Metal-Free Radical Dendrimers as MRI Contrast Agents for Glioblastoma Diagnosis: Ex Vivo and In Vivo Approaches.

Simultaneously being a nonradiative and noninvasive technique makes magnetic resonance imaging (MRI) one of the highly required imaging approaches for the early diagnosis and follow-up of tumors, specifically for brain cancer. Paramagnetic gadolinium (Gd)-based contrast agents (CAs) are the most widely used ones in brain MRI acquisitions with special interest when assessing blood-brain barrier (BBB) integrity, a characteristic of high-grade tumors. However, alternatives to Gd-based contrast agents (CAs) are highly required to overcome their established toxicity. Organic radicals anchored on a dendrimer macromolecule surface (radical dendrimers) are promising alternatives since they also exhibit paramagnetic properties and can act as T1 CAs like Gd-based CAs while being organic species (mitigating concerns about toxic metal accumulation). Here, we studied the third generation of a water-soluble family of poly(phosphorhydrazone) radical dendrimers, with 48 PROXYL radical units anchored on their branches, exploring their potential of ex vivo and in vivo contrast enhancement in brain tumors (in particular, of immunocompetent, orthotopic GL261 murine glioblastoma (GB)). Remarkably, this radical species provides suitable contrast enhancement on murine GL261 GB tumors, which was comparable to that of commercial Gd-based CAs (at standard dose 0.1 mmol/kg), even at its 4 times lower administered dose (0.025 mmol/kg). Importantly, no signs of toxicity were detected in vivo. In addition, it showed a selective accumulation in brain tumor tissues, exhibiting longer retention within the tumor, which allows performing imaging acquisition over longer time frames (≥2.5 h) as opposed to Gd chelates. Finally, we observed high stability of the radicals in biological media, on the order of hours instead of minutes, characteristic of the isolated radicals. All of these features allow us to suggest that the G3-Tyr-PROXYL-ONa radical dendrimer could be a viable alternative to metal-based MRI contrast agents, particularly on MRI analysis of GB, representing, to the best of our knowledge, the first case of organic radical species used for this purpose and one of the very few examples of these types of radical species working as MRI CAs in vivo.

Metronomic treatment in immunocompetent preclinical GL261 glioblastoma: effects of cyclophosphamide and temozolomide.

Glioblastoma (GBM) causes poor survival in patients even when applying aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment, but resistance always ensues. In previous years, efforts have focused on new therapeutic regimens with conventional drugs to activate immune responses that may enhance tumor regression and prevent regrowth, for example the "metronomic" approaches. In metronomic scheduling studies, cyclophosphamide (CPA) in GL261 GBM growing subcutaneously in C57BL/6 mice was shown not only to activate antitumor CD8+ T-cell response, but also to induce long-term specific T-cell tumor memory. Accordingly, we have evaluated whether metronomic CPA or TMZ administration could increase survival in orthotopic GL261 in C57BL/6 mice, an immunocompetent model. Longitudinal in vivo studies with CPA (140 mg/kg) or TMZ (range 140-240 mg/kg) metronomic administration (every 6 days) were performed in tumor-bearing mice. Tumor evolution was monitored at 7 T with MRI (T2 -weighted, diffusion-weighted imaging) and MRSI-based nosological images of response to therapy. Obtained results demonstrated that both treatments resulted in increased survival (38.6 ± 21.0 days, n = 30) compared with control (19.4 ± 2.4 days, n = 18). Best results were obtained with 140 mg/kg TMZ (treated, 44.9 ± 29.0 days, n = 12, versus control, 19.3 ± 2.3 days, n = 12), achieving a longer survival rate than previous group work using three cycles of TMZ therapy at 60 mg/kg (33.9 ± 11.7 days, n = 38). Additional interesting findings were, first, clear edema appearance during chemotherapeutic treatment, second, the ability to apply the semi-supervised source analysis previously developed in our group for non-invasive TMZ therapy response monitoring to detect CPA-induced response, and third, the necropsy findings in mice cured from GBM after high TMZ cumulative dosage (980-1400 mg/kg), which demonstrated lymphoma incidence. In summary, every 6 day administration schedule of TMZ or CPA improves survival in orthotopic GL261 GBM with respect to controls or non-metronomic therapy, in partial agreement with previous work on subcutaneous GL261.

