Pharmacological preclinical comparison of tenecteplase and alteplase for the treatment of acute stroke.

Alteplase (rtPA) remains the standard thrombolytic drug for acute ischemic stroke. However, new rtPA-derived molecules, such as tenecteplase (TNK), with prolonged half-lives following a single bolus administration, have been developed. Although TNK is currently under clinical evaluation, the limited preclinical data highlight the need for additional studies to elucidate its benefits. The toxicities of rtPA and TNK were evaluated in endothelial cells, astrocytes, and neuronal cells. In addition, their in vivo efficacy was independently assessed at two research centers using an ischemic thromboembolic mouse model. Both therapies were tested via early (20 and 30 min) and late administration (4 and 4.5 h) after stroke. rtPA, but not TNK, caused cell death only in neuronal cultures. Mice were less sensitive to thrombolytic therapies than humans, requiring doses 10-fold higher than the established clinical dose. A single bolus dose of 2.5 mg/kg TNK led to an infarct reduction similar to perfusion with 10 mg/kg of rtPA. Early administration of TNK decreased the hemorrhagic transformations compared to that by the early administration of rtPA; however, this result was not obtained following late administration. These two independent preclinical studies support the use of TNK as a promising reperfusion alternative to rtPA.

New Perspectives in Neuroprotection for Ischemic Stroke.

The constant failure of new neuroprotective therapies for ischemic stroke has partially halted the search for new therapies in recent years, mainly because of the high investment risk required to develop a new treatment for a complex pathology, such as stroke, with a narrow intervention window and associated comorbidities. However, owing to recent progress in understanding the stroke pathophysiology, improvement in patient care in stroke units, development of new imaging techniques, search for new biomarkers for early diagnosis, and increasingly widespread use of mechanical recanalization therapies, new opportunities have opened for the study of neuroprotection. This review summarizes the main protective agents currently in use, some of which are already in the clinical evaluation phase. It also includes an analysis of how recanalization therapies, new imaging techniques, and biomarkers have improved their efficacy.

Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

BACKGROUND: Intravenous administration of fibrinolytic drugs, such as recombinant tissue plasminogen activator (rtPA) is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and risk of hemorrhagic transformations. Platelet membrane-based nanocarriers have received increasing attention for ischemic stroke therapies, as they have natural thrombus-targeting activity, can prolong half-life of the fibrinolytic therapy, and reduce side effects. In this study we have gone further in developing platelet-derived nanocarriers (defined as cellsomes) to encapsulate and protect rtPA from degradation. Following lyophilization and characterization, their formulation properties, biocompatibility, therapeutic effect, and risk of hemorrhages were later investigated in a thromboembolic model of stroke in mice. RESULTS: Cellsomes of 200 nm size and loaded with rtPA were generated from membrane fragments of human platelets. The lyophilization process did not influence the nanocarrier size distribution, morphology, and colloidal stability conferring particle preservation and long-term storage. Encapsulated rtPA in cellsomes and administered as a single bolus showed to be as effective as a continuous clinical perfusion of free rtPA at equal concentration, without increasing the risk of hemorrhagic transformations or provoking an inflammatory response. CONCLUSIONS: This study provides evidence for the safe and effective use of lyophilized biomimetic platelet-derived nanomedicine for precise thrombolytic treatment of acute ischemic stroke. In addition, this new nanoformulation could simplify the clinical use of rtPA as a single bolus, being easier and less time-consuming in an emergency setting than a treatment perfusion, particularly in stroke patients. We have successfully addressed one of the main barriers to drug application and commercialization, the long-term storage of nanomedicines, overcoming the potential chemical and physical instabilities of nanomedicines when stored in an aqueous buffer.

Perfusion-weighted software written in Python for DSC-MRI analysis.

