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Female Dark Agouti rats were immunized with increasing doses of myelin oligodendrocyte glycoprotein (MOG) to develop experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis. Typical EAE motor impairments were assessed daily and noninvasive visual evoked potentials (VEPs) were recorded at baseline and 5 weeks after immunization, with final histopathology of optic nerves (ONs). Immunized rats exhibited a relapsing-remitting clinical course. Both VEP and histological abnormalities were detected in a MOG dose-dependent gradient. Increasing MOG dosage augmented visual function impairment in EAE, which could be monitored with VEP recording to assess demyelination and axonal loss along ONs.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Molecular imaging techniques are essential tools for better investigating biological processes and detecting disease biomarkers with improvement of both diagnosis and therapy monitoring. Often, a single imaging technique is not sufficient to obtain comprehensive information at different levels. Multimodal diagnostic probes are key tools to enable imaging across multiple scales. The direct registration of in vivo imaging markers with ex vivo imaging at the cellular level with a single probe is still challenging. Fluorinated (19F) probes have been increasingly showing promising potentialities for in vivo cell tracking by 19F-MRI. Here we present the unique features of a bioorthogonal 19F-probe that enables direct signal correlation of MRI with Raman imaging. In particular, we reveal the ability of PERFECTA, a superfluorinated molecule, to exhibit a remarkable intense Raman signal distinct from cell and tissue fingerprints. Therefore, PERFECTA combines in a single molecule excellent characteristics for both macroscopic in vivo 19F-MRI, across the whole body, and microscopic imaging at tissue and cellular levels by Raman imaging.

Fluorine-19 (

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor characterized by a strongly immunosuppressive tumor microenvironment (TME) that represents a barrier also for the development of effective immunotherapies. The possibility to revert this hostile TME by immunoactivating cytokines is hampered by the severe toxicity associated with their systemic administration. Here, we exploited a lentiviral vector–based platform to engineer hematopoietic stem cells ex vivo with the aim of releasing, via their tumor-infiltrating monocyte/macrophage progeny, interferon-α (IFN-α) or interleukin-12 (IL-12) at the tumor site with spatial and temporal selectivity. Taking advantage of a syngeneic GBM mouse model, we showed that inducible release of IFN-α within the TME achieved robust tumor inhibition up to eradication and outperformed systemic treatment with the recombinant protein in terms of efficacy, tolerability, and specificity. Single-cell RNA sequencing of the tumor immune infiltrate revealed reprogramming of the immune microenvironment toward a proinflammatory and antitumoral state associated with loss of a macrophage subpopulation shown to be associated with poor prognosis in human GBM. The spatial and temporal control of IL-12 release was critical to overcome an otherwise lethal hematopoietic toxicity while allowing to fully exploit its antitumor activity. Overall, our findings demonstrate a potential therapeutic approach for GBM and set the bases for a recently launched first-in-human clinical trial in patients with GBM.

Experimental autoimmune encephalomyelitis (EAE) is the primary disease model of multiple sclerosis (MS), one of the most diffused neurological diseases characterized by fatigue, muscle weakness, vision loss, anxiety and depression. EAE can be induced through injection of myelin peptides to susceptible mouse or rat strains. In particular, EAE elicited by the autoimmune reaction against myelin oligodendrocyte glycoprotein (MOG) presents the common features of human MS: inflammation, demyelination and axonal loss. Optic neuritis affects visual pathways in both MS and in several EAE models. Neurophysiological evaluation through visual evoked potential (VEP) recording is useful to check visual pathway dysfunctions and to test the efficacy of innovative treatments against optic neuritis. For this purpose, we investigate the extent of VEP abnormalities in the dark agouti (DA) rat immunized with MOG, which develops a relapsing–remitting disease course. Together with the detection of motor signs, we acquired VEPs during both early and late stages of EAE, taking advantage of a non-invasive recording procedure that allows long follow-up studies. The validation of VEP outcomes was determined by comparison with ON histopathology, aimed at revealing inflammation, demyelination and nerve fiber loss. Our results indicate that the first VEP latency delay in MOG-EAE DA rats appeared before motor deficits and were mainly related to an inflammatory state. Subsequent VEP delays, detected during relapsing EAE phases, were associated with a combination of inflammation, demyelination and axonal loss. Moreover, DA rats with atypical EAE clinical course tested at extremely late time points, manifested abnormal VEPs although motor signs were mild. Overall, our data demonstrated that non-invasive VEPs are a powerful tool to detect visual involvement at different stages of EAE, prompting their validation as biomarkers to test novel treatments against MS optic neuritis.