Assessment of biodistribution using mesenchymal stromal cells: Algorithm for study design and challenges in detection methodologies.

BACKGROUND AIMS: Biodistribution of candidate cell-based therapeutics is a critical safety concern that must be addressed in the preclinical development program. We aimed to design a decision tree based on a series of studies included in actual dossiers approved by competent regulatory authorities, noting that the design, execution and interpretation of pharmacokinetics studies using this type of therapy is not straightforward and presents a challenge for both developers and regulators. METHODS: Eight studies were evaluated for the definition of a decision tree, in which mesenchymal stromal cells (MSCs) were administered to mouse, rat and sheep models using diverse routes (local or systemic), cell labeling (chemical or genetic) and detection methodologies (polymerase chain reaction [PCR], immunohistochemistry [IHC], fluorescence bioimaging, and magnetic resonance imaging [MRI]). Moreover, labeling and detection methodologies were compared in terms of cost, throughput, speed, sensitivity and specificity. RESULTS: A decision tree was defined based on the model chosen: (i) small immunodeficient animals receiving heterologous MSC products for assessing biodistribution and other safety aspects and (ii) large animals receiving homologous labeled products; this contributed to gathering data not only on biodistribution but also on pharmacodynamics. PCR emerged as the most convenient technique despite the loss of spatial information on cell distribution that can be further assessed by IHC. DISCUSSION: This work contributes to the standardization in the design of biodistribution studies by improving methods for accurate assessment of safety. The evaluation of different animal models and screening of target organs through a combination of techniques is a cost-effective and timely strategy.

Real-time assessment of ¹³C metabolism reveals an early lactate increase in the brain of rats with acute liver failure.

Intracranial hypertension is a severe complication of acute liver failure (ALF) secondary to brain edema. The pathogenesis of cerebral edema in ALF is not clear, but seems to be related to energy metabolism in which lactate may have an important role. The aim of this study was to follow the synthesis of brain lactate using a novel in vivo metabolic technology in a rat model of ALF. Time-resolved (13) C MRS of hyperpolarized (13) C1 -pyruvate was used to quantitatively follow the in vivo conversion of pyruvate to its substrates in a model of devascularized ALF in rats. Rats with ALF showed a significant increase in the lactate to pyruvate ratio from 36% to 69% during the progression of liver disease relative to rats with portocaval anastomosis. Rats with ALF also showed a significant increase in the alanine to pyruvate ratio from 72% to 95%. These increases were detectable at very early stages (6 h) when animals had no evident disease signs in their behavior (without loss of righting or corneal reflexes). This study shows the dynamic consequences of cerebral in vivo (13) C metabolism at real time in rats with ALF. The early detection of the de novo synthesis of lactate suggests that brain lactate is involved in the physiopathology of ALF. Hyperpolarization is a potential non-invasive technique to follow the in vivo metabolism, and both the development and optimization of (13) C-labeled substrates can clarify the mechanism involved in ALF.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

