Magnetite Nanoparticles for Stem Cell Labeling with High Efficiency and Long-Term in Vivo Tracking.

Superparamagnetic iron oxide nanoparticles (SPIO-PAA), ultrasmall iron oxide nanoparticles (USPIO-PAA), and glucosamine-modified iron oxide nanoparticles (USPIO-PAA-GlcN) were studied as mesenchymal stem cell (MSCs) labels for cell tracking applications by magnetic resonance imaging (MRI). Pronounced differences were found in the labeling performance of the three samples in terms of cellular dose and labeling efficiency. In combination with polylysine, SPIO-PAA showed nonhomogeneous cell internalization, while for USPIO-PAA no uptake was found. On the contrary, USPIO-PAA-GlcN featured high cellular uptake and biocompatibility, and sensitive detection in both in vitro and in vivo experiments was found by MRI, showing that glucosamine functionalization can be an efficient strategy to increase cell uptake of ultrasmall iron oxide nanoparticles by MSCs.

Vectorized nanodelivery systems for ischemic stroke: a concept and a need.

Neurological diseases of diverse aetiologies have significant effects on the quality of life of patients. The limited self-repairing capacity of the brain is considered to be the origin of the irreversible and progressive nature of many neurological diseases. Therefore, neuroprotection is an important goal shared by many clinical neurologists and neuroscientists. In this review, we discuss the main obstacles that have prevented the implementation of experimental neuroprotective strategies in humans and propose alternative avenues for the use of neuroprotection as a feasible therapeutic approach. Special attention is devoted to nanotechnology, which is a new approach for developing highly specific and localized biomedical solutions for the study of the multiple mechanisms involved in stroke. Nanotechnology is contributing to personalized neuroprotection by allowing us to identify mechanisms, determine optimal therapeutic windows, and protect patients from brain damage. In summary, multiple aspects of these new players in biomedicine should be considered in future in vivo and in vitro studies with the aim of improving their applicability to clinical studies.

Quick adjustment of imaging tracer payload, for in vivo applications of theranostic nanostructures in the brain.

In order to provide sufficient sensibility for detection, selection of an adequate payload of imaging probe is critical, during the design of MRI theranostic nanoplatforms. This fact is particularly crucial for in vivo applications in the brain, where delivery of macromolecules is limited by the blood-brain barrier. Here we report a simple and quick process for the estimation of adequate payloads of gadolinium in liposomes with potential to act as theranostic agents, for in vivo MRI applications in the brain. Our studies show that an excessive payload of gadolinium in liposomes may actually have a negative influence on in vivo T1 contrast. By preparing and characterizing 4 different liposomal compositions of increasing Gadolinium loads, we show that a superior sensitivity for in vivo detection of MRI theranostic molecules can be quickly improved by adjusting the payload of imaging probe in the molecules. FROM THE CLINICAL EDITOR: This team of authors report the development of a simple and quick process for the estimation of adequate payloads of gadolinium in liposomes as theranostic agents for in vivo brain MRI studies, using a rodent model.

Light-Emitting Diode Photobiomodulation After Cerebral Ischemia.

Photobiomodulation (PBM) therapy is a promising therapeutic approach for several pathologies, including stroke. The biological effects of PBM for the treatment of cerebral ischemia have previously been explored as a neuroprotective strategy using different light sources, wavelengths, and incident light powers. However, the capability of PBM as a novel alternative therapy to stimulate the recovery of the injured neuronal tissue after ischemic stroke has been poorly explored. The aim of this study was to investigate the low-level light irradiation therapy by using Light Emitting Diodes (LEDs) as potential therapeutic strategy for stroke. The LED photobiomodulation (continuous wave, 830 nm, 0.2-0.6 J/cm2) was firstly evaluated at different energy densities in C17.2 immortalized mouse neural progenitor cell lines, in order to observe if this treatment had any effect on cells, in terms of proliferation and viability. Then, the PBM-LED effect (continuous wave, 830 nm, 0.28 J/cm2 at brain cortex) on long-term recovery (12 weeks) was analyzed in ischemic animal model by means lesion reduction, behavioral deficits, and functional magnetic resonance imaging (fMRI). Analysis of cellular proliferation after PBM was significantly increased (1 mW) in all different exposure times used; however, this effect could not be replicated in vivo experimental conditions, as PBM did not show an infarct reduction or functional recovery. Despite the promising therapeutic effect described for PBM, further preclinical studies are necessary to optimize the therapeutic window of this novel therapy, in terms of the mechanism associated to neurorecovery and to reduce the risk of failure in futures clinical trials.

Blood glutamate EAAT2-cell grabbing therapy in cerebral ischemia.

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

Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia.

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.

Easy and Efficient Cell Tagging with Block Copolymer-Based Contrast Agents for Sensitive MRI Detection in Vivo.

