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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.

To analyse the influence of socioeconomic status on the clinical profile of patients undergoing non-traumatic lower-limb amputation.Retrospective study of 697 lower-limb amputee patients in an Angiology and Vascular Surgery Department during a 5-year period. Patients were classified according to their socioeconomic status (low, medium and high). We analysed demographic (age and gender) and clinical variables (cause of amputation, comorbidity, cardiovascular risk factors and amputation level).Mean age was 70.5 ± 11.9 years, and the median was 72 years. The low socioeconomic status group presented a higher frequency of amputations in men. Cardiovascular risks factors were more frequent in this socioeconomic group, and the difference was statistically significant for diabetes (85.8% low, 69.3% medium, 65% high; P.01) and obesity (31.4% low, 22.6% medium, 12.5% high, P.01). Diabetic retinopathy was the only comorbidity with a significant association with low socioeconomic status (21.1% low, 15.3% medium, 12.5% high, P.03). Regarding the cause for amputation, there was no difference in terms of socioeconomic status. The low socioeconomic level showed a higher frequency of major amputation, which was a significant difference (63.6% low, 41.2% medium, 55% high, P.04) and a higher predisposition for this amputation level.The low socioeconomic status has been shown to determine an unfavourable vascular risk profile in lower-limb non-traumatic amputees and a higher predisposition of a major amputation. This socioeconomic level demonstrates a negative influence on these patients' diabetes, obesity and diabetic retinopathy.

Resumen Introduccion Analizar la influencia del nivel socioeconomico (NSE) sobre el perfil clinico del paciente amputado de miembro inferior por causa no traumatica. Metodos Estudio retrospectivo con 697 pacientes amputados de miembro inferior, en un Servicio de Angiologia y Cirugia Vascular, durante un periodo de 5 anos. Los pacientes fueron divididos segun NSE (bajo, medio y alto). Se analizaron variables demograficas (edad y genero) y clinicas (causa, comorbilidad, factores de riesgo cardiovascular y nivel de amputacion) Resultados La edad media fue 70,5 ± DE 11,9 anos y la mediana 72 anos. El NSE bajo presento mayor frecuencia de hombres amputados. Todos los factores de riesgo cardiovascular fueron mas frecuentes en este nivel, aunque solo fue estadisticamente significativo en diabetes (85,8% bajo, 69,3% medio, 65% alto; p Conclusiones El NSE bajo determina un perfil de riesgo vascular mas desfavorable en los pacientes amputados de miembro inferior por causa no traumatico y una mayor predisposicion a sufrir amputacion mayor. Este NSE muestra una influencia negativa sobre diabetes, obesidad y retinopatia diabetica en estos pacientes.

Background: Magnetic resonance imaging (MRI) is currently used for the study of intracerebral hemorrhage (ICH) in animal models. However, ultrasound is an inexpensive, non-invasive and rapid technique that could facilitate the diagnosis and follow-up of ICH. This study aimed to evaluate the feasibility and reliability of B-mode ultrasound as an alternative tool for in vivo monitoring of ICH volume and brain structure displacement in an animal model.Methods: A total of 31 male and female Sprague-Dawley rats were subjected to an ICH model using collagenase-IV in the striatum following stereotaxic references. The animals were randomly allocated into 3 groups: healthy (n = 10), sham (n = 10) and ICH (n = 11). B-mode ultrasound studies with a 13-MHz probe were performed pre-ICH and at 5 h, 48 h, 4 d and 1 mo post-ICH for the assessment of ICH volume and displacement of brain structures, considering the distance between the subarachnoid cisterns and the dura mater. The same variables were studied by MRI at 48 h and 1 mo post-ICH.Results: Both imaging techniques showed excellent correlation in measuring ICH volume at 48 h (r = 0.905) and good at 1 mo (r = 0.656). An excellent correlation was also observed in the measured distance between the subarachnoid cisterns and the dura mater at 1 mo between B-mode ultrasound and MRI, on both the ipsilateral (r = 0.870) and contralateral (r = 0.906) sides of the lesion.Conclusion: B-mode ultrasound imaging appears to be a reliable tool for in vivo assessment of ICH volume and displacement of brain structures in animal models.

Background and purpose The aim was to identify whether post-stroke hyperglycaemia (PSH) influences the levels of circulating biomarkers of brain damage and repair, and to explore whether these biomarkers mediate the effect of PSH on the ischaemic stroke (IS) outcome. Methods This was a secondary analysis of the Glycaemia in Acute Stroke II study. Biomarkers of inflammation, prothrombotic activity, endothelial dysfunction, blood-brain barrier rupture, cell death and brain repair processes were analysed at 24-48 h (baseline) and 72-96 h (follow-up) after IS. The associations of the biomarkers and stroke outcome (modified Rankin Scale score at 3 months) based on the presence of PSH were compared. Results A total of 174 patients participated in this sub-study. Brain-derived neurotrophic factor (BDNF) at admission was negatively correlated with glucose levels. PSH was associated with a trend toward higher levels of endothelial progenitor cells (EPCs) at baseline. The EPCs in the PSH group then decreased in the follow-up samples (-8.5 ± 10.3) compared with the non-PSH group (4.7 ± 7.33; P = 0.024). However, neither BDNF nor EPC values had correlation with the 3-month outcome. Higher interleukin-6 at follow-up was associated with poor outcomes (modified Rankin Scale > 2) independently of PSH. Conclusion Post-stroke hyperglycaemia appears to be associated with a negative regulation of BDNF and a different reaction in EPC levels. However, neither BDNF nor EPCs showed significant mediation of the PSH association with IS outcome, and only higher interleukin-6 in the follow-up samples (72-96 h) was related to poor outcomes, independently of PSH status. Further studies are needed to achieve definite conclusions.

Los autores agradecen al Servicio de Angiología y Cirugía Vascular del Hospital Universitario San Cecilio de Granada, España., Objetivo: Analizar las diferencias según el sexo en el perfil clínico de riesgo de los pacientes amputados de miembro inferior por causa no traumática. Método: Estudio retrospectivo descriptivo con 697 pacientes amputados de miembro inferior por causa no traumática, en un Servicio de Angiología y Cirugía Vascular, durante un periodo de 5 años y divididos en función del sexo. Se analizaron variables demográficas (edad) y clínicas (causa, comorbilidad, factores de riesgo cardiovascular y nivel de amputación). Resultados: La edad media (± desviación estándar) de las mujeres fue mayor (75.3 ± 12 frente a 68.9 ± 11 años; p < 0.001) y ellas presentaron mayor frecuencia de factores de riesgo cardiovascular concomitantes, tales como diabetes, hipertensión y obesidad, que los varones, mientras que ellos presentaron mayor frecuencia de tabaquismo. No hubo diferencia significativa entre sexos respecto a la distribución de las causas de amputación, siendo la principal causa la isquémica, destacando la arteriosclerosis. Las mujeres presentaron mayor frecuencia y predisposición para un nivel de amputación mayor. Conclusiones: En nuestro estudio, las mujeres presentaron un perfil de riesgo cardiovascular más desfavorable y el sexo femenino fue un predictor independiente de amputación mayor de miembro inferior por causa no traumática., Objectives: To analyse the possible gender differences within the clinical risk profile of lower-limb amputee patients who had a non-traumatic amputation. Method: A retrospective study with 697 lower-limb amputee patients, classified according to their gender, has been conducted at the Angiology and Vascular Surgery Department for 5 years. The variables considered were demographic variables (age and gender) and clinic variables (cause of the amputation, comorbidity, cardiovascular risk factors and the amputation level). Results: Regarding to the results, the average of women was older than men (75.3 ± 12 vs. 68.9 ± 11 years; p < 0.001), showing an increasing frequency to concomitant cardiovascular risk factors, such as diabetes, hypertension, and obesity. However, men present a higher frequency to smoking. There were no gender differences in the main cause of the amputation, the ischemic cause, being notably into this cause the subgroup of arteriosclerosis cause. Women showed a higher frequency and predisposition for major amputation. Conclusions: This study showed that women had a more unfavorable cardiovascular risk profile than men, being the female gender an independent predictor to a major lower-limb amputation with non-traumatic amputation.