The presence of circulating human apolipoprotein J reduces the occurrence of cerebral microbleeds in a transgenic mouse model with cerebral amyloid angiopathy.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is characterized by amyloid-ß (Aß) deposition in cerebral vessels, leading to lobar cerebral microbleeds (CMB) and intracerebral hemorrhages (ICH). Apolipoprotein J (ApoJ) is a multifunctional chaperone related to Aß aggregation and clearance. Our study investigated the vascular impact of chronic recombinant human Apolipoprotein J (rhApoJ) treatment in a transgenic mouse model of ß-amyloidosis with prominent CAA. METHODS: Twenty-month-old APP23 C57BL/6 mice received 25 doses of rhApoJ (1 mg/kg) (n = 9) or saline (n = 8) intraperitoneally for 13 weeks, while Wild-type (WT) mice received saline (n = 13). Postmortem brains underwent T2*-weighted magnetic resonance imaging (MRI) to detect hemorrhagic lesions. Aß levels and distribution, cerebral fibrinogen leakage, brain smooth muscle actin (sma), and plasma matrix metalloproteinases and inflammatory markers were analyzed after treatments. Additionally, plasma samples from 22 patients with lobar ICH were examined to determine the clinical relevance of the preclinical findings. RESULTS: rhApoJ-treated APP23 presented fewer cortical CMBs (50-300 µm diameter) (p = 0.012) and cortical larger hemorrhages (> 300 µm) (p = 0.002) than saline-treated mice, independently of Aß brain levels. MRI-detected hemorrhagic lesions correlated with fibrinogen cerebral extravasation (p = 0.011). Additionally, rhApoJ-treated mice presented higher number of sma-positive vessels than saline-treated mice (p = 0.038). In rhApoJ-treated mice, human ApoJ was detected in plasma and in occasional leptomeningeal vessels, but not in the parenchyma, suggesting that its mechanism of action operates through the periphery. The administration of rhApoJ induced an increase in plasma Groα (p = 0.035) and MIP-1α (p = 0.035) levels, while lower MMP-12 (p = 0.046) levels, compared to the saline-treated group. In acute lobar ICH patients, MMP-12 plasma levels correlated with larger hemorrhage volume (p = 0.040) and irregular ICH shape (p = 0.036). CONCLUSIONS: Chronic rhApoJ treatment in aged APP23 mice ameliorated CAA-related neurovascular damage by reducing the occurrence of CMB. We propose that rhApoJ may prevent blood-brain barrier (BBB) leakage and CMB appearance partly through circulating MMP-12 modulation.

Brain magnetic resonance in experimental acute-on-chronic liver failure.

BACKGROUND & AIM: Acute-on-chronic liver failure is the term that refers to sustained liver injury with acute decompensation, usually induced by a precipitating factor. A common link between ensuing failures of various organs is impairment of the vascular supply, which may also induce vasogenic oedema in the brain. The aim of this study was to perform magnetic resonance (MR) study of the brain in a rat model combining bile duct ligation (BDL) and lipopolysaccharide (LPS) administration to investigate brain oedema in liver failure. METHODS: Bile duct-ligated rats underwent in vivo brain MR imaging at 4, 5 and 6 weeks, and after superimposed administration of LPS. The MR techniques applied enabled assessment of brain metabolites, and intra- or extracellular water distribution. Brain water content was assessed by gravimetry. RESULTS: MR spectroscopy showed an increase in brain glutamine and a decrease in myo-inositol and choline in relation to progression of liver disease. BDL rats showed a slight, progressive increase in the amount of cortical brain water that was significant after LPS injection. These changes did not modify the apparent diffusion coefficient, supporting a mixed origin of brain oedema (vasogenic and cytotoxic). CONCLUSIONS: The mechanisms leading to the development of brain oedema in an experimental liver disease model were related to the time course of liver failure and to pro-inflammatory stimuli. MR findings support the presence of cytotoxic and vasogenic mechanisms in induced brain oedema in BDL rats exposed to LPS.

Efficient Tumor Eradication at Ultralow Drug Concentration via Externally Controlled and Boosted Metallic Iron Magnetoplasmonic Nanocapsules.