Myelin loss occurring in demyelinating diseases, including multiple sclerosis, is the leading cause of long-lasting neurological disability in adults. While endogenous remyelination, driven by resident oligodendrocyte precursor cells (OPCs), might partially compensate myelin loss in the early phases of demyelinating disorders, this spontaneous reparative potential fails at later stages. To investigate the cellular mechanisms sustaining endogenous remyelination in demyelinating disorders, we focused our attention on endogenous neural precursor cells (eNPCs) located within the subventricular zone (SVZ) since this latter area is considered one of the primary sources of new OPCs in the adult forebrain. First, we fate mapped SVZ-eNPCs in cuprizone-induced demyelination and found that SVZ endogenous neural stem/precursor cells are recruited during the remyelination phase to the corpus callosum (CC) and are capable of forming new oligodendrocytes. When we ablated SVZ-derived eNPCs during cuprizone-induced demyelination in female mice, the animals displayed reduced numbers of oligodendrocytes within the lesioned CC. Although this reduction in oligodendrocytes did not impact the ensuing remyelination, eNPC-ablated mice experienced increased axonal loss. Our results indicate that, in toxic models of demyelination, SVZ-derived eNPCs contribute to support axonal survival. SIGNIFICANCE STATEMENT One of the significant challenges in MS research is to understand the detrimental mechanisms leading to the failure of CNS tissue regeneration during disease progression. One possible explanation is the inability of recruited oligodendrocyte precursor cells (OPCs) to complete remyelination and to sustain axonal survival. The contribution of endogenous neural precursor cells (eNPCs) located in the subventricular zone (SVZ) to generate new OPCs in the lesion site has been debated. Using transgenic mice to fate map and to selectively kill SVZ-derived eNPCs in the cuprizone demyelination model, we observed migration of SVZ-eNPCs after injury and their contribution to oligodendrogenesis and axonal survival. We found that eNPCs are dispensable for remyelination but protect partially from increased axonal loss.

In the continuous search for multimodal systems with combined diagnostic and therapeutic functions, several efforts have been made to develop multifunctional drug delivery systems. In this work, through a covalent approach, a new class of fluorinated poly(lactic-co-glycolic acid) co-polymers (F-PLGA) were designed that contain an increasing number of magnetically equivalent fluorine atoms. In particular, two novel compounds, F

Fluorinated hyperbranched polyether copolymers of tuneable composition and architecture were designed and synthesized to develop drug nanocarriers as well as 19F MRI contrast agents. The performance of these nanomaterials in terms of nanoparticle formation and drug (dexamethasone) loading, as well as 19F-MRI detectability, was evaluated. These polymers were obtained through controlled ring opening multibranching polymerisation, and the presence of pendant fluorinated groups was obtained by copolymerisation of glycidol and the fluorinated glycidyl ether 2-[(2,2,2-trifluoroethoxy)methyl]oxirane. By varying polymer composition and architecture during the synthesis, we controlled the key properties of the nanomaterials, including the fluorine content, drug loading, nanoparticle size, and MRI signal. The macromolecules were cytocompatible, with the ability to deliver dexamethasone on damaged kidney glomerular cells in vitro; therefore they hold promise as new generation 19F MRI nanotheranostics.

In the continuous search for multimodal systems with combined diagnostic and therapeutic functions, several efforts have been made to develop multifunctional drug delivery systems. In this work, through a covalent approach, a new class of fluorinated poly(lactic-co-glycolic acid) co-polymers (F-PLGA) were designed that contain an increasing number of magnetically equivalent fluorine atoms. In particular, two novel compounds, F3 -PLGA and F9 -PLGA, were synthesized and their chemical structure and thermal stability were analyzed by solution NMR, DSC, and TGA. The obtained F-PLGA compounds were proven to form in aqueous solution colloidal stable nanoparticles (NPs) displaying a strong 19 F NMR signal. The fluorinated NPs also showed an enhanced ability to load hydrophobic drugs containing fluorine atoms compared to analogous pristine PLGA NPs. Preliminary in vitro studies showed high cell viability and the NP ability to intracellularly deliver and release a functioning drug.