Ultrasound is a noninvasive technique that provides real-time imaging with excellent resolution, and several studies demonstrated the potential of ultrasound in acute ischemic stroke monitoring. However, only a few studies were performed using animal models, of which many showed ultrasound to be a safe and effective tool also in therapeutic applications. The full potential of ultrasound application in experimental stroke is yet to be explored to further determine the limitations of this technique and to ensure the accuracy of translational research. This review covers the current status of ultrasound applied to monitoring and treatment in experimental animal models of stroke and examines the safety, limitations, and future perspectives., This research was funded by the Carlos III Health Institute Health Care Research Fund grant number FIS PI16/01052 and cofunded by the European Regional Development Fund (ERDF)–Miguel Servet (CP15/00069, CPII20/00002 to María Gutiérrez–Fernández; CP20/00024 to Laura Otero–Ortega) and predoctoral fellowship (FI17/00188 to Mari Carmen Gómez–de Frutos, FI18/00026 to Fernando Laso–García) and the INVICTUS PLUS Spanish Network (RD16/0019/0005) of the Carlos III Health Institute (ISCIII).

Background Ultrasound is a safe, non-invasive and affordable imaging technique for the visualization of internal structures and the measurement of blood velocity using Doppler imaging. However, despite all these advantages, no study has identified the structures of the rat brain using conventional ultrasound. Methods A 13 MHz high frequency transducer was used to identify brain structures in the rat. The enlargement of the transcranial window was performed gradually using the ultrasound directly on the skin of the animal, then against the skull, then through a delimited craniotomy and finally through a complete craniotomy. Results Our results showed that ultrasound allowed the identification of cerebral ventricles and subarachnoid cisterns, as well as the analysis of real-time monitoring of cerebral blood flow in the main brain arteries of the rat. Comparison with existing methods Ultrasound is a tool with the potential to identify brain structures and blood vessels. In contrast to MRI, transcranial ultrasound is a fast, non-invasive, well tolerated and low-cost method and can be done at the bedside. Conclusion In the present study, we described an atlas of the main brain structures as well as the main vasculature in the rat using ultrasound. This technique could be applied in animal models of various neurological diseases.

Acute ischemic stroke is currently a major cause of disability despite improvement in recanalization therapies. Stem cells represent a promising innovative strategy focused on reduction of neurologic sequelae by enhancement of brain plasticity. We performed a phase IIa, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. Patients aged ≥60 years with moderate to severe stroke (National Institutes of Health Stroke Scale [NIHSS] 8–20) were randomized (1:1) to receive intravenous adipose tissue–derived mesenchymal stem cells (AD-MSCs) or placebo within the first 2 weeks of stroke onset. The primary outcome was safety, evaluating adverse events (AEs), neurologic and systemic complications, and tumor development. The secondary outcome evaluated treatment efficacy by measuring modified Rankin Scale (mRS), NIHSS, infarct size, and blood biomarkers. We report the final trial results after 24 months of follow-up. Recruitment began in December 2014 and stopped in December 2017 after 19 of 20 planned patients were included. Six patients did not receive study treatment: two due to technical issues and four for acquiring exclusion criteria after randomization. The final study sample was composed of 13 patients (4 receiving AD-MSCs and 9 placebo). One patient in the placebo group died within the first week after study treatment delivery due to sepsis. Two non-treatment-related serious AEs occurred in the AD-MSC group and nine in the placebo group. The total number of AEs and systemic or neurologic complications was similar between the study groups. No injection-related AEs were registered, nor tumor development. At 24 months of follow-up, patients in the AD-MSC group showed a nonsignificantly lower median NIHSS score (interquartile range, 3 [3–5.5] vs 7 [0–8]). Neither treatment group had differences in mRS scores throughout follow-up visits up to month 24. Therefore, intravenous treatment with AD-MSCs within the first 2 weeks from ischemic stroke was safe at 24 months of follow-up.

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., Using a rodent model of ischemic stroke, Zahgmi et al. report on the neuro-protective effects of a bioconjugate form of the glutamate scavenger, glutamate-oxaloacetate transaminase (GOT). They found that a single dose of the bioconjugate was more effective in reducing the infarct size and improving motor function than the native GOT due to its higher and long lasting blood activity.

Background Mesenchymal stem cell-derived extracellular vesicles (EVs) are one of the most promising therapeutics in protective and/or regenerative therapy in animal models of stroke using a dose of 100 μg. However, whether EVs dose is related to outcomes is not known. This study aimed to identify the optimal effective dose of EVs from adipose tissue-derived mesenchymal stem cells that promote functional recovery in subcortical stroke. Materials and methods For this purpose, various doses of EVs were tested in an in vitro oxygen-glucose deprivation (OGD) model of oligodendrocytes and neuronal ischemia. At least 50 μg of EVs were necessary to induce proliferation and differentiation of oligodendrocyte and neurons in OGD conditions. For in vivo study, rats were subjected to subcortical stroke and various doses (50 μg, 100 μg, or 200 μg) of EVs were intravenously administered after 24 h. Results All the animals in the EV groups showed significant improvement in functional tests, with an increase in tract connectivity and brain repair-associated markers, and a decrease in cell death and in astrocyte-marker expression. Cell proliferation was increased in the groups receiving 50 μg and 100 μg doses. Only the 50-μg dose was associated with significant increases in brain-derived neurotrophic factor expression. Conclusion In conclusion, 50 μg of EVs appears to be the minimal effective dose to enhance protection, brain repair, and recovery in subcortical ischemic stroke.

Extracellular vesicles (EVs) are a heterogeneous group of bilayer membrane-wrapped molecules that play an important role in cell-to-cell communication, participating in many physiological processes and in the pathogenesis of several diseases, including multiple sclerosis (MS). In recent years, many studies have focused on EVs, with promising results indicating their potential role as biomarkers in MS and helping us better understand the pathogenesis of the disease. Recent evidence suggests that there are novel subpopulations of EVs according to cell origin, with those derived from cells belonging to the nervous and immune systems providing information regarding inflammation, demyelination, axonal damage, astrocyte and microglia reaction, blood–brain barrier permeability, leukocyte transendothelial migration, and ultimately synaptic loss and neuronal death in MS. These biomarkers can also provide insight into disease activity and progression and can differentiate patients’ disease phenotype. This information can enable new pathways for therapeutic target discovery, and consequently the development of novel treatments. Recent evidence also suggests that current disease modifying treatments (DMTs) for MS modify the levels and content of circulating EVs. EVs might also serve as biomarkers to help monitor the response to DMTs, which could improve medical decisions concerning DMT initiation, choice, escalation, and withdrawal. Furthermore, EVs could act not only as biomarkers but also as treatment for brain repair and immunomodulation in MS. EVs are considered excellent delivery vehicles. Studies in progress show that EVs containing myelin antigens could play a pivotal role in inducing antigen-specific tolerance of autoreactive T cells as a novel strategy for the treatment as “EV-based vaccines” for MS. This review explores the breakthrough role of nervous and immune system cell-derived EVs as markers of pathological disease mechanisms and potential biomarkers of treatment response in MS. In addition, this review explores the novel role of EVs as vehicles for antigen delivery as a therapeutic vaccine to restore immune tolerance in MS autoimmunity.