Introduction: Dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion studies in magnetic resonance imaging (MRI) provide valuable data for studying vascular cerebral pathophysiology in different rodent models of brain diseases (stroke, tumor grading, and neurodegenerative models). The extraction of these hemodynamic parameters via DSC-MRI is based on tracer kinetic modeling, which can be solved using deconvolution-based methods, among others. Most of the post-processing software used in preclinical studies is home-built and custom-designed. Its use being, in most cases, limited to the institution responsible for the development. In this study, we designed a tool that performs the hemodynamic quantification process quickly and in a reliable way for research purposes. Methods: The DSC-MRI quantification tool, developed as a Python project, performs the basic mathematical steps to generate the parametric maps: cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), signal recovery (SR), and percentage signal recovery (PSR). For the validation process, a data set composed of MRI rat brain scans was evaluated: i) healthy animals, ii) temporal blood-brain barrier (BBB) dysfunction, iii) cerebral chronic hypoperfusion (CCH), iv) ischemic stroke, and v) glioblastoma multiforme (GBM) models. The resulting perfusion parameters were then compared with data retrieved from the literature. Results: A total of 30 animals were evaluated with our DSC-MRI quantification tool. In all the models, the hemodynamic parameters reported from the literature are reproduced and they are in the same range as our results. The Bland-Altman plot used to describe the agreement between our perfusion quantitative analyses and literature data regarding healthy rats, stroke, and GBM models, determined that the agreement for CBV and MTT is higher than for CBF. Conclusion: An open-source, Python-based DSC post-processing software package that performs key quantitative perfusion parameters has been developed. Regarding the different animal models used, the results obtained are consistent and in good agreement with the physiological patterns and values reported in the literature. Our development has been built in a modular framework to allow code customization or the addition of alternative algorithms not yet implemented.

Circulating extracellular vesicles promote recovery in a preclinical model of intracerebral hemorrhage.

Circulating extracellular vesicles (EVs) are proposed to participate in enhancing pathways of recovery after stroke through paracrine signaling. To verify this hypothesis in a proof-of-concept study, blood-derived allogenic EVs from rats and xenogenic EVs from humans who experienced spontaneous good recovery after an intracerebral hemorrhage (ICH) were administered intravenously to rats at 24 h after a subcortical ICH. At 28 days, both treatments improved the motor function assessment scales score, showed greater fiber preservation in the perilesional zone (diffusion tensor-fractional anisotropy MRI), increased immunofluorescence markers of myelin (MOG), and decreased astrocyte markers (GFAP) compared with controls. Comparison of the protein cargo of circulating EVs at 28 days from animals with good vs. poor recovery showed down-expression of immune system activation pathways (CO4, KLKB1, PROC, FA9, and C1QA) and of restorative processes such as axon guidance (RAC1), myelination (MBP), and synaptic vesicle trafficking (SYN1), which is in line with better tissue preservation. Up-expression of PCSK9 (neuron differentiation) in xenogenic EVs-treated animals suggests enhancement of repair pathways. In conclusion, the administration of blood-derived EVs improved recovery after ICH. These findings open a new and promising opportunity for further development of restorative therapies to improve the outcomes after an ICH.

Fibrin-Targeted Nanoparticles for Finding, Visualizing and Characterizing Blood Clots in Acute Ischemic Stroke.

Recanalization of the occluded artery is the gold standard treatment for acute ischemic stroke, which includes enzymatic fibrinolytic treatment with the use of recombinant tissue plasminogen activators (rtPAs) to disrupt the occluding clot, the use of mechanical thrombectomy to physically remove the clot, or a combination of both. Fibrin is one of the main components of blood clots causing ischemic stroke and is the target of rtPA upon activation of plasminogen in the clot. In addition, fibrin content also influences the efficacy of mechanical thrombectomy. Current imaging methods can successfully identify occlusions in large vessels; however, there is still a need for contrast agents capable of visualizing small thrombi in ischemic stroke patients. In this work, we describe the synthesis and the in vitro characterization of a new diagnostic nanoparticle, as well as the in vivo evaluation in an animal model of thromboembolic stroke. Gd-labeled KCREKA peptides were synthesized and attached onto the surface of PEGylated superparamagnetic nanoparticles. Magnetic resonance imaging (MRI) of blood clots was performed in vitro and in vivo in animal models of thromboembolic stroke. KCREKA-NPs were synthesized by attaching the peptide to the amino (N) termini of the PEG-NPs. The sizes of the nanoparticles, measured via DLS, were similar for both KCREKA-NPs and PEG-NPs (23 ± 4 nm, PDI = 0.11 and 25 ± 8 nm, PDI = 0.24, respectively). In the same line, r2 relaxivities were also similar for the nanoparticles (149 ± 2 mM Fe s−1 and 151 ± 5 mM Fe s−1), whereas the r1 relaxivity was higher for KCREKA-NPs (1.68 ± 0.29 mM Fe s−1 vs. 0.69 ± 0.3 mM Fe s−1). In vitro studies showed that blood clots with low coagulation times were disrupted by rtPA, whereas aged clots were almost insensitive to the presence of rtPA. MRI in vitro studies showed a sharp decrease in the T1 × T2 signals measured for aged clots incubated with KCREKA-NPs compared with fresh clots (47% [22, 80] to 26% [15, 51]). Furthermore, the control blood showed a higher value of the T1 × T2 signal (39% [20, 61]), being the blood clots with low coagulation times the samples with the lowest values measured by MRI. In vivo studies showed a significant T1 × T2 signal loss in the clot region of 24% after i.v. injection of KCREKA-NPs. The thrombus age (2.5% ± 6.1% vs. 81.3% ± 19.8%, p < 0.01) confirmed our ability to identify in vivo fresh blood clots. In this study, we developed and tested a dual MRI nanoparticle, acting as T1 and T2 contrast agents in MRI analyses. The developed KCREKA-NPs showed affinity for the fibrin content of blood clots, and the MRI signals provided by the nanoparticles showed significant differences depending on the clot age. The developed KCREKA-NPs could be used as a tool to predict the efficacy of a recanalization treatment and improve the triage of ischemic stroke patients.

