Self-assembling dendrimer nanosystems for specific fluorine magnetic resonance imaging and effective theranostic treatment of tumors.

Fluorine magnetic resonance imaging (19F-MRI) is particularly promising for biomedical applications owing to the absence of fluorine in most biological systems. However, its use has been limited by the lack of safe and water-soluble imaging agents with high fluorine contents and suitable relaxation properties. We report innovative 19F-MRI agents based on supramolecular dendrimers self-assembled by an amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic dendron. Specifically, this amphiphilic dendrimer bears multiple negatively charged terminals with high fluorine content, which effectively prevented intra- and intermolecular aggregation of fluorinated entities via electrostatic repulsion. This permitted high fluorine nuclei mobility alongside good water solubility with favorable relaxation properties for use in 19F-MRI. Importantly, the self-assembling 19F-MRI agent was able to encapsulate the near-infrared fluorescence (NIRF) agent DiR and the anticancer drug paclitaxel for multimodal 19F-MRI and NIRF imaging of and theranostics for pancreatic cancer, a deadly disease for which there remains no adequate early detection method or efficacious treatment. The 19F-MRI and multimodal 19F-MRI and NIRF imaging studies on human pancreatic cancer xenografts in mice confirmed the capability of both imaging modalities to specifically image the tumors and demonstrated the efficacy of the theranostic agent in cancer treatment, largely outperforming the clinical anticancer drug paclitaxel. Consequently, these dendrimer nanosystems constitute promising 19F-MRI agents for effective cancer management. This study offers a broad avenue to the construction of 19F-MRI agents and theranostics, exploiting self-assembling supramolecular dendrimer chemistry.

Impact of the day/night cycle on functional connectome in ageing male and female mice.

To elucidate how time of day, sex, and age affect functional connectivity (FC) in mice, we aimed to examine whether the mouse functional connectome varied with the day/night cycle and whether it depended on sex and age. We explored C57Bl6/J mice (6♀ and 6♂) at mature age (5 ± 1 months) and middle-age (14 ± 1 months). Each mouse underwent Blood Oxygen-Level-Dependent (BOLD) resting-state functional MRI (rs-fMRI) on a 7T scanner at four different times of the day, two under the light condition and two under the dark condition. Data processing consisted of group independent component analysis (ICA) and region-level analysis using resting-state networks (RSNs) derived from literature. Linear mixed-effect models (LMEM) were used to assess the effects of sex, lighting condition and their interactions for each RSN obtained with group-ICA (RSNs-GICA) and six bilateral RSNs adapted from literature (RSNs-LIT). Our study highlighted new RSNs in mice related to day/night alternation in addition to other networks already reported in the literature. In mature mice, we found sex-related differences in brain activation only in one RSNs-GICA comprising the cortical, hippocampal, midbrain and cerebellar regions of the right hemisphere. In males, brain activity was significantly higher in the left hippocampus, the retrosplenial cortex, the superior colliculus, and the cerebellum regardless of lighting condition; consistent with the role of these structures in memory formation and integration, sleep, and sex-differences in memory processing. Experimental constraints limited the analysis to the impact of light/dark cycle on the RSNs for middle-aged females. We detected significant activation in the pineal gland during the dark condition, a finding in line with the nocturnal activity of this gland. For the analysis of RSNs-LIT, new variables "sexage" (sex and age combined) and "edges" (pairs of RSNs) were introduced. FC was calculated as the Pearson correlation between two RSNs. LMEM revealed no effect of sexage or lighting condition. The FC depended on the edges, but there were no interaction effects between sexage, lighting condition and edges. Interaction effects were detected between i) sex and lighting condition, with higher FC in males under the dark condition, ii) sexage and edges with higher FC in male brain regions related to vision, memory, and motor action. We conclude that time of day and sex should be taken into account when designing, analyzing, and interpreting functional imaging studies in rodents.

Quantitative MRI of Gd-DOTA Accumulation in the Mouse Brain After Intraperitoneal Administration: Validation by Mass Spectrometry.