Cell-based therapy has demonstrated safety and efficacy in experimental animal models of stroke, as well as safety in stroke patients. However, various questions remain regarding the therapeutic window, dosage, route of administration, and the most appropriate cell type and source, as well as mechanisms of action and immune-modulation to optimize treatment based on stem cell therapy. Various delivery routes have been used in experimental stroke models, including intracerebral, intraventricular, subarachnoid, intra-arterial, intraperitoneal, intravenous, and intranasal routes. From a clinical point of view, it is necessary to demonstrate which is the most feasible, safest, and most effective for use with stroke patients. Therefore, further experimental studies concerning the safety, efficacy, and mechanisms of action involved in these therapeutic effects are required to determine their optimal clinical use.

Background: Pre-clinical studies on adipose tissue-derived mesenchymal stem cells (AD-MSC) suggest its efficacy and safety for ischemic stroke (IS). Our aim was to evaluate the safety of a single dose of AD-MSC administered intravenously in acute IS patients. Methods: A phase II, randomised, double-blind, placebo-controlled, single-center, pilot clinical trial. We included patients aged ≥ 60 years, with moderate to severe IS. Patients were randomly assigned (1:1 ratio) to receive either 1M cells/kg AD-MSC or placebo within the first two weeks after the onset of an IS. The primary outcome measure was safety (adverse events, neurological and systemic complications, and tumor developments). Secondary outcomes explored efficacy (modified Rankin Scale (mRS) and NIHSS). Here we report the preliminary results at 6 months of follow-up. EudraCT: 2011-003551-18; NCT01678534. Findings: Recruitment was stopped in December 2017 after including 19 out of the 20 planned patients. Six patients did not receive the study treatment (3 for technical reasons and 3 because they presented exclusion criteria after randomisation). The final sample was composed of 13 patients (4 in the AD-MSC and 9 in the placebo group). One patient from the placebo group died within the first 7 days due to sepsis. Serious adverse events at 6 months were reported in 5.1% of the AD-MSC group and in 6.6% in the placebo group. No injection-related adverse events or tumour developments were reported. After 6 months of follow-up there were no significant differences between the groups in median NIHSS scores [2.5 (IQR 1.75, 3.50) vs. 5 (IQR 4.0, 7.50)] and median mRS [3 (IQR 3.0, 3.25) vs. 4 (IQR 3.0, 4.0)]. Interpretation: Intravenous administration of AD-MSC within the first 2 weeks from IS onset seems to be safe at 6-months of follow-up and shows a trend toward improving neurological and functional recovery. Trial Registration: The study was registered at Clinicaltrials.gov, under the number NCT01678534. Funding Statement: Spanish Ministry of Health (EC2010/171). Declaration of Interests: B Fuentes reports grants from Spanish Ministry of Health and European Commission (Horizon2020 program), during the conduct of the study; other from Daichi-Sankyo, other from Bayer, other from BMS-Pfizer, other from Novonordisk, other from Novartis, outside the submitted work; M Gutierrez-Fernandez rports grants from Spanish Ministry of Health and European Commission (Horizon2020 program), during the conduct of the study; non-financial support from Cellerix/Tigenix, non-financial support from Histocell, outside the submitted work.AM Borobia reports grants from Instituto de Salud Carlos III, during the conduct of the study; personal fees from Mundipharma, personal fees from Menarini, outside the submitted work; and Principal Investigator in Clinical Trials for GSK and Daiichi Sankyo. E Diez-Tejedor reports grants from Spanish Ministry of Health and European Commission (Horizon2020 program), during the conduct of the study; non-financial support from Cellerix/Tigenix, non-financial support from Histocell, other from Daichi-Sankyo, other from Bayer, other from Sanofy Aventis, other from Novartis outside the submitted work. R Gutierrez-Zuniga, M Alonso de Lecinana, JC Martinez Avila, G Ruiz-Ares, MC Gomez-de Frutos, F Laso-Garcia and L Otero-Ortega report nothing to disclosure. Ethics Approval Statement: It was approved by the La Paz University Hospital Research Ethics Committee and by the Spanish Agency of Medicines and Health Products (EudraCT: 2011-003551-18).

Approximately, 16 million strokes occur worldwide each year, causing 6 million deaths and considerable disability, implying an enormous social, individual health, and economic burden. Due to this high incidence, strategies to promote stroke recovery are urgently needed. Research into new therapeutic approaches for stroke has determined that intravenous administration of mesenchymal stem cells (MSCs) is a good strategy to improve recovery by amplifying mechanisms implicated in brain plasticity. Recent studies have demonstrated the efficacy of MSCs in stroke, with no need for them to reach the area of brain injury. Although the mechanisms by which they mediate restorative effects are still unknown, the evidence suggests that MSCs might use specialised communication by sending and receiving biological information included in elements called exosomes. Exosomes are nanosized extracellular vesicles released into physical environments, and they have recently been suggested to mediate restorative stem cell effects. Moreover, after stroke, exosomes can also be synthesised and released from brain cells, passing through the blood-brain barrier (BBB), and can be detected in peripheral blood or in cerebrospinal fluid. Thus, exosomes could possibly be biomarkers that reflect pathological progress and promote stroke recovery. This review discusses the translational aspects of MSC-derived exosomes and their various roles in brain repair and as circulating biomarkers in stroke.

Aim: To assess the efficacy of 3 doses of exosomes from adipose tissue derived human mesenchymal stem cells to identify the minimal effective dose to enhance brain repair and recovery after subcortical stroke in rats. Methods: Male and female Sprague-Dawley rats (250gr) were injected with endothelin-1 to induce subcortical stroke. 24h after stroke, exosomes were administrated by the tail vein. Study groups were: Sham (n=10): without stroke neither treatment; control: stroke+saline (n=8); stroke+low dose (50μg) (n=10); stroke+intermediate dose (100μg) (n=9); stroke+high dose (200μg) (n=10). We evaluated: motor function (Roger’s, Rotarod and Walking beam test) at 24h, 7 and 28d, lesion volume and tract connectivity were studied by magnetic resonance image at 24h and 28d, brain repair markers: GFAP (Glial Fibrillary Acidic Protein), MOG (Myelin Oligodendrocyte Glycoprotein), MBP (Myelin Basic Protein) by immunofluorescence at 28d. Results: Animals treated with 50μg, 100μg or 200μg of exosomes showed a significant improvement in functional evaluation compared to control (p Conclusion: Intravenous administration of different doses of exosomes (50μg, 100μg and 200μg) improves the functional recovery, tract connectivity and expression of brain repair markers. Low dose is as effective as intermediate and high doses. Therefore, 50μg of exosomes is the minimal effective dose to enhance brain repair and recovery in stroke.From a translational point of view, this dose decreases the cost and risk.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication and as possible therapeutic agents in inflammation-mediated demyelinating diseases, including multiple sclerosis (MS). In the present study, we investigated whether intravenously administered EVs derived from mesenchymal stem cells (MSCs) from human adipose tissue might mediate recovery in Theiler’s murine encephalomyelitis virus (TMEV)induced demyelinating disease, a progressive model of MS. SJL/J mice were subjected to EV treatment once the disease was established. We found that intravenous EV administration improved motor deficits, reduced brain atrophy, increased cell proliferation in the subventricular zone and decreased inflammatory infiltrates in the spinal cord in mice infected with TMEV. EV treatment was also capable of modulating neuroinflammation, given glial fibrillary acidic protein and Iba-1 staining were reduced in the brain, whereas myelin protein expression was increased. Changes in the morphology of microglial cells in the spinal cord suggest that EVs also modulate the activation state of microglia. The clear reduction in plasma cytokine levels, mainly in the Th1 and Th17 phenotypes, in TMEV mice treated with EVs confirms the immunomodulatory ability of intravenous EVs. According to our results, EV administration attenuates motor deficits through immunomodulatory actions, diminishing brain atrophy and promoting remyelination. Further studies are necessary to establish EV delivery as a possible therapy for the neurodegenerative phase of MS., This study was supported by Red Española de Esclerosis Múltiple Grants RD12/ 0032/0008 and RD16/0015/0021 (sponsored by the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional [FEDER]) to C.G.

