote long-term disability. Accordingly, in vitro (mouse primary brain cultures of endothelial and glial cells stimulated with lipopolysaccharide) and in vivo (adult mice intraperitoneally injected with lipopolysaccharide) data revealed that exogenous Ago2 application (downregulated in brain vasculature in these conditions) normalized endothelial markers associated with inflammatory-induced damage which, in turn, reestablished previously compromised glial and neuronal function. Then, after confirming the potential of Ago2 for cerebral ischemia studies, a strategy to enhance the Ago2 intracellular levels (also downregulated in the ischemic context) and to promote endothelial cell survival was defined. The controlled release of retinoic acid (a signaling molecule with defined anti-inflammatory and pro-angiogenic properties) via polymeric nanoparticles arose as a promising approach. In vitro data (mouse primary brain cultures of endothelial cells subjected to oxygen and glucose deprivation) revealed the protective potential of RA-NP in ischemic conditions, only observed by restoring basal levels of Ago2 protein (an effect mediated by the nitric oxide signaling), highlighting the importance of Ago2 in vascular function. Additionally, in vivo data (adult mice subjected to transient occlusion of the middle cerebral artery) showed that the systemic administration of this formulation promoted tissue survival, normalized inflammatory response, and promoted neurovascular repair (with a robust pro-angiogenic effect) in the peri-infarct region after stroke. However, further studies are needed to establish a correlation between the observed in vivo outcome and the Ago2 protein, and of RA-NP treatment and to decipher interactions between this protein and retinoic acid. Altogether, the experimental research developed throughout this Thesis provided evidence that the modulation of the Ago2 endogenous mechanism can be envisioned as a neuroprotective approach in post-stroke recovery. Furthermore, it revealed a safe nanotechnological tool able to modulate this system, suitable for intravenous administration, and compatible with pre-existing therapies.

Stroke is the third leading cause of mortality and disability in industrial countries. Treatment with herbs with antioxidant properties has been reported to be an alternative to the conventional treatments. This study was conducted to investigate the effect of Anchusa italica extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in the rat. To do so, 50 rats were randomly assigned to five groups; control, sham, ischemia, and 50 or 100 mg/kg of Anchusa italica treated animals. Ischemia was induced by occlusion of carotid artery for 30 minutes. Afterward, behavioral tests and biochemical analyses were conducted. Induction of ischemia/reperfusion caused a decline in learning and passive avoidance memory in rats. Moreover, Anchusa italica caused an increase in learning and improved the passive avoidance memory. Induction of ischemia/reperfusion caused a decrease in the antioxidant capacity of the brain and serum as well as an increase in the malondialdehyde of the brain and serum. Anchusa italica led to an increase in the antioxidant capacity of the brain and serum and decrease in the malondialdehyde of the brain and serum. Overall, because of its protective effects on spatial memory, passive avoidance learning, antioxidant capacity, and lipid peroxidation during ischemia/reperfusion, Anchusa italica might be beneficial in ischemic patients.

Stroke is one of the most important health concerns worldwide. Calcium ions accumulation in the nerve cells and increase in the catecholamines level of the brain following cerebral ischemia/reperfusion (I/R) are accompanied by damaging effects. Therefore, the present study aimed to evaluate the effects of diltiazem, as a calcium channel blocker, and metoprolol, as a β-adrenoceptors antagonist, on I/R injury. In this study, 30 male Wistar rats were divided into control, I/R, metoprolol, diltiazem, and metoprolol plus diltiazem groups (n=6 in each). Metoprolol (1 mg/kg/day) and diltiazem (5 mg/kg/day) were injected intraperitoneally (i.p.) for 7 days before I/R induction. On day 8, the animals underwent ischemia by bilateral common carotid arteries occlusion for 20 min. Histopathological analysis showed a significant reduction in leukocyte infiltration in diltiazem, metoprolol, and diltiazem plus metoprolol treated rats compared with the I/R group (P<0.05, P<0.01, P<0.01, respectively). In addition, in all treated groups, myeloperoxidase activity and malondialdehyde levels in the brain tissue significantly declined compared with the I/R group (P<0.001). Furthermore, pre-treatment with diltiazem and metoprolol alone or in co-administration remarkably reduced infarct size following I/R (P<0.001). Overall, the results indicate the considerable neuroprotective effects of metoprolol and diltiazem in cerebral I/R.