Surrogate biomarkers of outcome for wake-up ischemic stroke.

BACKGROUND: Wake-up ischemic stroke (IS) has been usually excluded from acute stroke therapy options for being outside of the safe treatment window. We identified risk factors, and clinical or molecular biomarkers that could be therapeutic targets for wake-up stroke prevention, thus hopefully leading to a decrease in its mortality and disability in medium to long-term outcome. METHODS: 4251 ischemic stroke (IS) patients from a prospectively registered database were recruited; 3838 (90.3%) had known onset-symptom time, and 413 (9.7%) were wake-up strokes. The main endpoint was to analyze the association between different serum biomarkers with wake-up IS episodes and their progression. Leukocytes count, serum levels of C-reactive protein, fibrinogen, interleukin 6 (IL-6), and vitamin D were analyzed as inflammation biomarkers; N-terminal pro-B-type Natriuretic-Peptide and microalbuminuria, used as atrial/endothelial dysfunction biomarkers; finally, glutamate levels as excitotoxicity biomarker. In addition, demographic, clinical and neuroimaging variables associated with the time-evolution of wake-up IS patients and functional outcome at 3 months were evaluated. Good and poor functional outcome were defined as mRS ≤2 and mRS > 2 at 3 months, respectively. RESULTS: Wake-up IS showed a poorer outcome at 3-months than in patients with known on-set-symptom time (59.1% vs. 48.1%; p < 0.0001). Patients with wake-up IS had higher levels of inflammation biomarkers; IL-6 levels at admission (51.5 ± 15.1 vs. 27.8 ± 18.6 pg/ml; p < 0.0001), and low vitamin D levels at 24 h (5.6 ± 5.8 vs. 19.2 ± 9.4 ng/ml; p < 0.0001) are worthy of attention. In a logistic regression model adjusted for vitamin D, OR was 15.1; CI 95%: 8.6-26.3, p < 0.0001. However, we found no difference in vitamin D levels between patients with or without clinical-DWI mismatch (no: 18.95 ± 9.66; yes: 17.84 ± 11.77 ng/mL, p = 0.394). No difference in DWI volume at admission was found (49.3 ± 96.9 ml in wake-up IS patients vs. 51.7 ± 98.2 ml in awake IS patients; p = 0.895). CONCLUSIONS: Inflammatory biomarkers are the main factors that are strongly associated with wake-up IS episodes. Wake-up IS is associated with lower vitamin D levels. These data indicate that vitamin D deficiency could become a therapeutic target to reduce wake-up IS events.

New Perspectives for Developing Therapeutic Bioconjugates of Metabolite-Depleting Enzymes: Lessons Learned Combating Glutamate Excitotoxicity.