BACKGROUND: In mice, intraperitoneal (ip) contrast agent (CA) administration is convenient for mapping microvascular parameters over a long-time window. However, continuous quantitative MRI of CA accumulation in brain over hours is still missing. PURPOSE: To validate a quantitative time-resolved MRI technique for mapping the CA kinetics in brain upon ip administration. STUDY TYPE: Prospective, animal model. SPECIMEN: 25 C57Bl/6JRj mice underwent MRI. FIELD STRENGTH/SEQUENCE: 7-T, gradient echo sequence. ASSESSMENT: Gd-DOTA concentration was monitored by MRI (25 s/repetition) over 135 minutes with (N = 15) and without (N = 10) ip mannitol challenge (5 g/kg). After the final repetition, the brains were sampled to quantify gadolinium by mass spectrometry (MS). Upon manual brain segmentation, the average gadolinium concentration was compared with the MS quantification in transcardially perfused (N = 20) and unperfused (N = 5) mice. Precontrast T1 -maps were acquired in 8 of 25 mice. STATISTICAL TESTS: One-tailed Spearman and Pearson correlation between gadolinium quantification by MRI and by MS, D'Agostino-Pearson test for normal distribution, Bland-Altman analysis to evaluate the agreement between MRI and MS. Significance was set at P-value <0.05. RESULTS: MRI showed that ip administered CA reached the blood compartment (>5 mM) within 10 minutes and accumulated continuously for 2 hours in cerebrospinal fluid (>1 mM) and in brain tissue. The MRI-derived concentration maps showed interindividual differences in CA accumulation (from 0.47 to 0.81 mM at 2 hours) with a consistent distribution resembling the pathways of the glymphatic system. The average in-vivo brain concentration 2 hours post-CA administration correlated significantly (r = 0.8206) with the brain gadolinium quantification by MS for N = 21 paired observations available. DATA CONCLUSION: The presented experimental and imaging protocol may be convenient for monitoring the spatiotemporal pattern of CA uptake and clearance in the mouse brain over 2 hours. The quantification of the CA from the MRI signal in brain is corroborated by MS. EVIDENCE LEVEL: N/A TECHNICAL EFFICACY: Stage 1.

MALDI-TOF MS as an innovative tool for detection of Plasmodium parasites in Anopheles mosquitoes.

BACKGROUND: Malaria is still a major public health issue worldwide, and one of the best approaches to fight the disease remains vector control. The current methods for mosquito identification include morphological methods that are generally time-consuming and require expertise, and molecular methods that require laboratory facilities with relatively expensive running costs. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology, routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of the present study was to assess whether MALDI-TOF MS could successfully distinguish Anopheles stephensi mosquitoes according to their Plasmodium infection status. METHODS: C57BL/6 mice experimentally infected with Plasmodium berghei were exposed to An. stephensi bites. For the determination of An. stephensi infection status, mosquito cephalothoraxes were dissected and submitted to mass spectrometry analyses and DNA amplification for molecular analysis. Spectra were grouped according to mosquitoes' infection status and spectra quality was validated based on intensity and reproducibility within each group. The in-lab MALDI-TOF MS arthropod reference spectra database, upgraded with representative spectra from both groups (infected/non-infected), was subsequently queried blindly with cephalothorax spectra from specimens of both groups. RESULTS: The MALDI TOF MS profiles generated from protein extracts prepared from the cephalothorax of An. stephensi allowed distinction between infected and uninfected mosquitoes. Correct classification was obtained in blind test analysis for (79/80) 98.75% of all mosquitoes tested. Only one of 80 specimens, an infected mosquito, was misclassified in the blind test analysis. CONCLUSIONS: Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry appears to be a promising, rapid and reliable tool for the epidemiological surveillance of Anopheles vectors, including their identification and their infection status.

Modular Self-Assembling Dendrimer Nanosystems for Magnetic Resonance and Multimodality Imaging of Tumors.