Mononuclear cell activity was monitored in vivo after the temporary inhibition of colony-stimulating factor-1 receptor by using multispectral fluorine MRI with the administration of two fluorine pr...

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination. Electronic supplementary material The online version of this article (10.1007/s00401-019-02049-1) contains supplementary material, which is available to authorized users.

Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis. We assessed motor signs and recorded VEPs for five or eight weeks with epidural or epidermal electrodes, respectively, with final histopathology of optic nerves (ONs). Injected rats exhibited motor deficits a week after immunization. VEP delays arose from the 2nd to the 5th week, when a recovery occurred in epidermal-recorded rats. ON damage appeared in epidural-, but not in epidermal-recorded rats, probably due to a remyelination process. VEP could be exploited as neurophysiological marker to test novel treatments against neurodegeneration involving ONs.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

A magnetic resonance imaging contrast agent based on a tetrameric Gd-DTPA-like system linked to a fibrin-targeting peptide (Gd-F) has been designed for in vivo tumor characterization.Gd-F was synthesized following Fmoc-SPPS strategy. Binding was measured using soluble fibrin DD(E) fragment and a dried fibrin assay. Contrast efficiency was tested on human and mouse clots and in vivo on Neuro2A tumor model. An anti-thrombotic drug was used to evaluate Gd-F sensitivity for changes in fibrin availability at the tumor site.The high relaxivity of Gd-F (42 mM(-1) s(-1), per molecule) yielded a strong signal enhancement in human and murine clots. High contrast was also measured in vivo in Neuro2A tumors, with a persistent enhancement in tumor rim and stroma. Upon treatment with an anti-thrombotic drug, the contrast uptake was significantly reduced in the tumor area confirming the specificity of the probe.Gd-F resulted to be an efficient probe for tumor delineation and for monitoring fibrin deposits during tumor progression and anti-thrombotic therapy.

Local acidosis is associated with neuro-inflammation and can have significant effects in several neurological disorders, including multiple sclerosis, brain ischemia, spinal cord injury and epilepsy. Despite local acidosis has been implicated in numerous pathological functions, very little is known about the modulatory effects of pathological acidosis on the activity of neuronal networks and on synaptic structural properties. Using non-invasive MRI spectroscopy we revealed protracted extracellular acidosis in the CNS of Experimental Autoimmune Encephalomyelitis (EAE) affected mice. By multi-unit recording in cortical neurons, we established that acidosis affects network activity, down-sizing firing and bursting behaviors as well as amplitudes. Furthermore, a protracted acidosis reduced the number of presynaptic terminals, while it did not affect the postsynaptic compartment. Application of the diarylamidine Diminazene Aceturate (DA) during acidosis significantly reverted both the loss of neuronal firing and bursting and the reduction of presynaptic terminals. Finally, in vivo DA delivery ameliorated the clinical disease course of EAE mice, reducing demyelination and axonal damage. DA is known to block acid-sensing ion channels (ASICs), which are proton-gated, voltage-insensitive, Na(+) permeable channels principally expressed by peripheral and central nervous system neurons. Our data suggest that ASICs activation during acidosis modulates network electrical activity and exacerbates neuro-degeneration in EAE mice. Therefore pharmacological modulation of ASICs in neuroinflammatory diseases could represent a new promising strategy for future therapies aimed at neuro-protection.

Objective Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine-1-phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte–neuron interaction and NO synthesis in vivo. Methods Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S1P receptors, interleukin (IL) 1R, IL17RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmune encephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model was established to study the effects of S1P, IL1, and IL17 stimulation on NFkB translocation and NO production in astrocytes, on spinal neuron survival, and on astrocyte–neuron interaction. Furthermore, fingolimod efficacy in blocking astrocyte-mediated neurodegeneration was evaluated. Results We found coordinated upregulation of IL1R, IL17RA, S1P1, and S1P3 together with nitrosative markers in astrocytes within MS and EAE lesions. In vitro studies revealed that S1P, IL17, and IL1 induced NFκB translocation and NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingolimod blocked the 2 activation events evoked in astrocytes by either S1P or inflammatory cytokines, resulting in inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE mice hampered astrocyte activation and NO production. Interpretation A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyte activation steps. Ann Neurol 2014;76:325–337