Glutamate, the main excitatory neurotransmitter in the central nervous system, plays an essential role in several cognitive activities such as memorizing and learning. Excessive glutamate release and disturbance of glutamate homeostasis participates in multiple neuronal pathologies including cerebral ischemia (inadequate blood supply), traumatic brain injury (e.g., from a fall or an accident), multiple sclerosis, epilepsy, migraine, fetal hypoxia, or Alzheimer's disease. Attenuating excitotoxicity by, for example, targeting glutamate receptors has proved to be beneficial in animal models but has largely failed in clinical trials because of toxic side effects. New therapeutic concepts have been explored to reduce the excitotoxic effect caused by the excessive glutamate release by using or stimulating glutamate-depleting enzymes in the bloodstream. These enzymes indirectly act upon the brain by depleting glutamate in the bloodstream, which is believed to siphon it out of the brain. Recent studies have shown that bioconjugate approaches applied to such enzymes exacerbate this therapeutic effect but raise additional questions for future research. This Perspective provides an overview of lessons learned by our group when exploring bioconjugate approaches for combatting glutamate excitotoxicity as an illustration of how research on therapeutic bioconjugates is evolving.

Stress Granules and Acute Ischemic Stroke: Beyond mRNA Translation.

Ischemic stroke is a leading cause of death and disability worldwide. Following an ischemic insult, cells undergo endoplasmic reticulum (ER) stress, which increases the ER's protein-folding and degradative capacities and blocks the global synthesis of proteins by phosphorylating the eukaryotic translation initiation factor 2-alpha (eIF2α). Phosphorylation of eIF2α is directly related to the dynamics of stress granules (SGs), which are membraneless organelles composed of RNA-binding proteins and mRNA. SGs play a critical role in mRNA metabolism and translational control. Other translation factors are also linked to cellular pathways, including SG dynamics following a stroke. Because the formation of SGs is closely connected to mRNA translation, it is interesting to study the relationship between SG dynamics and cellular outcome in cases of ischemic damage. Therefore, in this review, we focus on the role of SG dynamics during cerebral ischemia.

Associations between RNA-Binding Motif Protein 3, Fibroblast Growth Factor 21, and Clinical Outcome in Patients with Stroke.

BACKGROUND: RNA-binding motif protein 3 (RBM3) is a cold-induced marker of good functional outcome of ischemic stroke that is promising as a protective target. Fibroblast growth factor 21 (FGF21) is an obesity- and temperature-related hormone that upregulates the expression of RBM3, which is beneficial as a recombinant treatment and has been tested under different experimental pathological conditions, including stroke. However, the interaction between RBM3 and FGF21 has not yet been tested for clinical stroke conditions. METHODS: In a sample of 66 stroke patients, we analyzed the associations between the FGF21 and RBM3 serum concentrations on admission and at 72 h, body weight, maximum temperature during the first 24 h, and the outcome of patients at 3 months. We also analyzed their association with biomarkers of obesity (adiponectin and leptin) and inflammation (interleukin-6 (IL-6) and interleukin (IL-10)). RESULTS: Higher concentrations of FGF21 on admission and RBM3 at 72 h were associated with good outcomes. Serum FGF21 and RBM3 were directly related to body mass index and inversely related to the maximum temperature during the first 24 h. We found a positive association between the FGF21 concentrations in obese patients with leptin and a negative correlation with adiponectin in non-obese participants. CONCLUSIONS: This clinical study demonstrates the association between RBM3 and FGF21 levels and the outcome of stroke patients. Although further investigations are required, these data support the pharmacological induction of RBM3 as a promising protective therapy.

Protein content of blood-derived extracellular vesicles: An approach to the pathophysiology of cerebral hemorrhage.

Introduction: Extracellular vesicles (EVs) participate in cell-to-cell paracrine signaling and can be biomarkers of the pathophysiological processes underlying disease. In intracerebral hemorrhage, the study of the number and molecular content of circulating EVs may help elucidate the biological mechanisms involved in damage and repair, contributing valuable information to the identification of new therapeutic targets. Methods: The objective of this study was to describe the number and protein content of blood-derived EVs following an intracerebral hemorrhage (ICH). For this purpose, an experimental ICH was induced in the striatum of Sprague-Dawley rats and EVs were isolated and characterized from blood at baseline, 24 h and 28 days. The protein content in the EVs was analyzed by mass spectrometric data-dependent acquisition; protein quantification was obtained by sequential window acquisition of all theoretical mass spectra data and compared at pre-defined time points. Results: Although no differences were found in the number of EVs, the proteomic study revealed that proteins related to the response to cellular damage such as deubiquitination, regulation of MAP kinase activity (UCHL1) and signal transduction (NDGR3), were up-expressed at 24 h compared to baseline; and that at 28 days, the protein expression profile was characterized by a higher content of the proteins involved in healing and repair processes such as cytoskeleton organization and response to growth factors (COR1B) and the regulation of autophagy (PI42B). Discussion: The protein content of circulating EVs at different time points following an ICH may reflect evolutionary changes in the pathophysiology of the disease.