Bioimaging is a powerful tool for diagnosing tumors but remains limited in terms of sensitivity and specificity. Nanotechnology-based imaging probes able to accommodate abundant imaging units with different imaging modalities are particularly promising for overcoming these limitations. In addition, the nanosized imaging agents can specifically increase the contrast of tumors by exploiting the enhanced permeability and retention effect. A proof-of-concept study is performed on pancreatic cancer to demonstrate the use of modular amphiphilic dendrimer-based nanoprobes for magnetic resonance (MR) imaging (MRI) or MR/near-infrared fluorescence (NIRF) multimodality imaging. Specifically, the self-assembly of an amphiphilic dendrimer bearing multiple Gd3+ units at its terminals, generates a nanomicellar agent exhibiting favorable relaxivity for MRI with a good safety profile. MRI reveals an up to two-fold higher contrast enhancement in tumors than in normal muscle. Encapsulating the NIRF dye within the core of the nanoprobe yields an MR/NIRF bimodal imaging agent for tumor detection that is efficient both for MRI, at Gd3+ concentrations 1/10 the standard clinical dose, and for NIRF imaging, allowing over two-fold stronger fluorescence intensities. These self-assembling dendrimer nanosystems thus constitute effective probes for MRI and MR/NIRF multimodality imaging, offering a promising nanotechnology platform for elaborating multimodality imaging probes in biomedical applications.

Olesoxime accelerates myelination and promotes repair in models of demyelination.

OBJECTIVE: Multiple sclerosis is a neurodegenerative disease characterized by episodes of immune attack of oligodendrocytes leading to demyelination and progressive functional deficit. One therapeutic strategy to address disease progression could consist in stimulating the spontaneous regenerative process observed in some patients. Myelin regeneration requires endogenous oligodendrocyte progenitor migration and activation of the myelination program at the lesion site. In this study, we have tested the ability of olesoxime, a neuroprotective and neuroregenerative agent, to promote remyelination in the rodent central nervous system in vivo. METHODS: The effect of olesoxime on oligodendrocyte progenitor cell (OPC) differentiation and myelin synthesis was tested directly in organotypic slice cultures and OPC-neuron cocultures. Using naive animals and different mouse models of demyelination, we morphologically and functionally assessed the effect of the compound on myelination in vivo. RESULTS: Olesoxime accelerated oligodendrocyte maturation and enhanced myelination in vitro and in vivo in naive animals during development and also in the adult brain without affecting oligodendrocyte survival or proliferation. In mouse models of demyelination and remyelination, olesoxime favored the repair process, promoting myelin formation with consequent functional improvement. INTERPRETATION: Our observations support the strategy of promoting oligodendrocyte maturation and myelin synthesis to enhance myelin repair and functional recovery. We also provide proof of concept that olesoxime could be useful for the treatment of demyelinating diseases.

In the eye of experimental cerebral malaria.

Cerebral malaria is the most severe complication of Plasmodium falciparum infection, accounting for 1 million deaths per year. We characterized the murine disease using in vivo magnetic resonance imaging (MRI) at 4.7 T, proving that ischemic edema is responsible for fatality. The aim of the present study was to identify early markers of experimental cerebral malaria using very high field conventional MRI (11.75 T). CBA/J mice infected with Plasmodium berghei ANKA were observed at an early stage of the disease, before the onset of detectable brain swelling and at the most acute stage of cerebral malaria. Herein, we report the first detection of damage to the optic and trigeminal nerves on T(2)-weighted MRI. The trigeminal nerves appeared hypointense, with significantly reduced diameter and cross-sectional area. The optic nerves were hypointense and often not visible. In addition, the internerve distance between the optic nerves was significantly and progressively reduced between the early and severest stages. Cranial nerve injury was the earliest anatomic hallmark of the disease, visible before brain edema became detectable. Thus, cranial nerve damage may manifest in neurologic signs, which may assist in the early recognition of cerebral malaria.

Clinical and Preclinical Imaging of Hepatosplenic Schistosomiasis.