Superparamagnetic iron oxide nanoparticles (MNPs) together with magnetic resonance imaging (MRI) are the preferred tools for monitoring the fate and biodistribution of administered cells in stem cell therapy studies. Commercial MNPs need transfection agents and long incubation times for sufficient cell labeling and further in vivo cell detection. In this work, we have synthesized MNPs coated with pluronic F127 and tetronic 908, and validated their applicability as contrast agents for MRI cell detection on two different cell types: rat mesenchymal stem cells (MSCs) and multipotent neural progenitor cell line from mice (C17.2). No transfection agent was needed for a complete MNP internalization, and the uptake was only dependent on MNP concentration in medium and limited on the incubation time. By combining in vivo MRI and ex vivo histology microscopy, we have demonstrated the MRI signal detected corresponded exclusively to labeled cells and not to free particles. Pluronic F127- and tetronic 908-coated MNPs represent promising contrast agents for stem cell tracking due to their ease of use in preparation, their efficiency for cell labeling, and their high sensitivity for in vivo cell detection.

Influence of the separation procedure on the properties of magnetic nanoparticles: Gaining in vitro stability and T1-T2 magnetic resonance imaging performance.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) coated with polyacrylic acid (PAA) were synthesized by a hydrothermal method in gram-scale quantity and extensively characterized. Only the nanoparticles subjected to an additional centrifugation step showed narrow size distribution, high polymeric coverage, and ideal superparamagnetism. In addition to improved physico-chemical properties, these nanoparticles feature high stability in vitro as well as dual T1-T2 performance as contrast agents (CAs) for magnetic resonance imaging (MRI), highlighting the importance of the additional separation step in obtaining material with the desired properties.

Blood glutamate grabbing does not reduce the hematoma in an intracerebral hemorrhage model but it is a safe excitotoxic treatment modality.

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.

In vitro and in vivo ocular safety and eye surface permanence determination by direct and Magnetic Resonance Imaging of ion-sensitive hydrogels based on gellan gum and kappa-carrageenan.

Gellan gum, kappa-carrageenan and alginates are natural polysaccharides able to interact with different cations that can be used to elaborate ion-activated in situ gelling systems for different uses. The interaction between fluid solutions of these polysaccharides and cations presents into the tear made these biopolymers very interesting to elaborate ophthalmic drug delivery systems. The main purpose of this study is to evaluate the ability of mixtures of these polymers to obtain ion-activated ophthalmic in situ gelling systems with optimal properties for ocular use. To achieve this purpose different proportion of the biopolymers were analyzed using a mixture experimental design evaluating their transparency, mechanical properties and bioadhesion in the absence and presence of simulated tear fluid. Tear induces a rapid sol-to-gel phase transition in the mixtures forming a consistent hydrogel. The solution composed by 80% of gellan gum and 20% kappa-carrageenan showed the best mechanical and mucoadhesive properties. This mixture was evaluated for rheological behavior, microstructure, cytotoxicity, acute corneal irritancy, ex-vivo and in vivo ocular toxicity and in vivo corneal contact time using Magnetic Resonance Images (MRI) techniques. Result indicates that the system is safe at ophthalmic level and produces an extensive ocular permanence higher than 6h.

Sensitive in vivo cell detection using size-optimized superparamagnetic nanoparticles.

Magnetic nanoparticle (MNP) enabled cell visualization with magnetic resonance imaging (MRI) is currently an intensively studied area of research. In the present study, we have synthesized polyethylene glycolated (PEG) MNPs and validated their suitability as MR cell labeling agents in in vitro and in vivo experiments. The labeling of therapeutic potent mesenchymal stem cells (MSCs) with small core and large core MNPs was evaluated. Both MNPs were, in combination with a transfection agent, stably internalized into the MSCs and didn't show an effect on cell metabolism. The labeled cells showed high contrast in MRI phantom studies. For quantification purposes, the MRI contrast generating properties of cells labeled with small core MNPs were compared with large core MNPs and with the commercial contrast agent Endorem. MSCs labeled with the large core MNPs showed the highest contrast generating properties in in vitro phantom studies and in in vivo intracranial stereotactic injection experiments, confirming the size-relaxivity relationship in biological systems. Finally, the distribution of MSCs pre-labeled with large core PEGylated MNPs was visualized non-invasively with MRI in a glioma model.

In vivo theranostics at the peri-infarct region in cerebral ischemia.