With the aim to locally enhance the efficacy of cancer nanotherapies, here we present metal iron based magnetoplasmonic drug-loaded nanocapsules (MAPSULES), merging powerful external magnetic concentration in the tumor and efficient photothermal actuation to locally boost the drug therapeutic action at ultralow drug concentrations. The MAPSULES are composed of paclitaxel-loaded polylactic-co-glycolic acid (PLGA) nanoparticles partially coated by a nanodome shape iron/silica semishell. The iron semishell has been designed to present a ferromagnetic vortex for incorporating a large quantity of ferromagnetic material while maintaining high colloidal stability. The large iron semishell provides very strong magnetic manipulation via magnetophoretic forces, enabling over 10-fold higher trapping efficiency in microfluidic channels than typical superparamagnetic iron oxide nanoparticles. Moreover, the iron semishell exhibits highly damped plasmonic behavior, yielding intense broadband absorbance in the near-infrared biological windows and photothermal efficiency similar to the best plasmonic nanoheaters. The in vivo therapeutic assays in a mouse xenograft tumor model show a high amplification of the therapeutic effects by combining magnetic concentration and photothermal actuation in the tumor, leading to a complete eradication of the tumors at ultralow nanoparticle and drug concentration (equivalent to only 1 mg/kg PLGA nanoparticles containing 8 µg/kg of paclitaxel, i.e., 100-500-fold lower than the therapeutic window of the free and PLGA encapsulated drug and 13-3000-fold lower than current nanotherapies combining paclitaxel and light actuation). These results highlight the strength of this externally controlled and amplified therapeutic approach, which could be applied to locally boost a wide variety of drugs for different diseases.

In vivo assessment of neurodegeneration in Niemann-Pick type C mice by quantitative T2 mapping and diffusion tensor imaging.

PURPOSE: To quantitatively and noninvasively assess neurological disease progression in a mouse model of Niemann-Pick type C (NPC) disease by measuring white matter status with magnetic resonance imaging (MRI) techniques of T2 mapping and diffusion tensor imaging (DTI). MATERIALS AND METHODS: Quantitative T2 and DTI experiments were performed in vivo in NPC disease model and control mice at three timepoints to quantify differences and changes in white matter with measurements of T2 relaxation and DTI parameters. Histological staining for myelin content was also performed at two timepoints to compare with the MRI findings. RESULTS: The results of the T2 and DTI measurements show significant differences in white matter areas of the brain in the NPC disease model compared to control mice at several timepoints, and were seen to change over time in both groups. CONCLUSION: The findings of this study suggest that quantitative MRI measurements may be suitable in vivo biomarkers of disease status for future studies of NPC disease models. The changes in white matter measurements between timepoints in both control and NPC disease groups suggest that white matter structures continue to change and develop over time in the NPC model and can be tracked with MRI techniques.

Diffusion tensor imaging supports the cytotoxic origin of brain edema in a rat model of acute liver failure.

BACKGROUND & AIMS: Brain edema is a severe complication of acute liver failure (ALF) that has been related to ammonia concentrations. Two mechanisms have been proposed in the pathogenesis: vasogenic edema that is secondary to the breakdown of the blood-brain barrier and cytotoxic edema caused by ammonia metabolites in astrocytes. METHODS: We applied magnetic resonance techniques to assess the intracellular or extracellular distribution of brain water and metabolites in a rat model of devascularized ALF. The brain water content was assessed by gravimetry and blood-brain barrier permeability was determined from the transfer constant of (14)C-labeled sucrose. RESULTS: Rats with ALF had a progressive decrease in the apparent diffusion coefficient (ADC) in all brain regions. The average decrease in ADC was significant in precoma (-14%) and coma stages (-20%). These changes, which indicate an increase of the intracellular water compartment, were followed by a significant increase in total brain water (coma 82.4% +/- 0.3% vs sham 81.6% +/- 0.3%; P = .0001). Brain concentrations of glutamine (6 hours, 540%; precoma, 851%; coma, 1086%) and lactate (6 hours, 166%; precoma, 998%; coma, 3293%) showed a marked increase in ALF that paralleled the decrease in ADC and neurologic outcome. In contrast, the transfer constant of (14)C-sucrose was unaltered. CONCLUSIONS: The pathogenesis of brain edema in an experimental model of ALF involves a cytotoxic mechanism: the metabolism of ammonia in astrocytes induces an increase of glutamine and lactate that appears to mediate cellular swelling. Therapeutic measures should focus on removing ammonia and improving brain energy metabolism.

Bioinspired Theranostic Coordination Polymer Nanoparticles for Intranasal Dopamine Replacement in Parkinson's Disease.