Schistosomiasis, a neglected tropical disease, is a major cause of chronic morbidity and disability, and premature death. The hepatosplenic form of schistosomiasis is characterized by hepatosplenomegaly, liver fibrosis, portal hypertension, and esophageal varices, whose rupture may cause bleeding and death. We review currently available abdominal imaging modalities and describe their basic principles, strengths, weaknesses, and usefulness in the assessment of hepatosplenic schistosomiasis (HSS). Advanced imaging methods are presented that could be of interest for hepatosplenic schistosomiasis evaluation by yielding morphological, functional, and molecular parameters of disease progression. We also provide a comprehensive view of preclinical imaging studies and current research objectives such as parasite visualization in hosts, follow-up of the host's immune response, and development of noninvasive quantitative methods for liver fibrosis assessment.

Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.

The first in vivo magnetic resonance study of experimental cerebral malaria is presented. Cerebral involvement is a lethal complication of malaria. To explore the brain of susceptible mice infected with Plasmodium berghei ANKA, multimodal magnetic resonance techniques were applied (imaging, diffusion, perfusion, angiography, spectroscopy). They reveal vascular damage including blood-brain barrier disruption and hemorrhages attributable to inflammatory processes. We provide the first in vivo demonstration for blood-brain barrier breakdown in cerebral malaria. Major edema formation as well as reduced brain perfusion was detected and is accompanied by an ischemic metabolic profile with reduction of high-energy phosphates and elevated brain lactate. In addition, angiography supplies compelling evidence for major hemodynamics dysfunction. Actually, edema further worsens ischemia by compressing cerebral arteries, which subsequently leads to a collapse of the blood flow that ultimately represents the cause of death. These findings demonstrate the coexistence of inflammatory and ischemic lesions and prove the preponderant role of edema in the fatal outcome of experimental cerebral malaria. They improve our understanding of the pathogenesis of cerebral malaria and may provide the necessary noninvasive surrogate markers for quantitative monitoring of treatment.

Magnetic resonance imaging and the detection of fetal brain anomalies, injury, and physiologic adaptations.

PURPOSE OF REVIEW: Magnetic resonance imaging is playing an increasingly prominent role in depicting brain maturation, especially gyral formation that follows a temporospatial pattern, and in detecting developmental abnormalities of the cortex and other brain sectors. Knowledge of the technical advantages and limitations of in-utero magnetic resonance imaging techniques, relative to those of the postnatal period, is essential to optimize magnetic resonance sequences for early diagnosis. This includes an understanding of the changes in both brain anatomy and magnetic resonance signals that occur with an increase in gestational age. RECENT FINDINGS: Magnetic resonance imaging has evolved has an important adjunct in the diagnosis of brain malformations, particularly in the late-second or third trimester. Noxious conditions elicit more of a chronic rather than acute response in the fetal brain, which differs from that observed postnatally. Clinical applications of proton magnetic resonance spectroscopy may help elucidate fetal brain maturation and its abnormalities from a metabolic point of view. SUMMARY: Indications for fetal brain magnetic resonance imaging have increased because of improvements in magnetic resonance techniques and the ability to detect subtle changes within the cerebral parenchyma, especially in fetuses at increased risk of brain damage.

MRS of normal and impaired fetal brain development.

Cerebral maturation in the human fetal brain was investigated by in utero localized proton magnetic resonance spectroscopy (MRS). Spectra were acquired on a clinical MR system operating at 1.5 T. Body phased array coils (four coils) were used in combination with spinal coils (two coils). The size of the nominal volume of interest (VOI) was 4.5 cm(3) (20 mm x 15 mm x 15 mm). The MRS acquisitions were performed using a spin echo sequence at short and long echo times (TE = 30 ms and 135 ms) with a VOI located within the cerebral hemisphere at the level of the centrum semiovale. A significant reduction in myo-inositol and choline and an increase in N-acetylaspartate were observed with progressive age. The normal MR spectroscopy data reported here will help to determine whether brain metabolism is altered, especially when subtle anatomic changes are observed on conventional images. Some examples of impaired fetal brain development studied by MRS are illustrated.

Creatine, Glutamine plus Glutamate, and Macromolecules Are Decreased in the Central White Matter of Premature Neonates around Term.