The use of theranostics in neurosciences has been rare to date because of the limitations imposed on the free delivery of substances to the brain by the blood-brain barrier. Here we report the development of a theranostic system for the treatment of stroke, a leading cause of death and disability in developed countries. We first performed a series of proteomic, immunoblotting and immunohistological studies to characterize the expression of molecular biomarkers for the so-called peri-infarct tissue, a key region of the brain for stroke treatment. We confirmed that the HSP72 protein is a suitable biomarker for the peri-infarct region, as it is selectively expressed by at-risk tissue for up to 7 days following cerebral ischemia. We also describe the development of anti-HSP72 vectorized stealth immunoliposomes containing imaging probes to make them traceable by conventional imaging techniques (fluorescence and MRI) that were used to encapsulate a therapeutic agent (citicoline) for the treatment of cerebral ischemia. We tested the molecular recognition capabilities of these nano-platforms in vitro together with their diagnostic and therapeutic properties in vivo, in an animal model of cerebral ischemia. Using MRI, we found that 80% of vectorized liposomes were located on the periphery of the ischemic lesion, and animals treated with citicoline encapsulated on these liposomes presented lesion volumes up to 30% smaller than animals treated with free (non-encapsulated) drugs. Our results show the potential of nanotechnology for the development of effective tools for the treatment of neurological diseases.

Serial MRI study of the enhanced therapeutic effects of liposome-encapsulated citicoline in cerebral ischemia.

Liposome encapsulation of active principles enhances their bioavailability to the brain. We investigated whether encapsulation of citicoline in liposomes increases its therapeutic effects in ischemia, performing a longitudinal MRI study of lesion volumes and edema in an animal model of stroke. Nineteen rats were submitted to permanent occlusion of the middle cerebral artery and treated with: (1) saline, (2) intraperitoneal citicoline (500mg/kg), (3) intravenous citicoline (48mg/kg), and (4) intravenous liposome-encapsulated citicoline (48mg/kg). Lesion volumes were measured by MRI at days 0, 1, 3 and 7 following surgery. Encapsulation in liposomes increased the therapeutic effects of citicoline, as reflected by a 32% reduction of the infarct sizes at day 7, in contrast with controls where infarct sizes at day 7 increased by 39%, respect to values at day 0. Intravenously injected citicoline reduced infarct sizes by 9% while intraperitoneal citicoline resulted in an increase of infarct sizes by 10%. A slight (not significant) reduction of edema formation was observed for animals treated with citicoline, in all of its delivery forms. Liposome-encapsulated citicoline causes a noticeable reduction in lesion volumes as compared to free citicoline (either i.p. or i.v.) at days 1, 3 and 7 following permanent stroke.

Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study.

As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain-blood gradient. In this regard, the ability of glutamate oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.

Técnicas de imagen para el estudio de la recuperación funcional tras el ictus: I. Aspectos metodológicos / Imaging techniques for studying functional recovery following a stroke: I. Methodological aspects

Resumen. Muchos pacientes que sobreviven a un ictus se enfrentan a serias discapacidades funcionales durante el resto de sus vidas, lo que supone un drama personal para sí mismos y para sus allegados, y un elevado coste para la sociedad. Por ello, la recuperación funcional del sujeto tras el ictus debería ser un objetivo esencial que se debería considerar en el desarrollo de nuevas aproximaciones terapéuticas. En esta serie de dos trabajos, revisamos las estrategias y herramientas disponibles hoy en día para la evaluación de múltiples aspectos relacionados con la función cerebral (tanto en humanos como en animales de experimentación), y que están ayudando a los neurocientíficos a entender mejor los procesos de restauración y reorganización de la función cerebral que se inician tras un ictus. Hemos puesto especial énfasis en las aplicaciones de la resonancia magnética, probablemente la técnica de neuroimagen más versátil disponible hoy en día, y que aún no ha dejado de evolucionar y proporcionar nuevas y excitantes aplicaciones. Pero también abordamos otras técnicas alternativas y complementarias, puesto que una aproximación multidisciplinar proporciona una perspectiva más completa de los mecanismos que subyacen bajo los mecanismos de reparación tisular, de reorganización plástica del cerebro, y de los compensatorios que se desencadenan tras un ictus. El primer trabajo de esta serie se centra en aspectos metodológicos que nos ayudarán a comprender cómo es posible caracterizar la función cerebral basándonos en diferentes principios físicos y fisiológicos. El segundo trabajo se centrará en técnicas complementarias y en diversos aspectos prácticos relacionados con la aplicación de las técnicas aquí comentadas (AU)

Summary. Many patients that survive stroke have to face serious functional disabilities for the rest of their lives, which is a personal drama for themselves and their relatives, and an elevated charge for society. Thus functional recovery following stroke should be a key objective for the development of new therapeutic approaches. In this series of two works we review the strategies and tools available nowadays for the evaluation of multiple aspects related to brain function (both in humans and research animals), and how they are helping neuroscientist to better understand the processes of restoration and reorganization of brain function that are triggered following stroke. We have mainly focused on magnetic resonance applications, probably the most versatile neuroimaging technique available nowadays, and that everyday surprises us with new and exciting applications. But we tackle other alternative and complementary techniques, since a multidisciplinary approach allows a wider perspective over the underlying mechanisms behind tissue repair, plastic reorganization of the brain and compensatory mechanisms that are triggered after stroke. The first of the works of this series is focused on methodological aspects that will help us to understand how it is possible to assess brain function based on different physical and physiological principles. In the second work we will focus on different practical issues related to the application of the techniques here discussed (AU)