Quantitative and qualitative defects of high-density lipoprotein (HDL) are important in atherogenesis. In this study, we investigated whether antibodies against HDL components had additional value to conventional cardiovascular risk factors for the diagnosis of ischaemic stroke (IS) and coronary artery disease (CAD). Cross-sectional study was conducted on 53 patients with IS, 51 with CAD and 55 healthy controls, and in vitro studies to validate findings of the clinical study. We determined serum immunoglobulin G (IgG) antibodies against HDL (aHDL), apolipoproteins (aApoA-I, aApoA-II and aApoC-I) and paraoxonase-1 (aPON1) as well as PON1 activity (PON1a), total antioxidant capacity and biomarkers of endothelial activation (serum nitric oxide metabolites, 3-nitrotyrosine, VCAM-1 and ICAM-1); in vitro assays tested the capacity of IgG aHDL purified from high titer patients to inhibit PON1a and to reverse protective effect of HDL on endothelial cells. IgG aHDL, aApoA-I and aPON1 were higher in IS and CAD than controls (p < 0.001), predicted negatively PON1a and positively VCAM-1 and ICAM-1. By adding IgG aHDL and aApoA-I to a traditional cardiovascular risk factors model for IS and by adding IgG aHDL in a similar model for CAD, we obtained better discrimination of IS and CAD from healthy controls. IgG aHDL purified from IS and CAD inhibited PON1a by 38% (p < 0.01) and abrogated the protective effect of HDL on VCAM-1 expression by 126% compared with non-specific human IgG (p < 0.001). IgG against HDL components interfere with the antioxidant and anti-inflammatory properties of HDL and may represent novel biomarkers for vascular disease that need to be investigated in prospective studies.

BACKGROUND AND PURPOSE: The first medical contact of an acute stroke victim is often a nonneurologist. Validation of stroke diagnosis made by these medical doctors is poorly known. The present study seeks to validate the stroke diagnoses made by general practitioners (GPs) and hospital emergency service physicians (ESPs). METHODS: Validation through direct interview and examination by a neurologist was performed for diagnoses of stroke made by GPs in patients under their care and doctors working at the emergency departments of 3 hospitals. RESULTS: Validation of the GP diagnosis was confirmed in 44 cases (85%); 3 patients (6%) had transient ischemic attacks and 5 (9%) suffered from noncerebrovascular disorders. Validation of the ESP diagnosis was confirmed in 169 patients (91%); 16 (9%) had a noncerebrovascular diagnosis. Overall, the most frequent conditions misdiagnosed as stroke were neurological in nature (cerebral tumor, 3; subdural hematoma, 1; seizure, 1; benign paroxysmal postural vertigo, 1; peripheral facial palsy, 2; psychiatric condition, 6; and other medical disorders, 7). CONCLUSIONS: In the majority of cases, nonneurologists (either GPs or ESPs) can make a correct diagnosis of acute stroke. Treatment of acute stroke with drugs that do not cause serious side effects can be started before evaluation by a neurologist and CT scan.