Circulating Extracellular Vesicle Proteins and MicroRNA Profiles in Subcortical and Cortical-Subcortical Ischaemic Stroke.

In order to investigate the role of circulating extracellular vesicles (EVs), proteins, and microRNAs as damage and repair markers in ischaemic stroke depending on its topography, subcortical (SC), and cortical-subcortical (CSC) involvement, we quantified the total amount of EVs using an enzyme-linked immunosorbent assay technique and analysed their global protein content using proteomics. We also employed a polymerase chain reaction to evaluate the circulating microRNA profile. The study included 81 patients with ischaemic stroke (26 SC and 55 CSC) and 22 healthy controls (HCs). No differences were found in circulating EV levels between the SC, CSC, and HC groups. We detected the specific expression of C1QA and Casp14 in the EVs of patients with CSC ischaemic stroke and the specific expression of ANXA2 in the EVs of patients with SC involvement. Patients with CSC ischaemic stroke showed a lower expression of miR-15a, miR-424, miR-100, and miR-339 compared with those with SC ischaemic stroke, and the levels of miR-339, miR-100, miR-199a, miR-369a, miR-424, and miR-15a were lower than those of the HCs. Circulating EV proteins and microRNAs from patients with CSC ischaemic stroke could be considered markers of neurite outgrowth, neurogenesis, inflammation process, and atherosclerosis. On the other hand, EV proteins and microRNAs from patients with SC ischaemic stroke might be markers of an anti-inflammatory process and blood-brain barrier disruption reduction.

Characterization of a Temporal Profile of Biomarkers as an Index for Ischemic Stroke Onset Definition.

BACKGROUND AND PURPOSE: Stroke is a dynamic process in terms of molecular mechanisms, with prominent glutamate-mediated excitotoxicity at the onset of symptoms followed by IL-6-mediated inflammation. Our aim was to study a serum glutamate/IL-6 ratio as an index for stroke onset definition. METHODS: A total of 4408 ischemic stroke patients were recruited and then subdivided into four quartiles according to latency time in minutes (0-121, 121-185, 185-277 and >277). Latency time is defined as the time between stroke onset and treatment at the neurological unit. The primary endpoint of the study was the association of early latency times with different clinical aspects and serum markers. Serum glutamate and interleukin-6 (IL-6) levels at admission were selected as the main markers for excitotoxicity and inflammation, respectively. RESULTS: Glutamate serum levels were significantly higher in the earlier latency time compared with the higher latency times (p < 0.0001). IL-6 levels were lower in early latency times (p < 0.0001). Patients with a glutamate/IL-6 index on admission of >5 were associated with a latency time of <121 min from the onset of symptoms with a sensitivity of 88% and a specificity of 80%. CONCLUSIONS: The glutamate/IL-6 index allows the development of a ratio for an easy, non-invasive early identification of the onset of ischemic stroke symptoms, thus offering a new tool for selecting early stroke patient candidates for reperfusion therapies.

sTWEAK as Predictor of Stroke Recurrence in Ischemic Stroke Patients Treated With Reperfusion Therapies.