Growing evidence indicates that alterations within the peripheral nervous system (PNS) are involved at an early stage in the amyotrophic lateral sclerosis (ALS) pathogenetic cascade. In this study, magnetic resonance imaging (MRI), neurophysiologic analyses, and histologic analyses were used to monitor the extent of PNS damage in the hSOD-1 ALS rat model. The imaging signature of the disease was defined using in vivo MRI of the sciatic nerve. Initial abnormalities were detected in the nerves by an increase in T2 relaxation time before the onset of clinical disease; diffusion MRI showed a progressive increase in mean and radial diffusivity and reduction of fractional anisotropy at advanced stages of disease. Histologic analysis demonstrated early impairment of the blood-nerve barrier followed by acute axonal degeneration associated with endoneurial edema and macrophage response in motor nerve compartments. Progressive axonal degeneration and motor nerve fiber loss correlated with MRI and neurophysiologic changes. These functional and morphologic investigations of the PNS might be applied in following disease progression in preclinical therapeutic studies. This study establishes the PNS signature in this rat ALS model (shedding new light into pathogenesis) and provides a rationale for translating into future systematic MRI studies of PNS involvement in patients with ALS.

The approach to molecular imaging of enzymes by MRI typically relies upon imaging probes composed of an enzymecleavable moiety conjugated with a paramagnetic imaging reporter, such as a GdIII chelate.[1] Upon enzymatic processing, the probe is transformed into a fragment with an altered relaxivity, leading to a different capability to enhance contrast in MR images with respect to the parent species. Ideally, the unprocessed (intact) form of the probe should be completely silent while the processed (cleaved) form should have a high relaxivity (that is, high contrast enhancement). In such a way the appearance of contrast within images can be unambiguously attributed to the result of enzymatic activity and not to dynamic changes of tissue probe concentration. However, gadolinium-based agents as enzyme responsive agents are never completely silent and both forms (unprocessed and processed) contribute to the overall contrast enhancement as a function of their respective relaxivities and tissue local concentrations.[2] Exact knowledge of the total concentration of Gd is essential to translate image contrast enhancement into the molar ratio of unprocessed versus processed forms, and thus into true enzyme activity maps. Aviable solution to the concentration problem can be provided by the R2p/R1p.

To assess brachial plexus magnetic resonance (MR) imaging features and limb-girdle muscle abnormalities as signs of muscle denervation in patients with amyotrophic lateral sclerosis (ALS).This study was approved by the local ethical committees on human studies, and written informed consent was obtained from all subjects before enrollment. By using an optimized protocol of brachial plexus MR imaging, brachial plexus and limb-girdle muscle abnormalities were evaluated in 23 patients with ALS and clinical and neurophysiologically active involvement of the upper limbs and were compared with MR images in 12 age-matched healthy individuals. Nerve root and limb-girdle muscle abnormalities were visually evaluated by two experienced observers. A region of interest-based analysis was performed to measure nerve root volume and T2 signal intensity. Measures obtained at visual inspection were analyzed by using the Wald χ(2) test. Mean T2 signal intensity and volume values of the regions of interest were compared between groups by using a hierarchical linear model, accounting for the repeated measurement design.The level of interrater agreement was very strong (κ = 0.77-1). T2 hyperintensity and volume alterations of C5, C6, and C7 nerve roots were observed in patients with ALS (P.001 to .03). Increased T2 signal intensity of nerve roots was associated with faster disease progression (upper-limb Medical Research Council scale progression rate, r = 0.40; 95% confidence interval: 0.001, 0.73). Limb-girdle muscle alterations (ie, T2 signal intensity alteration, edema, atrophy) and fat infiltration also were found, in particular, in the supraspinatus muscle, showing more frequent T2 signal intensity alterations and edema (P = .01) relative to the subscapularis and infraspinatus muscles.Increased T2 signal intensity and volume of brachial nerve roots do not exclude a diagnosis of ALS and suggest involvement of the peripheral nervous system in the ALS pathogenetic cascade. MR imaging of the peripheral nervous system and the limb-girdle muscle may be useful for monitoring the evolution of ALS and distinguishing patients with ALS from those with inflammatory neuropathy, respectively.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd–DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd–K11) or through a hydrophilic spacer (compd Gd–K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd–K11 and Gd–K11N have a good affinity for β-cyclodextrins (KD 310 and 670 µ m respectively) and for serum albumin (KD 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd–K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs. Copyright © 2012 John Wiley & Sons, Ltd.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