Recent studies have shown that blood glutamate grabbing is an effective strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. The purpose of the study was to investigate the effect of two of the most efficient blood glutamate grabbers (oxaloacetate and recombinant glutamate oxaloacetate transaminase 1: rGOT1) in a rat model of intracerebral hemorrhage (ICH). Intracerebral hemorrhage was produced by injecting collagenase into the basal ganglia. Three treatment groups were developed: a control group treated with saline, a group treated with oxaloacetate, and a final group treated with human rGOT1. Treatments were given 1 hour after hemorrhage. Hematoma volume (analyzed by magnetic resonance imaging (MRI)), neurologic deficit, and blood glutamate and GOT levels were quantified over a period of 14 days after surgery. The results observed showed that the treatments used induced a significant reduction of blood glutamate levels; however, they did not reduce the hematoma, nor did they improve the neurologic deficit. In the present experimental study, we have shown that this novel therapeutic strategy is not effective in case of ICH pathology. More importantly, these findings suggest that blood glutamate grabbers are a safe treatment modality that can be given in cases of suspected ischemic stroke without previous neuroimaging.

Blocking axonal growth inhibitor NogoA has been of great interest for promoting axonal recovery from neurological diseases. The present study investigates the therapeutic effects of blocking NogoA, inducing functional recovery and promoting white matter repair in an experimental animal model of stroke. Adult male rats were subjected to white matter injury by subcortical ischemic stroke. Twenty-four hours after surgery, 250 ug of anti-NogoA or anti-IgG-1 were administered through the tail vein. The quantity of NogoA protein was determined by immunohistochemistry in the brain and peripheral organs. In addition, functional status, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers were analyzed. Moreover, an in vitro study was performed in order to strengthen the results obtained in vivo. A lower quantity of NogoA protein was found in the brain and peripheral organs of the animals that received anti-NogoA treatment. The animals receiving anti-NogoA treatment showed significantly better results in terms of functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group at 28 days. White matter integrity was in part restored by antibody-mediated inhibition of NogoA administration in those animals that were subjected to an axonal injury by subcortical stroke. This white matter restoration triggered functional recovery.

Exosomes are gaining importance because they show great promise in therapeutic applications for several diseases. Particularly in stroke, exosomes derived from mesenchymal stem cell (MSC) therapy work as paracrine effectors responsible for promoting neurovascular remodeling and functional recovery. Adult male rats were subjected to intracerebral hemorrhage (ICH) by intrastriatal injection of collagenase type IV; 24 h after surgery, MSC-derived exosomes were administered through the tail vein. The rats were euthanized at 7 or 28 days after treatment. Functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers, biodistribution of exosomes and secretome proteomics were analyzed. DiI-labeled exosomes were found in the brains of the ICH-treated group and in the liver, lung and spleen. Animals receiving treatment with exosomes showed significantly better results in terms of functional recovery, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group 28 days after stroke. Proteomics analysis of the exosomes identified more than 2000 proteins that could be implicated in brain repair function. In conclusion, white matter integrity was partly restored by exosome administration mediated by molecular repair factors. Exosomes as a treatment could be a heterogeneous process by nature and presents many factors that can influence brain plasticity in an adaptable and versatile manner. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study has been partially supported by grants from PS15/01318 and INVICTUS (RD12/0014) (Spanish Neurovascular Network), Miguel Servet (CP15/00069 to Marıa Gutierrez-Fernandez) and a Sara Borrell postdoctoral fellowship (CD12/00706, to Laura Otero-Ortega) from the Research Institute Carlos III, the Spanish Ministry of Science and Innovation and the European Regional Development Fund. This study was also co-funded within the Roche Farma project. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Sí

Angiotensin type 1 receptor blockers (ARBs) have induced improved functional recovery and reduced infarct volume in experimental animal models of stroke. Clinical data have indicated a positive correlation between prestroke treatment with ARBs and reduced stroke severity and better outcomes; however, the mechanisms of these beneficial effects are not yet well understood. This study compares the protective and possible reparative effects of continuous oral treatment with olmesartan (OLM) with OLM pretreatment and withdrawal after permanent middle cerebral artery occlusion (pMCAO) in rats. Fifty-two Sprague-Dawley rats were randomly assigned to five groups: MCAO–/OLM (OLM 10 mg/kg/day for 14 days after infarct), MCAOOLM/OLM (OLM 10 mg/kg/day for 7 days before and 14 days after infarct), MCAOOLM/– (OLM 10 mg/kg/day for 7 days before infarct), sham, and control. We analyzed functional recovery; lesion size; cell death; expression of the pro-oxidant enzyme NADPH oxidase 4 (NOX-4); isolectin-B4; and repair markers such as glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). All of the OLM-treated groups showed significantly better functional scores and reduced infarct sizes and cell death compared with the control group 14 days after pMCAO. Levels of NOX-4, VEGF, and BDNF were significantly lower in the brains of the MCAOOLM/OLM and sham groups compared with the other groups. OLM treatment improved functional recovery and reduced lesion size and cell death after cerebral ischemia. Only the continuous administration of OLM before and after stroke reduced oxidative stress levels, with better tissue preservation, without triggering brain repair marker activation. © 2014 Wiley Periodicals, Inc.

Background Few studies have evaluated the possible beneficial effect of the administration of stem cells in the early stages of stroke. Intravenous administration of allogeneic mesenchymal stem cells (MSCs) from adipose tissue in patients with acute stroke could be a safe therapy for promoting neurovascular unit repair, consequently supporting better functional recovery. We aim to assess the safety and efficacy of MSC administration and evaluate its potential as a treatment for cerebral protection and repair. Materials A Phase IIa, prospective, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. Twenty patients presenting acute ischemic stroke will be randomized in a 1:1 proportion to treatment with allogeneic MSCs from adipose tissue or to placebo (or vehicle) administered as a single intravenous dose within the first 2 weeks after the onset of stroke symptoms. The patients will be followed up for 2 years. Primary outcomes for safety analysis: adverse events (AEs) and serious AEs; neurologic and systemic complications, and tumor development. Secondary outcomes for efficacy analysis: modified Rankin Scale; NIHSS; infarct size; and biochemical markers of brain repair (vascular endothelial growth factor, brain-derived neurotrophic factor, and matrix metalloproteinases 9). Results and Conclusions To our knowledge, this is the first, phase II, pilot clinical trial to investigate the safety and efficacy of intravenous administration of allogeneic MSCs from adipose tissue within the first 2 weeks of stroke. In addition, its results will help us define the best criteria for a future phase III study.