Aim: The purpose of this study was to investigate clinical and neuroimaging factors associated with stroke recurrence in reperfused ischemic stroke patients, as well as the influence of specific biomarkers of inflammation and endothelial dysfunction. Methods: We conducted a retrospective analysis on a prospectively registered database. Of the 875 patients eligible for this study (53.9% males; mean age 69.6 ± 11.8 years vs. 46.1% females; mean age 74.9 ± 12.6 years), 710 underwent systemic thrombolysis, 87 thrombectomy and in 78, systemic or intra-arterial thrombolysis together with thrombectomy was applied. Plasma levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) were analyzed as markers of inflammation, and soluble tumor necrosis factor-like inducer of apoptosis (sTWEAK) as an endothelial dysfunction marker. The main outcome variables of the study were the presence and severity of leukoaraiosis (LA) and stroke recurrence. Results: The average follow-up time of the study was 25 ± 13 months, during which 127 patients (14.5%) showed stroke recurrence. The presence and severity of LA was more severe in the second stroke episode (Grade III of the Fazekas 28.3 vs. 52.8%; p < 0.0001). IL-6 levels at the first admission and before reperfusion treatment in patients with and without subsequent recurrence were similar (9.9 ± 10.4 vs. 9.1 ± 7.0 pg/mL, p = 0.439), but different for TNFα (14.7 ± 5.6 vs. 15.9 ± 5.7 pg/mL, p = 0.031) and sTWEAK (5,970.8 ± 4,330.4 vs. 8,660.7 ± 5,119.0 pg/mL, p < 0.0001). sTWEAK values ≥7,000 pg/mL determined in the first stroke were independently associated to recurrence (OR 2.79; CI 95%: 1.87-4.16, p < 0.0001). Conclusions: The severity and the progression of LA are the main neuroimaging factors associated with stroke recurrence. Likewise, sTWEAK levels were independently associated to stroke recurrence, so further studies are necessary to investigate sTWEAK as a therapeutic target.

Mild systemic inflammation enhances response to OnabotulinumtoxinA in chronic migraineurs.

The anti-inflammatory effect of OnabotulinumtoxinA (OnabotA) has been a matter of discussion for many years. In chronic migraine, however, increased pro-inflammatory state is associated with good response to OnabotA. We aimed to investigate whether a mild systemic inflammatory state elicited by a common oral infection (periodontitis) could enhance treatment response to OnabotA. In this study, we included 61 chronic migraineurs otherwise healthy treated with OnabotA of which 7 were poor responders and 54 good responders. Before receiving OnabotA therapy, all participants underwent a full-mouth periodontal examination and blood samples were collected to determine serum levels of calcitonin gene-related peptide (CGRP), interleukin 6 (IL-6), IL-10 and high sensitivity C-reactive protein (hs-CRP). Periodontitis was present in 70.4% of responders and 28.6% of non-responders (P = 0.042). Responders showed greater levels of inflammation than non-responders (IL-6: 15.3 ± 8.7 vs. 9.2 ± 4.7 ng/mL, P = 0.016; CGRP: 18.8 ± 7.6 vs. 13.0 ± 3.1 pg/mL, P = 0.002; and hs-CRP: 3.9 ± 6.6 vs. 0.9 ± 0.8 mg/L, P = 0.003). A linear positive correlation was found between the amount of periodontal tissue inflamed in the oral cavity and markers of inflammation (IL-6: r = 0.270, P = 0.035; CGRP: r = 0.325, P = 0.011; and hs-CRP: r = 0.370, P = 0.003). This report shows that in presence of elevated systemic inflammatory markers related to periodontitis, OnabotA seems to reduce migraine attacks. The changes of scheduled inflammatory parameters after treatment and subsequent assessment during an adequate period still need to be done.

Inhibition of endogenous blood glutamate oxaloacetate transaminase enhances the ischemic damage.

Glutamate oxaloacetate transaminase 1 (GOT1) enzyme plays a critical role in the cell metabolism by participating in the carbohydrate and amino acid metabolism. In ischemic stroke, we have demonstrated that recombinant GOT1 acts as a novel neuroprotective treatment against the excess of extracellular glutamate that accumulates in the brain following ischemic stroke. In this study, we investigated the inhibitory effect of GOT1 on brain metabolism and on the ischemic damage in a rat model of ischemic stroke by means of a specific antibody developed against this enzyme. Inhibition of GOT1 caused higher brain glutamate and lactate levels and this response was associated with larger ischemic lesion. This study represents the first demonstration that the inhibition of the blood GOT1 activity leads to more severe ischemic damage and poorer outcome and supports the protective role of GOT1 against ischemic insults.

Similarities and Differences in Extracellular Vesicle Profiles between Ischaemic Stroke and Myocardial Infarction.