Dopamine (DA) is one of the main neurotransmitters found in the central nervous system and has a vital role in the function of dopaminergic (DArgic) neurons. A progressive loss of this specific subset of cells is one of the hallmarks of age-related neurodegenerative disorders such as Parkinson's disease (PD). Symptomatic therapy for PD has been centered in the precursor l-DOPA administration, an amino acid precursor of DA that crosses the blood-brain barrier (BBB) while DA does not, although this approach presents medium- to long-term side effects. To overcome this limitation, DA-nanoencapsulation therapies are actively being searched as an alternative for DA replacement. However, overcoming the low yield of encapsulation and/or poor biodistribution/bioavailability of DA is still a current challenge. Herein, we report the synthesis of a family of neuromelanin bioinspired polymeric nanoparticles. Our system is based on the encapsulation of DA within nanoparticles through its reversible coordination complexation to iron metal nodes polymerized with a bis-imidazol ligand. Our methodology, in addition to being simple and inexpensive, results in DA loading efficiencies of up to 60%. In vitro, DA nanoscale coordination polymers (DA-NCPs) exhibited lower toxicity, degradation kinetics, and enhanced uptake by BE(2)-M17 DArgic cells compared to free DA. Direct infusion of the particles in the ventricle of rats in vivo showed a rapid distribution within the brain of healthy rats, leading to an increase in striatal DA levels. More importantly, after 4 days of nasal administrations with DA-NCPs equivalent to 200 µg of the free drug per day, the number and duration of apomorphine-induced rotations was significantly lower from that in either vehicle or DA-treated rats performed for comparison purposes. Overall, this study demonstrates the advantages of using nanostructured DA for DA-replacement therapy.

Nicotinamide Protects Against Diet-Induced Body Weight Gain, Increases Energy Expenditure, and Induces White Adipose Tissue Beiging.

SCOPE: Interventions that boost NAD+ availability are of potential therapeutic interest for obesity treatment. The potential of nicotinamide (NAM), the amide form of vitamin B3 and a physiological precursor of nicotinamide adenine dinucleotide (NAD)+ , in preventing weight gain has not previously been studied in vivo. Other NAD+ precursors have been shown to decrease weight gain; however, their impact on adipose tissue is not addressed. METHODS AND RESULTS: Two doses of NAM (high dose: 1% and low dose: 0.25%) are given by drinking water to C57BL/6J male mice, starting at the same time as the high-fat diet feeding. NAM supplementation protects against diet-induced obesity by augmenting global body energy expenditure in C57BL/6J male mice. The manipulation markedly alters adipose morphology and metabolism, particularly in inguinal (i) white adipose tissue (iWAT). An increased number of brown and beige adipocyte clusters, protein abundance of uncoupling protein 1 (UCP1), mitochondrial activity, adipose NAD+ , and phosphorylated AMP-activated protein kinase (P-AMPK) levels are observed in the iWAT of treated mice. Notably, a significant improvement in hepatic steatosis, inflammation, and glucose tolerance is also observed in NAM high-dose treated mice. CONCLUSION: NAM influences whole-body energy expenditure by driving changes in the adipose phenotype. Thus, NAM is an attractive potential treatment for preventing obesity and associated complications.

Changes in water content and distribution in Quercus ilex leaves during progressive drought assessed by in vivo 1H magnetic resonance imaging.