Mesenchymal stem cells have previously been shown to mediate brain repair after stroke; they secrete 50-100 nm complexes called extracellular vesicles (EVs), which could be responsible for provoking neurovascular repair and functional recovery. EVs have been observed by electron microscopy and NanoSight, and they contain associated proteins such as CD81 and Alix. This purified, homogeneous population of EVs was administered intravenously after subcortical stroke in rats. To evaluate the EVs effects, we studied the biodistribution, proteomics analysis, functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers. We found that a single administration of EVs improved functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers in an experimental animal model of subcortical stroke. EVs were found in the animals' brain and peripheral organs after euthanasia. White matter integrity was in part restored by EVs administration mediated by molecular repair factors implicated in axonal sprouting, tract connectivity, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery. This novel role for EVs presents a new perspective in the development of biologics for brain repair., This study has been partially supported by grants from PS15/01318 and INVICTUS (RD12/0014) (Spanish Neurovascular Network), Miguel Servet (CP15/00069 to María Gutiérrez-Fernández) and a Sara Borrell postdoctoral fellowship (CD12/00706, to Laura Otero-Ortega) from Research Institute Carlos III, Ministry of Science and Innovation of Spain and European Regional Development Fund

Patients with acute ischemic stroke (AIS) suffer from infections associated with mortality. The relevance of the innate immune system, and monocytes in particular, has emerged as an important factor in the evolution of these infections. The study enrolled 14 patients with AIS, without previous treatment, and 10 healthy controls. In the present study, we show that monocytes from patients with AIS exhibit a refractory state or endotoxin tolerance. The patients were unable to orchestrate an inflammatory response against LPS and expressed three factors reported to control the evolution of human monocytes into a refractory state: IL-1R–associated kinase-M, NFkB2/p100, and hypoxia-inducible factor-1α. The levels of circulating mitochondrial DNA (mtDNA) in patients with AIS correlated with impaired inflammatory response of isolated monocytes. Interestingly, the patients could be classified into two groups: those who were infected and those who were not, according to circulating mtDNA levels. This finding was validated in an independent cohort of 23 patients with AIS. Additionally, monocytes from healthy controls, cultured in the presence of both sera from patients and mtDNA, reproduced a refractory state after endotoxin challenge. This effect was negated by either a TLR9 antagonist or DNase treatment. The present data further extend our understanding of endotoxin tolerance implications in AIS. A putative role of mtDNA as a new biomarker of stroke-associated infections, and thus a clinical target for preventing poststroke infection, has also been identified.

Brain repair involves a compendium of natural mechanisms that are activated following stroke. From a therapeutic viewpoint, reparative therapies that encourage cerebral plasticity are needed. In the last years, it has been demonstrated that modulatory treatments for brain repair such as trophic factor- and stem cell-based therapies can promote neurogenesis, gliogenesis, oligodendrogenesis, synaptogenesis and angiogenesis, all of which having a beneficial impact on infarct volume, cell death and, finally, and most importantly, on the functional recovery. However, even when promising results have been obtained in a wide range of experimental animal models and conditions these preliminary results have not yet demonstrated their clinical efficacy. Here, we focus on brain repair modulatory treatments for ischaemic stroke, that use trophic factors, drugs with trophic effects and stem cell therapy. Important and still unanswered questions for translational research ranging from experimental animal models to recent and ongoing clinical trials are reviewed here, Supported by research grants from PI060575 and PS09/01606 (Spanish Ministry of Science), CIDEM, RENEVAS (RD07/0026/2003) (Spanish Neurovascular Network) and FEDER. Research Institute Carlos III, Ministry Science and Innovation. Also, we have obtained research grants from Ferrer Group, EVER Neuro Pharma and Cellerix

Use of thrombolysis in acute ischaemic stroke may be limited by a narrow benefit/risk ratio. Pharmacological inhibition of the ischaemic cascade may constitute an effective and safer approach to stroke treatment. This study compared the effects of high doses of cytidine diphosphate-choline (CDP-choline; 1000 mg/kg) with recombinant tissue plasminogen activator (rt-PA; 5 mg/kg) in an experimental animal model of embolic stroke. Fifteen rats were embolized in the right internal carotid artery with an autologous clot and were divided into three groups: (1) infarct; (2) intravenous rt-PA 5 mg/kg 30 minutes post-embolization; and (3) CDP-choline 1000 mg/kg, intraperitoneal, three doses, 30 minutes, 24 hours, and 48 hours post-embolization. Functional evaluation scores were evaluated using Rogers test, lesion volume by haematoxylin and eosin staining, cell death with transferase-mediated dUTP nick-end labelling, and plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha with enzyme-linked immunosorbent assay. In this study, CDP-choline and rt-PA produced a significant reduction in brain damage considering infarct volume, cell death, and inflammatory cytokines (tumour necrosis factor-alpha and IL-6) compared with the infarct group. Additionally, CDP-choline significantly decreased infarct volume, cell death, and IL-6 levels with respect to the rt-PA group. From these results, we conclude that high-dose CDP-choline may be an effective treatment for acute ischaemic stroke even in absence of thrombolysis.

Introduction: Extracellular vesicles such as exosomes has opened a new field of research. Exosomes are able to transfer DNAs, mRNAs, microRNAs, non-coding RNAs, proteins, trophic factors and lipids associated with brain plasticity enhancement after stroke. Aim: To investigate white matter repair after exosomes administration in two experimental models of subcortical stroke: ischemic and hemorrhage. Material/Methods: Subcortical ischemic stroke was induced by Endothelin-1 and Collagenase IV was used to induce subcortical hemorrhagic stroke into striatum. Intravenous exosomes or saline only were administrated at 24h after cerebral infarct as treatment. Exosomes were isolated from culture of adipose mesenchymal stem cell and they were characterized by Nanoshight, Electronic microscope, Western blot and Immunofluorescence. Proteins contained into exosomes were analyzed by Orbitrab. We analyzed functional recovery by Rotarod, beam walking and Rogers tests. Lesion volume and tract connectivity were studied by magnetic resonance image. Anterograde and retrograde tracers were used to analyze axonal sprouting. Myelin formation was analyzed by cryomielin. Results: Proteomics analysis of exosomes identified more than 1400 proteins, many of them involved in intercellular communication. DiI labeled-Exosomes were detected in brain and peripheral organs (liver, lung and spleen). After 28 days, treated groups showed smaller functional deficit compared to control groups in both hemorrhagic and ischemic models. Moreover, treated group showed an increase in tract connectivity at 7 and 28 days compared to control groups. Also, animals which received exosomes showed an increase axonal sprouting and myelin formation at 28 days after stroke in both hemorrhagic and ischemic stroke. The treated groups also showed higher levels of white matter-associated markers in the injured area than the control groups. Conclusion: White matter integrity in different subcortical strokes is in part restored by exosomes treatment, probably mediated by repair molecular factors implicated in axonal sprouting, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery in both kinds of strokes.

Ultrasound-targeted microbubble destruction (UTMD) has been shown to be a promising tool to deliver proteins to select body areas. This study aimed to analyze whether UTMD was able to deliver brain-derived neurotrophic factor (BDNF) to the brain, enhancing functional recovery and white matter repair, in an animal model of subcortical stroke induced by endothelin (ET)-1. UTMD was used to deliver BDNF to the brain 24 h after stroke. This technique was shown to be safe, given there were no cases of hemorrhagic transformation or blood brain barrier (BBB) leakage. UTMD treatment was associated with increased brain BDNF levels at 4 h after administration. Targeted ultrasound delivery of BDNF improved functional recovery associated with fiber tract connectivity restoration, increasing oligodendrocyte markers and remyelination compared to BDNF alone administration in an experimental animal model of white matter injury.