BACKGROUND: Limited observational data are available on endovascular treatment in acute ischemic stroke due to cervical artery dissection. Three studies comparing endovascular treatment with standard medical therapy or intravenous thrombolysis in cervical artery dissection-related acute ischemic stroke did not demonstrate superiority of endovascular treatment. Efficacy and the choice of endovascular treatment technique in this setting remain to be established. AIMS: To assess the potential efficacy and safety of endovascular treatment compared to intravenous thrombolysis alone or to no revascularization treatment in our center. METHODS: We selected all consecutive patients with cervical artery dissection-related acute ischemic stroke and intracranial occlusion from the Acute STroke Registry and Analysis of Lausanne between 2003 and 2017. We compared clinical and neuroimaging data of patients treated by endovascular treatment versus patients receiving intravenous thrombolysis or patients without revascularization treatment. Safety analysis included symptomatic intracranial hemorrhage, major radiological hemorrhages (parenchymal hematoma 1, parenchymal hematoma 2, and subarachnoid hemorrhage) and mortality within seven days. We assessed favorable clinical outcome (modified Rankin Scale 0-2) at three months using a binary logistic regression model. RESULTS: Of the 109 patients included, 24 had endovascular treatment, 38 received intravenous thrombolysis alone, and 47 had no revascularization treatment. Endovascular treatment patients had a higher rate of recanalization at 24 h. Major radiological hemorrhages occurred more often in endovascular treatment patients (all with bridging therapy) than in patients without revascularization treatment (p = 0.026), with no differences in symptomatic intracranial hemorrhage or mortality within seven days. Favorable clinical outcome at three months did not differ between groups (endovascular treatment versus intravenous thrombolysis p = 0.407; endovascular treatment versus no revascularization treatment p = 0.580). CONCLUSIONS: In this single-center cohort of cervical artery dissection-related acute ischemic stroke with intracranial occlusion, endovascular treatment with prior intravenous thrombolysis may increase the risk of major radiological but not symptomatic intracranial hemorrhage. Despite the lack of clear superiority in our cohort, endovascular treatment should currently not be withheld in these patients.

BACKGROUND: Citicoline is approved in some countries for the treatment of acute ischaemic stroke. The drug has shown some evidence of efficacy in a pooled analysis. We sought to confirm the efficacy of citicoline in a larger trial. METHODS: We undertook a randomised, placebo-controlled, sequential trial in patients with moderate-to-severe acute ischaemic stroke admitted at university hospitals in Germany, Portugal, and Spain. Using a centralised minimisation process, patients were randomly assigned in a 1:1 ratio to receive citicoline or placebo within 24 h after the onset of symptoms (1000 mg every 12 h intravenously during the first 3 days and orally thereafter for a total of 6 weeks [2×500 mg oral tablets given every 12 h]). All study participants were masked. The primary outcome was recovery at 90 days measured by a global test combining three measures of success: National Institutes of Health Stroke Scale ≤1, modified Rankin score ≤1, and Barthel Index ≥95. Safety endpoints included symptomatic intracranial haemorrhage in patients treated with recombinant tissue plasminogen activator, neurological deterioration, and mortality. This trial is registered, NCT00331890. RESULTS: 2298 patients were enrolled into the study from Nov 26, 2006, to Oct 27, 2011. 37 centres in Spain, 11 in Portugal, and 11 in Germany recruited patients. Of the 2298 patients who gave informed consent and underwent randomisation, 1148 were assigned to citicoline and 1150 to placebo. The trial was stopped for futility at the third interim analysis on the basis of complete data from 2078 patients. The final randomised analysis was based on data for 2298 patients: 1148 in citicoline group and 1150 in placebo group. Global recovery was similar in both groups (odds ratio 1·03, 95% CI 0·86-1·25; p=0·364). No significant differences were reported in the safety variables nor in the rate of adverse events. INTERPRETATION: Under the circumstances of the ICTUS trial, citicoline is not efficacious in the treatment of moderate-to-severe acute ischaemic stroke.

The ubiquitin–proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitincontaining proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review.

Cerebral ischemia is characterized by an early disruption of GABAergic neurotransmission contributing to an imbalance of the excitatory/inhibitory equilibriumand neuronal death, but the molecular mechanisms involved are not fully understood. Here we report a downregulation of GABAA receptor (GABAAR) expression, affecting both mRNA and protein levels of GABAAR subunits, in hippocampal neurons subjected to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Similar alterations in the abundance of GABAAR subunits were observed in in vivo brain ischemia. OGD reduced the interaction of surface GABAAR with the scaffold protein gephyrin, followed by clathrin-dependent receptor internalization. Internalization of GABAARwas dependent on glutamate receptor activation and mediated by dephosphorylation of the β3 subunit at serine 408/409. Expression of phospho-mimetic mutant GABAAR β3 subunits prevented receptor internalization and protected hippocampal neurons from ischemic cell death. The results show a key role for β3 GABAAR subunit dephosphorylation in the downregulation of GABAergic synaptic transmission in brain ischemia, contributing to neuronal death. GABAAR phosphorylation might be a therapeutic target to preserve synaptic inhibition in brain ischemia.