BACKGROUND: Drought is a common stressor in many regions of the world and current climatic global circulation models predict further increases in warming and drought in the coming decades in several of these regions, such as the Mediterranean basin. The changes in leaf water content, distribution and dynamics in plant tissues under different soil water availabilities are not well known. In order to fill this gap, in the present report we describe our study withholding the irrigation of the seedlings of Quercus ilex, the dominant tree species in the evergreen forests of many areas of the Mediterranean Basin. We have monitored the gradual changes in water content in the different leaf areas, in vivo and non-invasively, by 1H magnetic resonance imaging (MRI) using proton density weighted (rhow) images and spin-spin relaxation time (T2) maps. RESULTS: Rhow images showed that the distal leaf area lost water faster than the basal area and that after four weeks of similar losses, the water reduction was greater in leaf veins than in leaf parenchyma areas and also in distal than in basal leaf area. There was a similar tendency in all different areas and tissues, of increasing T2 values during the drought period. This indicates an increase in the dynamics of free water, suggesting a decrease of cell membranes permeability. CONCLUSIONS: The results indicate a non homogeneous leaf response to stress with a differentiated capacity to mobilize water between its different parts and tissues. This study shows that the MRI technique can be a useful tool to follow non-intrusively the in vivo water content changes in the different parts of the leaves during drought stress. It opens up new possibilities to better characterize the associated physiological changes and provides important information about the different responses of the different leaf areas what should be taken into account when conducting physiological and metabolic drought stress studies in different parts of the leaves during drought stress.

Self-assembled multifunctional Fe/MgO nanospheres for magnetic resonance imaging and hyperthermia.

A one-step process for the production of nanoparticles presenting advanced magnetic properties can be achieved using vapor condensation. In this article, we report on the fabrication of Fe particles covered by a uniform MgO epitaxial shell. MgO has a lower surface energy than Fe, which results in a core-shell crystal formation. The particles satisfy a few of technical requirements for the practical use in real clinics, such as a high biocompatibility in living cells in-vitro, an injection through blood vessels without any clothing problems in murine model, a high absorption rate for magnetic hyperthermia at small particle concentration, and the potential to be used as contrast agent in the field of diagnostic magnetic imaging. They are also able to be used in drug delivery and magnetic-activated cell sorting. FROM THE CLINICAL EDITOR: In this paper, the authors report on the synthesis of Fe particles covered by a uniform MgO epitaxial shell resulting in a core-shell crystal formation. The particles are proven to be useful as contrast agents for magnetic resonance imaging and have the potential to be useful as heating mediators for cancer therapy through hyperthermia. They also might be used in drug delivery and magnetic-activated cell sorting.

Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides.

BACKGROUND: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. METHODS: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. FINDINGS: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. INTERPRETATION: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. FUNDING: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Microwave-Assisted Synthesis of SPION-Reduced Graphene Oxide Hybrids for Magnetic Resonance Imaging (MRI).

Magnetic resonance imaging (MRI) is a useful tool for disease diagnosis and treatment monitoring. Superparamagnetic iron oxide nanoparticles (SPION) show good performance as transverse relaxation (T2) contrast agents, thus facilitating the interpretation of the acquired images. Attachment of SPION onto nanocarriers prevents their agglomeration, improving the circulation time and efficiency. Graphene derivatives, such as graphene oxide (GO) and reduced graphene oxide (RGO), are appealing nanocarriers since they have both high surface area and functional moieties that make them ideal substrates for the attachment of nanoparticles. We have employed a fast, simple and environmentally friendly microwave-assisted approach for the synthesis of SPION-RGO hybrids. Different iron precursor/GO ratios were used leading to SPION, with a median diameter of 7.1 nm, homogeneously distributed along the RGO surface. Good relaxivity (r2*) values were obtained in MRI studies and no significant toxicity was detected within in vitro tests following GL261 glioma and J774 macrophage-like cells for 24 h with SPION-RGO, demonstrating the applicability of the hybrids as T2-weighted MRI contrast agents.

MRI detects therapeutic effects in weanling Niemann-Pick type C mice.

To noninvasively evaluate the early effects of Niemann-Pick type C (NPC) disease, diffusion tensor imaging (DTI) was carried out in the brains of very young (23-day-old) mice. The diffusion of water in white matter tracts of Npc1(-/-) mice at this young age was already abnormal, exhibiting decreased anisotropy, as quantified by fractional anisotropy (FA), compared with their wild-type littermates, the controls. Postmortem histological staining revealed myelin deficiencies in Npc1(-/-) mice, consistent with the reduction in FA measured in vivo. Beneficial effects of treatment with allopregnanolone and/or 2 hydroxypropyl-beta-cyclodextrin was also detectable at this age by FA, which correlated with increased myelination as seen by histology. This is the earliest detection of a therapeutic effect in Npc1(-/-) mice.