Introduction: our primary objective was to analyze the efficacy of a Telestroke system in terms of an increase in the number of patients treated with intravenous thrombolysis (IVT) and a shorter door-to-needle time, in the metropolitan area of Madrid, Spain. Our secondary objective was to analyze the safety of Telestroke in terms of mortality and symptomatic hemorrhagic transformation (SHT). Methods: prospective observational cohort study: 1) cohort exposed to Telestroke: acute ischemic stroke patients attended at Infanta Sofia University Hospital (community hospital) connected by Telestroke with the Stroke Center at La Paz University Hospital; 2) Non-exposed cohort: acute stroke patients who are attended at Severo Ochoa University Hospital (community hospital) connected by phone with the Stroke Center at Ramon y Cajal University Hospital. The number of patient attended, number of IVT, times (min), 3-months outcome (mRS), the number of unnecessary transfers and safety parameters (mortality and SHT by ECAS III criteria) were recorded. We analyzed the first 34 months of the Telestroke (March 2011-december 2013). Results: in the Telestroke cohort the system was activated in 59 patients, being stroke code in 42 cases. In the non-exposed cohort 45 phone calls were made, being stroke code in 42 cases. In the Telestroke cohort, IVT were administrated in 61.9% (26/42) of patients whereas in the non-exposed only 28.6% (12/42) received this treatment (P=0,002). The door-to-needle time was shorter in Telestroke cohort than in the non-exposed cohort (mean [SD]) (86.9 [41.7] vs. 149.7 [68.2], P=0.001). The 3-month outcomes were better in the Telestroke cohort (mRS 0-3: 92.9% vs. 73.8%, P=0.038). Mortality (4,8% vs. 4.8%) and SHT (0% vs. 4.8%) were similar in both groups (P = NS). In addition, there were fewer unnecessary secondary transfers in the Telestroke cohort (4.3% vs. 20%, P=0.027). Conclusion: the implementation of a Telestroke system in a metropolitan area is effective and safe, increasing the number of IV thrombolysis and reducing the door-to-needle times. Furthermore, the Telestroke reduces the number of unnecessary secondary transfers.

et al., [Background and Purpose]: Translational research is beginning to reveal the importance of trophic factors as a therapy for cellular brain repair. The purpose of this study was to analyze whether brain-derived neurotrophic factor (BDNF) administration could mediate oligodendrogenesis and remyelination after white matter injury in subcortical stroke. [Methods]: Ischemia was induced in rats by injection of endothelin-1. At 24 hours, 0.4 μg/kg of BDNF or saline was intravenously administered to the treatment and control groups, respectively. Functional evaluation, MRI, and fiber tract integrity on tractography images were analyzed. Proliferation (KI-67) and white matter repair markers (A2B5, 2,3-cyclic-nucleotide 3-phosphodiesterase [CNPase], adenomatous polyposis coli [APC], platelet-derived growth factor receptor alpha [PDGFR-α], oligodendrocyte marker O4 [O4], oligodendrocyte transcription factor [Olig-2], and myelin basic protein [MBP]) were analyzed at 7 and 28 days. [Results]: The BDNF-treated animals showed less functional deficit at 28 days after treatment than the controls (P, This study was supported by research grants PS12/01754 (P.I.: Dr Díez-Tejedor), INVICTUS Spanish Neurovascular Network RD12/0014/0006 (Dr Fuentes and J. Ramos-Cejudo) and Sara Borrell postdoctoral fellowship CD12/00706 (Dr Otero-Ortega) from the Research Institute Carlos III, Ministry of Science and Innovation of Spain.

Introduction: Based on the positive results observed in experimental animal models, adipose tissue-derived mesenchymal stem cells (AD-MSCs) constitute a promising therapy for stroke treatment. However, several aspects need to be clarified to identify the optimal conditions for successful clinical translation. Areas covered: This review focuses on AD-MSC treatment for ischemic and hemorrhagic stroke in experimental animal models. In addition, we will explore the optimization of treatment conditions including AD-MSC production, administration routes and therapeutic windows for their appropriate use in patients, and we will provide an update on clinical trials on this therapy. Expert opinion: Compared with other cell types, AD-MSCs have been less investigated in stroke studies. Currently, experimental animal models have shown safety and efficacy with this treatment after stroke. Due to several advantages of AD-MSCs, such as their abundance and accessibility, they can be considered a promising strategy for use in patients. However, many questions are still to be resolved regarding their mechanisms of action, immune system modulation and the effects of AD-MSCs on all components of the brain that may be affected after ischemic and hemorrhagic strokes, This project is supported by research grants PS12/01754 (Spanish Ministry of Science) and INVICTUS (RD12/0014/0006) (Spanish Neurovascular Network), and Research Institute Carlos III, Ministry Science and Innovation

Background Long non-coding RNAs (lncRNAs) have been claimed as key molecular players in gene expression regulation, being involved in diverse epigenetic processes. They are aberrantly expressed in various tumors, but their exact role in bladder cancer is still obscure. We have recently found a major role of the Polycomb repression complex in recurrence of non-muscle-invasive bladder cancer. Here, we report the xpression of Polycomb-related lncRNAs:antisense noncoding RNA in the INK4 locus (ANRIL) and HOX antisense intergenic RNA (HOTAIR) in these tumors. Findings We studied a dataset of non-invasive bladder cancer samples by quantitative reverse transcription PCR (RT-qPCR) and analyzed also invasive bladder cancer samples using TCGA data. Our results showed that, while ANRIL seemed not to have a determining role, an increased HOTAIR expression appeared in recurrent and high-graded tumors associated with poor prognosis. In addition, through genome-wide transcriptome analyses, we observed that HOTAIR-EZH2-complex-regulated genes can efficiently discriminate between non-tumoral, recurrent, and non-recurrent bladder cancer samples. We also observed a significant correlation between EZH2 and HOTAIR expression levels. Using overexpression, knockdown, and pharmacological approaches in bladder cancer cell lines, we also observed that EZH2 regulates HOTAIR expression. Conclusions Our findings indicate that HOTAIR expression has prognostic value for bladder cancer progression, recurrence, and survival and suggest that HOTAIR plays active roles in modulating the cancer epigenome, becoming an interesting candidate as a target for cancer diagnosis and therapy. The observed HOTAIR regulation by EZH2 and the possibility of modulating EZH2 activity with specific inhibitors open new possible paths to be explored in bladder cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0141-x) contains supplementary material, which is available to authorized users.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Introduction]: An animal model of subcortical ischemic stroke with white matter affectation was induced in rats by injection of endothelin-1. At 24 hours, 2 × 10 6 ADMSC were administered intravenously to the treatment group. Functional evaluation, lesion size, fiber tract integrity, cell death, proliferation, white matter repair markers (Olig-2, NF, and MBP) and NogoA were all studied after sacrifice (7 days and 28 days). ADMSC migration and implantation in the brain as well as proteomics analysis and functions of the secretome were also analyzed. [Results]: Neither ADMSC migration nor implantation to the brain was observed after ADMSC administration. In contrast, ADMSC implantation was detected in peripheral organs. The treatment group showed a smaller functional deficit, smaller lesion area, less cell death, more oligodendrocyte proliferation, more white matter connectivity and higher amounts of myelin formation. The treated animals also showed higher levels of white matter-associated markers in the injured area than the control group. Proteomics analysis of the ADMSC secretome identified 2,416 proteins, not all of them previously described to be involved in brain plasticity. [Conclusions]: White matter integrity in subcortical stroke is in part restored by ADMSC treatment; this is mediated by repair molecular factors implicated in axonal sprouting, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery after stroke., This study was supported by research grants PS12/01754, PI11/00909 and INVICTUS (RD12/0014) (Spanish Neurovascular Network), SAF2010-37926, ProteoRed-PT13/0001/0017 and a Sara Borrell postdoctoral fellowship (CD12/00706, to LOO) from Research Institute Carlos III, Ministry of Science and Innovation of Spain. Furthermore, TS (CP12/03121) and FC (CP14/00154) are recipients of a research contract from Miguel Servet Program of Instituto de Salud Carlos III