Extracellular vesicles (EVs) are involved in intercellular signalling through the transfer of molecules during physiological and pathological conditions, such as ischaemic disease. EVs might therefore play a role in ischaemic stroke (IS) and myocardial infarction (MI). In the present study, we analysed the similarities and differences in the content of circulating EVs in patients with IS and MI. This prospective observational study enrolled 140 participants (81 patients with IS, 37 with MI and 22 healthy controls [HCs]). We analysed the protein and microRNA content from EVs using proteomics and reverse transcription quantitative real-time polymerase chain reaction and compared it between the groups. In the patients with IS and MI, we identified 14 common proteins. When comparing IS and MI, we found differences in the protein profiles (apolipoprotein B, alpha-2-macroglobulin, fibronectin). We also found lower levels of miR-340 and miR-424 and higher levels of miR-29b in the patients with IS and MI compared with the HCs. Lastly, we found higher miR-340 levels in IS than in MI. In conclusion, proteomic and miRNA analyses suggest a relationship between circulating EV content and the patient's disease state. Although IS and MI affect different organs (brain and heart) with distinct histological characteristics, certain EV proteins and miRNAs appear to participate in both diseases, while others are present only in patients with IS.

Sustained blood glutamate scavenging enhances protection in ischemic stroke.

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.

The presence of leukoaraiosis enhances the association between sTWEAK and hemorrhagic transformation.

OBJECTIVE: To investigate whether elevated serum levels of sTWEAK (soluble tumor necrosis factor-like inducer of apoptosis) might be involved in a higher frequency of symptomatic hemorrhagic transformation (HT) through the presence of leukoaraiosis (LA) in patients with acute ischemic stroke (IS) undergoing reperfusion therapies. METHODS: This is a retrospective observational study. The primary endpoint was to study the sTWEAK-LA-HT relationship by comparing results with biomarkers associated to HT and evaluating functional outcome at 3-months. Clinical factors, neuroimaging variables and biomarkers associated to inflammation, endothelial/atrial dysfunction or blood-brain barrier damage were also investigated. RESULTS: We enrolled 875 patients (mean age 72.3 ± 12.2 years; 46.0% women); 710 individuals underwent intravenous thrombolysis, 87 endovascular therapy and 78 both. HT incidence was 32%; LA presence was 75.4%. Patients with poor functional outcome at 3-months showed higher sTWEAK levels at admission (9844.2 [7460.4-12,542.0] vs. 2717.3 [1489.7-5852.3] pg/mL, P < 0.0001). By means of logistic regression models, PDGF-CC and sTWEAK were associated with mechanisms linked simultaneously to HT and LA. Serum sTWEAK levels at admission ≥6700 pg/mL were associated with an odds ratio of 13 for poor outcome at 3-months (OR: 13.6; CI 95%: 8.2-22.6, P < 0.0001). CONCLUSIONS: Higher sTWEAK levels are independently associated with HT and poor functional outcome in patients with IS undergoing reperfusion therapies through the presence of LA. sTWEAK could become a therapeutic target to reduce HT incidence in patients with IS.

Toll-like receptors as diagnostic targets in pellucid marginal degeneration.

The main purpose of this study is to evaluate the diagnostic role of Toll-like receptors 2 (TLR2) and 4 (TLR4) expression in corneal and conjunctival epithelial cells of eyes with pellucid marginal degeneration (PMD) compared to keratoconus patients (KC) and control subjects. A prospective case-control study in 29 PMD eyes, 109 KC eyes and 72 healthy eyes was done. All participants were subjected to a clinical, topographic, aberrometric and tomographic exam with extraction of corneal and conjunctival epithelial cells through scraping. The TLR2 and TLR4 expression was measured with flow cytometry. Receiver operating characteristic (ROC) curve analysis was used to determine the most appropriate cutoff point for predicting the risk of PMD and KC. Correlations between TLR2/TLR4 expression and the severity of PMD/KC were evaluated. A TLRs follow-up review was made 19 ± 4 months after to the first review. As result, mean expression of TLR2 and TLR4 in both corneal and conjunctival epithelial cells was significantly higher in eyes with corneal ectasia (PMD and KC) than in control eyes (all p < 0.05). Conjunctival TLR4 expression showed the highest capacity to diagnose the existence of PMD (odd ratio 42.84; 95% confidence interval:6.20-296.20; p < 0.0001) after adjusting by eye rubbing and steeper corneal meridian. Moreover, we found an association between the TLR2/TLR4 overexpression with the severity of the PMD and KC measured by corneal topographic, aberrometric and tomographic quantitative parameters (all p < 0.05). Differences on TLR2/TLR4 expression between study groups were maintained during the follow-up period. In conclusion, the TLR2/TLR4 overexpression in corneal and conjunctival epithelial cells of PMD and KC patients compared to healthy control subjects have demonstrated their role as diagnostic target in both corneal ectatic disorders.