Diffusion Tensor Imaging (DTI).

Diffusion Tensor Imaging is an MRI technique that allows in vivo noninvasive measurement of the translational motion of water, providing information about its anisotropy (or lack of it) in different tissues. DTI has been commonly used to quantitatively measure the integrity of tissues which may be compromised by neurological disease, such as white matter tracks of the brain, which normally impart significant anisotropy to water motion in healthy brains. However, this anisotropic effect is diminished when axonal or neuronal damage is present. This chapter describes a standard protocol for DTI data acquisition in preclinical studies.

Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aß scFv in 3xTg-AD mice.

Anti-Aß immunotherapy has emerged as a promising approach to treat Alzheimer's disease (AD). The single-chain variable fragment scFv-h3D6 is an anti-Aß antibody fragment that lacks the Fc region, which is associated with the induction of microglial reactivity by the full-length monoclonal antibody bapineuzumab. ScFv-h3D6 was previously shown to restore the levels of apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a triple-transgenic-AD (3xTg-AD) mouse model. Since apoE and apoJ play an important role in the development of AD, we aimed to study the in vivo effect of the combined therapy of scFv-h3D6 with apoE and apoJ mimetic peptides (MPs). Four-and-a-half-month-old 3xTg-AD mice were treated for six weeks with scFv-h3D6, apoE-MP, apoJ-MP, or a combination of scFv-h3D6 with each of the MPs, or a vehicle, and then the results were compared to non-transgenic mice. Magnetic Resonance Imaging showed a general tendency of the different treatments to protect against the reduction in brain volume. Aß burden decreased after treatment with scFv-h3D6, apoE-MP, or apoJ-MP, but the effect was not as evident with the combined therapies. In terms of glial reactivity, apoE-MP showed a potent anti-inflammatory effect that was eased by the presence of scFv-h3D6, whereas the combination of apoJ-MP and scFv-h3D6 was not detrimental. ScFv-h3D6 alone did not induce microglial reactivity, as full-length antibodies do; rather, it reduced it. Endogenous apoE and apoJ levels were decreased by scFv-h3D6, but the MPs lead to a simultaneous increase of both apolipoproteins. While apoE-MP and apoJ-MP demonstrated different effects in the combined therapies with scFv-h3D6, they did not improve the overall protective effect of scFv-h3D6 in reducing the Aß burden, apolipoproteins levels or microglial reactivity.

Metabolomics of Therapy Response in Preclinical Glioblastoma: A Multi-Slice MRSI-Based Volumetric Analysis for Noninvasive Assessment of Temozolomide Treatment.

Glioblastoma (GBM) is the most common aggressive primary brain tumor in adults, with a short survival time even after aggressive therapy. Non-invasive surrogate biomarkers of therapy response may be relevant for improving patient survival. Previous work produced such biomarkers in preclinical GBM using semi-supervised source extraction and single-slice Magnetic Resonance Spectroscopic Imaging (MRSI). Nevertheless, GBMs are heterogeneous and single-slice studies could prevent obtaining relevant information. The purpose of this work was to evaluate whether a multi-slice MRSI approach, acquiring consecutive grids across the tumor, is feasible for preclinical models and may produce additional insight into therapy response. Nosological images were analyzed pixel-by-pixel and a relative responding volume, the Tumor Responding Index (TRI), was defined to quantify response. Heterogeneous response levels were observed and treated animals were ascribed to three arbitrary predefined groups: high response (HR, n = 2), TRI = 68.2 ± 2.8%, intermediate response (IR, n = 6), TRI = 41.1 ± 4.2% and low response (LR, n = 2), TRI = 13.4 ± 14.3%, producing therapy response categorization which had not been fully registered in single-slice studies. Results agreed with the multi-slice approach being feasible and producing an inverse correlation between TRI and Ki67 immunostaining. Additionally, ca. 7-day oscillations of TRI were observed, suggesting that host immune system activation in response to treatment could contribute to the responding patterns detected.