et al., [Background]: After acute ischemia, the tissue that is at risk of infarction can be detected by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch but the time that is needed to process PWI limits its use. As DWI is highly sensitive to acute ischemic tissue damage, we hypothesized that different ADC patterns represent areas with a different potential for recovery. [Methods]: In a model of permanent middle cerebral artery occlusion (pMCAO), Sprague-Dawley rats were randomly distributed to sham surgery and pMCAO. We further separated the pMCAO group according to intralesional ADC pattern (homogeneous or heterogeneous). At 24 h after ischemia induction, we analyzed lesion size, functional outcome, cell death expression, and brain protection markers including ROS enzyme NOX-4. MRI included DWI (ADC maps), DTI (tractography), and PWI (CBF, CBV and MTT). [Results]: The lesion size was similar in pMCAO rats. Animals with a heterogeneous pattern in ADC maps showed better functional outcome in Rotarod test (p = 0.032), less expression of cell death (p = 0.014) and NOX-4 (p = 0.0063), higher intralesional CBF (p = 0.0026) and larger PWI/DWI mismatch (p = 0.007). [Conclusions]: In a rodent model for ischemic stroke, intralesional heterogeneity in ADC maps was related to better functional outcome in lesions of similar size and interval after pMCAO. DWI ADC maps may assist in the early identification of ischemic tissue with an increased potential for recovery as higher expression of acute protection markers, lower expression of cell death, increased PWI/DWI mismatch, and higher intralesional CBF were present in animals with a heterogeneous ADC pattern., This research was supported by the INVICTUS (Spanish Neurovascular Network) research grant (RD 12/0014/0006) from Research Institute Carlos III, Madrid, Spain, and by the Champalimaud Neuroscience Program (INDP2007), Champalimaud Center for the Unknown, Lisbon, Portugal.

Background: Rat adipose tissue-derived-mesenchymal stem cells (rAD-MSCs) have proven to be safe in experimental animal models of stroke. However, in order to use human AD-MSCs (hAD-MSCs) as a treatment for stroke patients, a proof of concept is needed. We analyzed whether the xenogeneic hAD-MSCs were as safe and effective as allogeneic rAD-MSCs in permanent Middle Cerebral Artery Occlusion (pMCAO) in rats. Methods: Sprague–Dawley rats were randomly divided into three groups, which were intravenously injected with xenogeneic hAD-MSCs (2 × 106), allogeneic rAD-MSCs (2 × 106) or saline (control) at 30 min after pMCAO. Behavior, cell implantation, lesion size and cell death were evaluated. Brain markers such as GFAP (glial fibrillary acid protein), VEGF (vascular endothelial growth factor) and SYP (synaptophysin) and tumor formation were analyzed. Results: Compared to controls, recovery was significantly better at 24 h and continued to be so at 14 d after IV administration of either hAD-MSCs or rAD-MSCs. No reduction in lesion size or migration/implantation of cells in the damaged brain were observed in the treatment groups. Nevertheless, cell death was significantly reduced with respect to the control group in both treatment groups. VEGF and SYP levels were significantly higher, while those of GFAP were lower in the treated groups. At three months, there was no tumor formation. Conclusions: hAD-MSCs and rAD-MSCs were safe and without side effects or tumor formation. Both treatment groups showed equal efficacy in terms of functional recovery and decreased ischemic brain damage (cell death and glial scarring) and resulted in higher angiogenesis and synaptogenesis marker levels, This research was supported by research grants FIS06/0575, FIS09/01606, FIS12/01754 and INVICTUS (RD12/0014/0006) (Spanish Neurovascular Network), Cellerix, and Research Institute Carlos III, Ministry of Science and Innovation of Spain.

Angiotensin type 1 receptor blockers (ARBs) have induced improved functional recovery and reduced infarct volume in experimental animal models of stroke. Clinical data have indicated a positive correlation between prestroke treatment with ARBs and reduced stroke severity and better outcomes; however, the mechanisms of these beneficial effects are not yet well understood. This study compares the protective and possible reparative effects of continuous oral treatment with olmesartan (OLM) with OLM pretreatment and withdrawal after permanent middle cerebral artery occlusion (pMCAO) in rats. Fifty-two Sprague-Dawley rats were randomly assigned to five groups: MCAO(-/OLM) (OLM 10 mg/kg/day for 14 days after infarct), MCAO(OLM/OLM) (OLM 10 mg/kg/day for 7 days before and 14 days after infarct), MCAO(OLM/-) (OLM 10 mg/kg/day for 7 days before infarct), sham, and control. We analyzed functional recovery; lesion size; cell death; expression of the pro-oxidant enzyme NADPH oxidase 4 (NOX-4); isolectin-B4; and repair markers such as glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). All of the OLM-treated groups showed significantly better functional scores and reduced infarct sizes and cell death compared with the control group 14 days after pMCAO. Levels of NOX-4, VEGF, and BDNF were significantly lower in the brains of the MCAO(OLM/OLM) and sham groups compared with the other groups. OLM treatment improved functional recovery and reduced lesion size and cell death after cerebral ischemia. Only the continuous administration of OLM before and after stroke reduced oxidative stress levels, with better tissue preservation, without triggering brain repair marker activation.

Few studies have evaluated the possible beneficial effect of the administration of stem cells in the early stages of stroke. Intravenous administration of allogeneic mesenchymal stem cells (MSCs) from adipose tissue in patients with acute stroke could be a safe therapy for promoting neurovascular unit repair, consequently supporting better functional recovery. We aim to assess the safety and efficacy of MSC administration and evaluate its potential as a treatment for cerebral protection and repair.A Phase IIa, prospective, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. Twenty patients presenting acute ischemic stroke will be randomized in a 1:1 proportion to treatment with allogeneic MSCs from adipose tissue or to placebo (or vehicle) administered as a single intravenous dose within the first 2 weeks after the onset of stroke symptoms. The patients will be followed up for 2 years. Primary outcomes for safety analysis: adverse events (AEs) and serious AEs; neurologic and systemic complications, and tumor development. Secondary outcomes for efficacy analysis: modified Rankin Scale; NIHSS; infarct size; and biochemical markers of brain repair (vascular endothelial growth factor, brain-derived neurotrophic factor, and matrix metalloproteinases 9).To our knowledge, this is the first, phase II, pilot clinical trial to investigate the safety and efficacy of intravenous administration of allogeneic MSCs from adipose tissue within the first 2 weeks of stroke. In addition, its results will help us define the best criteria for a future phase III study.

Introduction: Stroke is a major worldwide cause of death and disability. Currently, intravenous thrombolysis and reperfusion therapies, but not the so-called neuroprotectant drugs, have been shown to be effective for acute ischemic stroke. Thus, new strategies to promote brain plasticity are necessary. Stem cell administration is an attractive future therapeutic approach. Areas covered: Brain protection and repair mechanisms are activated after stroke. This article is focused on the capacity of stem cell-based therapy to enhance this postinfarct brain plasticity and recovery. Future therapeutic considerations and prospects for stroke are discussed. Expert opinion: Although cell therapy is promising in stroke treatment, mechanisms of action need to be characterized in detail. Further, the different mechanisms of axonal plasticity and remodeling involucrated in brain repair, not only in the gray but also in white matter, must be investigated through noninvasive techniques, and a multidisciplinary approach i...