Preterm birth represents a high risk of neurodevelopmental disabilities when associated with white-matter damage. Recent studies have reported cognitive deficits in children born preterm without brain injury on MRI at term-equivalent age. Understanding the microstructural and metabolic underpinnings of these deficits is essential for their early detection. Here, we used diffusion-weighted imaging and single-voxel 1H magnetic resonance spectroscopy (MRS) to compare brain maturation at term-equivalent age in premature neonates with no evidence of white matter injury on conventional MRI except diffuse excessive high-signal intensity, and normal term neonates. Thirty-two infants, 16 term neonates (mean post-conceptional age at scan: 39.8±1 weeks) and 16 premature neonates (mean gestational age at birth: 29.1±2 weeks, mean post-conceptional age at scan: 39.2±1 weeks) were investigated. The MRI/MRS protocol performed at 1.5T involved diffusion-weighted MRI and localized 1H-MRS with the Point RESolved Spectroscopy (PRESS) sequence. Preterm neonates showed significantly higher ADC values in the temporal white matter (P<0.05), the occipital white matter (P<0.005) and the thalamus (P<0.05). The proton spectrum of the centrum semiovale was characterized by significantly lower taurine/H2O and macromolecules/H2O ratios (P<0.05) at a TE of 30 ms, and reduced (creatine+phosphocreatine)/H2O and (glutamine+glutamate)/H2O ratios (P<0.05) at a TE of 135 ms in the preterm neonates than in full-term neonates. Our findings indicate that premature neonates with normal conventional MRI present a delay in brain maturation affecting the white matter and the thalamus. Their brain metabolic profile is characterized by lower levels of creatine, glutamine plus glutamate, and macromolecules in the centrum semiovale, a finding suggesting altered energy metabolism and protein synthesis.

In Vivo MRI Assessment of Hepatic and Splenic Disease in a Murine Model of Schistosomiasis [corrected].

BACKGROUND: Schistosomiasis (or bilharzia), a major parasitic disease, affects more than 260 million people worldwide. In chronic cases of intestinal schistosomiasis caused by trematodes of the Schistosoma genus, hepatic fibrosis develops as a host immune response to the helminth eggs, followed by potentially lethal portal hypertension. In this study, we characterized hepatic and splenic features of a murine model of intestinal schistosomiasis using in vivo magnetic resonance imaging (MRI) and evaluated the transverse relaxation time T2 as a non-invasive imaging biomarker for monitoring hepatic fibrogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CBA/J mice were imaged at 11.75 T two, six and ten weeks after percutaneous infection with Schistosoma mansoni. In vivo imaging studies were completed with histology at the last two time points. Anatomical MRI allowed detection of typical manifestations of the intestinal disease such as significant hepato- and splenomegaly, and dilation of the portal vein as early as six weeks, with further aggravation at 10 weeks after infection. Liver multifocal lesions observed by MRI in infected animals at 10 weeks post infection corresponded to granulomatous inflammation and intergranulomatous fibrosis with METAVIR scores up to A2F2. While most healthy hepatic tissue showed T2 values below 14 ms, these lesions were characterized by a T2 greater than 16 ms. The area fraction of increased T2 correlated (rS = 0.83) with the area fraction of Sirius Red stained collagen in histological sections. A continuous liver T2* decrease was also measured while brown pigments in macrophages were detected at histology. These findings suggest accumulation of hematin in infected livers. CONCLUSIONS/SIGNIFICANCE: Our multiparametric MRI approach confirms that this murine model replicates hepatic and splenic manifestations of human intestinal schistosomiasis. Quantitative T2 mapping proved sensitive to assess liver fibrogenesis non-invasively and may therefore constitute an objective imaging biomarker for treatment monitoring in diseases involving hepatic fibrosis.

A pancreatic tumor-specific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination.

Oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase (pBSDL) appear during human pancreatic oncogenesis and are detected by themonoclonal antibody J28 (mAbJ28). We aimed to identify murine counterparts onpancreatic ductal adenocarcinoma (PDAC) cells and tissue and investigate the potential of dendritic cells (DC) loaded with this unique pancreatic tumor antigen to promote immunotherapy in preclinical trials. Pathological BSDLs purified from pancreatic juices of patients with PDAC were cleaved to generate glycosylated C-terminal moieties (C-ter) containing mAbJ28-reactive glycoepitopes. Immunoreactivity of the murine PDAC line Panc02 and tumor tissue to mAbJ28 was detected by immunohistochemistry and flow cytometry. C-ter-J28+ immunization promoted Th1-dominated immune responses. In vitro C-ter-J28+-loaded DCskewed CD3+ T-cells toward Th1 polarization. C-ter-J28+-DC-vaccinations selectively enhanced cell immunoreactivity to Panc02, as demonstrated by CD4+- and CD8+-T-cell activation, increased percentages of CD4+- and CD8+-T-cells and NK1.1+ cells expressing granzyme B, and T-cell cytotoxicity. Prophylactic and therapeutic C-ter-J28+-DC-vaccinations reduced ectopic Panc02-tumor growth, provided long-lasting protection from Panc02-tumor development in 100% of micebut not from melanoma, and attenuated progression of orthotopic tumors as revealed by MRI. Thusmurine DC loaded with pancreatic tumor-specific glycoepitope C-ter-J28+ induce efficient anticancer adaptive immunity and represent a potential adjuvant therapy for patients afflicted with PDAC.

MR imaging and MR spectroscopy in rhizomelic chondrodysplasia punctata.

A case of rhizomelic chondrodysplasia punctata was investigated with MR imaging of the brain and hydrogen-1 MR spectroscopy of the brain and blood. Areas with abnormal signal hyperintensity on T2-weighted images or hypointensity on T1-weighted images were detected in the subcortical white matter. MR spectroscopy of the brain showed that normal-appearing white matter was characterized by increased levels of mobile lipids and myo-inositol, reduced levels of choline, and the presence of acetate. The importance of these metabolic anomalies is correlated to the deficiency in plasmalogen biosynthesis.

Melanin-concentrating hormone regulates beat frequency of ependymal cilia and ventricular volume.

Ependymal cell cilia help move cerebrospinal fluid through the cerebral ventricles, but the regulation of their beat frequency remains unclear. Using in vitro, high-speed video microscopy and in vivo magnetic resonance imaging in mice, we found that the metabolic peptide melanin-concentrating hormone (MCH) positively controlled cilia beat frequency, specifically in the ventral third ventricle, whereas a lack of MCH receptor provoked a ventricular size increase.

Deletion of TRAAK potassium channel affects brain metabolism and protects against ischemia.

Cerebral stroke is a worldwide leading cause of disability. The two-pore domain K⁺ channels identified as background channels are involved in many functions in brain under physiological and pathological conditions. We addressed the hypothesis that TRAAK, a mechano-gated and lipid-sensitive two-pore domain K⁺ channel, is involved in the pathophysiology of brain ischemia. We studied the effects of TRAAK deletion on brain morphology and metabolism under physiological conditions, and during temporary focal cerebral ischemia in Traak⁻/⁻ mice using a combination of in vivo magnetic resonance imaging (MRI) techniques and multinuclear magnetic resonance spectroscopy (MRS) methods. We provide the first in vivo evidence establishing a link between TRAAK and neurometabolism. Under physiological conditions, Traak⁻/⁻ mice showed a particular metabolic phenotype characterized by higher levels of taurine and myo-inositol than Traak⁺/⁺ mice. Upon ischemia, Traak⁻/⁻ mice had a smaller infarcted volume, with lower contribution of cellular edema than Traak⁺/⁺ mice. Moreover, brain microcirculation was less damaged, and brain metabolism and pH were preserved. Our results show that expression of TRAAK strongly influences tissue levels of organic osmolytes. Traak⁻/⁻ mice resilience to cellular edema under ischemia appears related to their physiologically high levels of myo-inositol and of taurine, an aminoacid involved in the modulation of mitochondrial activity and cell death. The beneficial effects of TRAAK deletion designate this channel as a promising pharmacological target for the treatment against stroke.