The aim of this study was to compare a pure macromolecular contrast agent (Gd-DTPA-albumin) with a new protein-binding blood pool contrast agent (B22956/1) in terms of their capacity to investigate the microvasculature in an experimental model of mammary carcinoma.Tumors were induced by subcutaneous injection of 5 x 10(5) BB1 cells into the backs of 5-7 week-old female FVB/neuNT233 mice. The animals were observed using DCE-MRI when the longest diameter of the tumor was 10.2+/-2.0 mm. DCE-MRI experiments were carried out using B22956/1 and (24 h later) Gd-DTPA-albumin.DCE-MRI data showed that vasculature in the tumor rim was characterized by greater fractional plasma volume and transendothelial permeability than vasculature in the tumor core as measured by both contrast agents. Permeability to Gd-DTPA-albumin in the tumor core was hardly measurable while permeability to B22956/1 was substantial. Histologically the tumor core showed areas of well vascularized, viable tissue surrounded by necrotic regions.DCE-MRI experiments performed with B22956/1 are useful in the investigation of vasculature in those tumor regions that are characterized by low permeability to macromolecules.

Objectives This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-IMilano phospholipid complex (recombinant apoA-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. Background A single high dose of recombinant apoA-IMilano has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. Methods Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. Results Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216–treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to −6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p Conclusions These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Positron Emission Tomography (PET) with 18 F-FDG is a promising tool for the detection and evaluation of active inflammation in animal models of neuroinflammation. MRI is a complementary imaging technique with high resolution and contrast suitable to obtain the anatomical data required to analyze PET data. To combine PET and MRI modalities, we developed a support bed system compatible for both scanners that allowed to perform imaging exams without animal repositioning. With this approach, MRI and PET data were acquired in mice with Experimental autoimmune encephalomyelitis (EAE). In this model, it was possible to measure a variation of 18 F-FDG uptake proportional to the degree of disease severity which is mainly related to Central Nervous System (CNS) inflammation. Against the low resolved PET images, the co-registered MRI/PET images allowed to distinguish the different brain structures and to obtain a more accurate tracer evaluation. This is essential in particular for brain regions whose size is of the order of the spatial resolution of PET.

High-resolution MRI of obese (fa/fa) Zucker rats was investigated to characterize and assess in vivo adipose tissue distribution. Thirty animals were gavaged with a placebo, a PPARgamma activator (pioglitazone), or a dual PPARalphagamma activator (LM 4156). At day 15, T1-weighted images were acquired in vivo using a 2TMRI system with a high in-plane spatial resolution (254 microm). Fat volumes of selected territories were measured by image segmentation, and the retroperitoneal fat was weighed post-mortem. Body-weight gain was significant with pioglitazone (101.8+/-5.9 g, p0.01 vs. placebo). The good quality of MR images allowed the delimitation and quantification of different fat territories. In response to pioglitazone, the retroperitoneal fat was more important compared to placebo (+23%, p0.01) while subcutaneous fat was not different. No significant effects were observed with LM 4156. In vivo measurements of fat volumes were strongly correlated with ex vivo tissue weights (r=0.91). High-resolution MRI provides an in vivo measurement of adipose tissue distribution in obese Zucker rats. Specific fat depots of regions that were particularly involved in drug response were determined in vivo. Fat remodeling was observed with pioglitazone but not with a dual PPARalphagamma activator (LM 4156).

[DOI:\hrefhttps://dx.doi.org/10.1002/mrm.1072210.1002/mrm.10722] [PubMed:\hrefhttps://www.ncbi.nlm.nih.gov/pubmed/1475566314755663]; International audience; Vessel-wall measurements from multicontrast MRI provide information on plaque structure and evolution. This requires the extraction of numerous contours. In this work a contour-extraction method is proposed that uses an active contour model (NLSnake) adapted for a wide range of MR vascular images. This new method employs length normalization for the purpose of deformation computation and offers the advantages of simplified parameter tuning, fast convergence, and minimal user interaction. The model can be initialized far from the boundaries of the region to be segmented, even by only one pixel. The accuracy and reproducibility of NLSnake endoluminal contours were assessed on vascular phantom MR angiography (MRA) and high-resolution in vitro MR images of rabbit aorta. An in vivo evaluation was performed on rabbit and clinical data for both internal and external vessel-wall contours. In phantoms with 95% stenoses, NLSnake measured 94.3% +/- 3.8%, and the accuracy was even better for milder stenoses. In the images of rabbit aorta, variability between NLSnake and experts was less than interobserver variability, while the maximum intravariability of NLSnake was equal to 1.25%. In conclusion, the NLSnake technique successfully quantified the vessel lumen in multicontrast MR images using constant parameters.