More recently, growing interests have brought cell therapy to the forefront of promising new approaches towards efficacious treatment for stroke. Of all cell-types, adipose tissue-derived mesenchymal stem cells (AD-MSC) are considered good candidates for stroke treatment because of their abundance and easy harvesting without invasive surgery from healthy donors. A wide number of experimental studies have demonstrated the potential of AD-MSC administration for improving functional deficits and have led to the first clinical trials in stroke patients. Trophic factor release and paracrine interactions, transdifferentiation potential, and immunomodulatory effects have all been cited as the main functional mechanisms involved in AD-MSC therapy. These properties make AD-MSC therapy of special interest in fomenting the enhancement of natural brain repair mechanisms such as neurogenesis, gliagenesis, oligodendrogenesis, axonal sprouting, nerve repair, and angiogenesis. This review will focus on studies showing promising results of AD-MSC in stroke treatment.

Introduction: Hydrogen sulphide (H2S) can regulate oxygen consumption by competing with O2 in binding to cytochrome C oxydase. At micro molar concentrations H2S interferes with antinecrotic and antiapoptotic mechanisms, including up regulation of cytoprotective genes. We studied the effect of H2S exposure on lesion and functional recovery after permanent middle cerebral artery occlusion (pMCAO) in rats. Methods: Young adult male and female Sprague- Dawley rats distributed into three groups: 1- Sham (control for craniotomy); 2-Control: Surgery + permanent MCAO; 3- Treated: Surgery + permanent MCAO + inhalation of 40 ppM hydrogen sulphide. We analyzed functional outcome (Rogers modified scale), lesion volume by MRI at 24h and 14 day and by hematoxyline-eosin, cell death by TUNEL, repair markers implicated in brain plasticity (cellular proliferation by BrdU, GFAP (glial fibrillar acis protein), VEGF (vascular endothelial growth factor), Synaptophysine) by western-blot and immunofluorescence at 14 days. Rats were sacrificed at day 14th. Results: Treated animals showed a benefit in functional outcome both at 24h (p = 0,004445) and sacrifice day (p = 0,000942). We observed a decrease in infarct size at day 14th on MRI (p= 0,0383 ) and in Hematoxyline Eosine infarct areas (p= 0.026672 ). At 24 hours there was no statistical difference on MRI (p= 0,380644). TUNEL analysis showed a decrease in the expression of marked cells in the perinfarct zone of treated animals (p= 0,048877). We did not observe significant differences in cellular proliferation (BrdU), GFAP, VEGF and synapthophysin levels between control and treated groups. Conclusions: Exposure to hydrogen sulphide in rats with cerebral infarct was effective in functional recovery associated to brain protection mechanisms with reduction in size volume and cell death and may be associated with reperfusion increased but not in repair effects.

We investigated the effect of CDP-choline on brain plasticity markers expression in the acute phase of cerebral infarct in an experimental animal model. Male Sprague–Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and treated or not with CDP-choline (500 mg/kg) daily for 14 days starting 30 min after pMCAO. Functional status was evaluated with Roger’s test; lesion volume with magnetic resonance imaging (MRI) and hematoxylin and eosin staining (HE cell death with TUNEL; cellular proliferation with BrdU immunohistochemistry; vascular endothelial growth factor (VEGF), synaptophysin, glial fibrillary acidic protein (GFAP) and low-density lipoprotein receptor-related protein (LRP) by immunofluorescence and Western-blot techniques. CDP-choline significantly improved functional recovery and decreased lesion volume on MRI, TUNEL-positive cell number and LRP levels at 14 days. In addition, CDP-choline significantly increased BrdU, VEGF and synaptophysin values and decreased GFAP levels in the peri-infarct zone compared with the infarct group. In conclusion, our data indicate that CDP-choline improved functional recovery after permanent middle cerebral artery occlusion in association with reductions in lesion volume, cell death and LRP expression. In fact, CDP-choline increased cell proliferation, vasculogenesis and synaptophysin levels and reduced GFAP levels in the peri-infarct area of the ischemic stroke.

Hematic administration of bone marrow-derived mesenchymal stem cells (MSCs) in acute ischemic stroke may not only be an effective reparative treatment but also a brain protective therapy that improves neurological recovery. Our purpose was to study whether either i.v. or intracarotid (i.c.) administration of allogenic MSCs during the acute phase were effective in improving neurological recovery and decreasing brain damage in an experimental rat model. In a model of permanent middle cerebral artery occlusion (pMCAO), we analyzed: neurological evaluation; MSCs migration and implantation; interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels; lesion volume; cell death; cellular proliferation; vascular endothelial growth factor (VEGF) expression and blood vessel number. Regardless of the administration route, treated groups showed better neurological recovery, without significant differences between the two groups. Migration and implantation of MSCs in the lesion area was observed in animals receiving i.c. but not i.v. treatment. The highest cytokine values were observed in the i.v. MSCs and i.c. control groups, and these levels were significantly different from the corresponding i.v. control and i.c. MSCs groups, respectively. In addition, there were significant differences between the i.v. MSCs and i.c. MSCs groups in IL-6 levels. Neither treatment reduced infarction volume. However, cell death, measured as TUNEL+ cells was decreased with significant differences between control groups. BrdU+ cells were also significantly increased in the peri-infarct zone at 14 days. VEGF expression was significantly higher in the i.c. MSCs group than in the i.c. control group and blood vessel number was significantly higher in treated groups than control groups with significant differences in the peri-infarct zone at 14 days. We conclude that allogenic MSCs administration shows therapeutic efficacy in our acute ischemic stroke model. Both routes demonstrably improved neurological recovery and provided brain protection.

CDP-choline has been widespread used in humans for decades as a treatment for many types of cognitive impairment. Despite this, its mechanism of action still remains unclear, but several experimental models in acute cerebral ischaemia suggest that it could have a brain repair action. Due to the lack of significant adverse effects and its high tolerability, there has been a growing interest for this molecule in recent years. In this article, a review of the most significant published clinical trials in cognitive decline has been made. A few Citicoline trials have studied its effects at medium and long-term on vascular cognitive impairment and Alzheimer's disease. Results show that Citicoline seems to have beneficial impact on several cognitive domains, but the methodological heterogeneity of the these studies makes it difficult to draw conclusions about these effects. New trials with a greater number of patients, uniform diagnostic criteria for inclusion and standardized neuropsychological assessment are needed to evidence with much more consistency Citicoline efficacy upon cognitive disorders. The use of new neuroimaging procedures in current trials could be of great interest.

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in paediatric patients after the first year of life. The aim of this study was to evaluate effects of locally administered allogeneic mesenchymal stem cells (MSC), in the acute period after a TBI.MSC were isolated from peritoneal fat of healthy rats, expanded in vitro and labelled with the green fluorescent protein. Rats were placed in one of three experimental groups: (1) CONTROL: TBI, (2) IP-CONTROL: TBI + local saline and (3) IP-Treat: TBI + 2 × 10(5) MSC 24 hours after receiving a moderate, unilateral, controlled cortical impact. Motor and cognitive behavioural tests were performed to evaluate functional recovery. Histological examination and immunohistochemistry were used to identify cell distribution.Improved performance was found on motor tests in the MSC-treated group compared to control groups. MSC were found in the perilesional area and their number decreased with time after transplantation. MSC treatment increased the cell density in the hippocampus (CA3 pyramidal cells and granule cells in the dentate gyrus) and enhanced neurogenesis in this area.MSC cell therapy resulted in better recovery of motor function compared with the control group. This cellular therapy might be considered for patients suffering from TBI.