PURPOSE To test whether a corneal injury can stimulate inflammation in the trigeminal ganglion (TG), a structure located in the brain. METHODS At 4 and 8 days after alkali burn induced in the right eyes of mice, in vivo magnetic resonance imaging (MRI) of the brain was done before and after ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) contrast to track macrophages. Trigeminal ganglia were stained for Prussian Blue and inflammatory cell markers. Interleukin-1β, TNF-α, and VEGF-A transcripts were quantified on days 1, 4, and 8, and 4 days after corneal topical anti-inflammatory treatment with 0.2% dexamethasone. The expression of Substance P and its receptor NK-1R was also measured in the TG on day 4. RESULTS Corneal alkali burn induced leukocyte infiltration, including T cells, in the right TG at 4 and 8 days. In vivo MRI showed an increased contrast uptake in the right TG, which peaked at day 8. Prussian Blue(+) USPIO(+) macrophages were observed in the right TG and exhibited an M2 phenotype. The M2-macrophage infiltration was preponderant in the TG after damage. The proinflammatory cytokines Substance P and NK-1R were significantly increased in both the TGs. The expression of IL-1β and VEGF-A was significantly reduced in the right TG with dexamethasone treatment. CONCLUSIONS We suggest, for the first time, inflammatory involvement of brain structures following ocular surface damage. Our findings support the hypothesis that the neuropeptide Substance P may be involved in the propagation of inflammation from the cornea to the TG through corneal nerves.

Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine-1-phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte-neuron interaction and NO synthesis in vivo.Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S1P receptors, interleukin (IL) 1R, IL17RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmune encephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model was established to study the effects of S1P, IL1, and IL17 stimulation on NFkB translocation and NO production in astrocytes, on spinal neuron survival, and on astrocyte-neuron interaction. Furthermore, fingolimod efficacy in blocking astrocyte-mediated neurodegeneration was evaluated.We found coordinated upregulation of IL1R, IL17RA, S1P1, and S1P3 together with nitrosative markers in astrocytes within MS and EAE lesions. In vitro studies revealed that S1P, IL17, and IL1 induced NFκB translocation and NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingolimod blocked the 2 activation events evoked in astrocytes by either S1P or inflammatory cytokines, resulting in inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE mice hampered astrocyte activation and NO production.A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyte activation steps.

This paper describes a magnetometer for temporal and spatial analysis of fields generated by eddy currents in superconducting magnets, mainly caused by switching gradients. The magnetic field distortions are measured from FID signals acquired and multi-exponentially fitted. The signals are taken from an array of small probes spatially distributed and switched by the magnetometer. An example is given using a 2 T horizontal superconducting magnet and showing the residual defects of an active-shielded gradient coil system.

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for β-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h ( Eif4h ) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1 , maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h . These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Objective: Atherosclerosis involves an inflammatory process characterized by cellular and molecular responses. A slow-clearance blood-pool paramagnetic agent (CMD-A2-Gd-DOTA: P717) chemically modified to create a functionalized product (F-P717) for targeting inflammation in vessel walls was evaluated in vivo in mice. Methods and Results: Carboxylate and sulfate groups were grafted onto the macromolecular paramagnetic Gd-DOTA-dextran backbone. Products were also fluorescently labeled with rhodamine isothiocyanate. Pre- and postcontrast MRI was performed on a 2-Tesla magnet in ApoE / and control C57BL/6 mice after P717 or F-P717 injection at a dose of 60 mol Gd/kg. Axial T1-weighted images of the abdominal aorta were obtained using a 2D multislice spin-echo sequence. F-P717 significantly enhanced the magnetic resonance imaging (MRI) signal in the abdominal aortic wall of ApoE / mice (50% signal-to-noise ratio increase between 10 and 30 minutes), but not of control mice. P717 produced only moderate (20%) MRI signal enhancement within the same time frame. The MRI data were correlated to histopathology. Immunofluorescence in ApoE / mice colocalized F-P717 but not P717 with the inflammatory area revealed by P-selectin labeling. Conclusion: This study demonstrates the efficacy of F-P717 as a new molecular imaging agent for noninvasive in vivo MRI location of inflammatory vascular tree lesions in ApoE / mice.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques.A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216.Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg.Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose.These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Experimental Autoimmune Encephalomyelitis (EAE) is a pre-clinical disease model of Multiple Sclerosis (MS), traditionally induced by injecting Myelin Oligodendrocyte Glycoprotein(MOG). Optic nerve involvement and VEP diagnostic reliability have been already shown in MOG induced EAE rat. In the present study we used spinal cord homogenate (SCH) to induce disease in Dark-Agouti (DA) rats, proposing to show optic nerve involvement and assess VEP usefulness as neurophysiological biomarker also in this model. Nineteen DA rats were used in the experiment: 7 immunized by SCH (EAE) and 12 healthy (H) controls. Flash VEPs from both eyes were recorded with epidural electrodes under sevoflurane volatile anesthesia once a week for 6 weeks (one day before immunization, days 6, 13, 20, 27, 34 post) with measurement P1 latency from N1-P1-N2 complex. Values exceeding 2.5 SD from control mean were considered abnormal. MRI analyses on both Optic Nerves were also performed using a 7 Tesla technology. After the last VEP session, 7 EAE and 3 H animals underwent histological analysis on a transverse section of both optic nerves. P1 latency in EAE group was significantly increased at days 13–20-27 post immunization and was abnormal in 10/14 eyes in EAE rats (p

International audience; To develop an MRI method for the evaluation of contrast enhancement in early atherosclerotic plaque development in the abdominal aorta of a mouse model. Male apoE(-/-) mice from three groups, respectively 4 (n = 6), 8 (n = 11) and 16 (n = 4) weeks were included. Axial T I spin echo images of the abdominal aorta were obtained above and below the renal arteries (90 mu m spatial resolution) before and over 1 h after the injection of a macromolecular contrast agent. Signal enhancement was measured in the vessel wall and compared to histological features. Maximal arterial wall signal enhancement was obtained from 16 to 32 min post injection. During this time, the signal-to-noise ratio increased by a factor up to 1.7 in 16 week mice and 2.7 and 2.4 in 8 and 4 weeks mice, respectively. The enhancement of the arterial wall appeared less pronounced in the oldest mice, 16 weeks old, exhibiting more advanced lesions. Using a macromolecular gadolinium agent, contrast uptake in atherogenesis varies with lesion stage and may be related to vessel-wall permeability. Dynamic contrast-enhanced MRI may be useful to evaluate the atherosclerotic plaque activity in mice.

[PubMed:\hrefhttps://www.ncbi.nlm.nih.gov/pubmed/1537793715377937]; International audience; Old ApoE-deficient mice were studied in vivo by magnetic resonance imaging (MRI) to prospectively evaluate vascular remodeling associated with atherosclerotic lesions.\ Old female ApoE-/- mice on a normal diet were followed by MRI at 2 Tesla for a 3-month period and killed for histopathology. Aortic dimensions were measured and compared.\ High-quality in vivo MR images were obtained at 2 Tesla with in plane spatial resolution of 86 X 86 microm2. On MRI, aortic lumen enlargement (>1.5-fold dilation) was seen in 10 of 13 mice, located predominantly in the suprarenal portion of the aorta. The mean maximal diameter of the aneurysms and of the aorta above and below the aneurysm were, respectively, 1.12 +/- 0.32 mm and 0.53 +/- 0.08 mm by MRI and 1.3+/- 0.41 mm and 0.55 +/- 0.15 mm by histology. Matched histologic cross-sections of the aortic wall showed medial degradation with rupture of the internal elastic lamina at multiple sites, associated with fibrolipidic plaque containing cholesterol crystals.\ Aortic lumen enlargement was diagnosed in old ApoE-/- mice at sites with advanced atherosclerotic plaques. MRI has potential both as an in vivo imaging technique for screening mouse models for vascular wall pathology and to follow arterial remodeling associated with the disease progression.