Longitudinal evaluation of neuroinflammation and oxidative stress in a mouse model of Alzheimer disease using positron emission tomography.

BACKGROUND: Validation of new biomarkers of Alzheimer disease (AD) is crucial for the successful development and implementation of treatment strategies. Additional to traditional AT(N) biomarkers, neuroinflammation biomarkers, such as translocator protein (TSPO) and cystine/glutamine antiporter system (xc-), could be considered when assessing AD progression. Herein, we report the longitudinal investigation of [18F]DPA-714 and [18F]FSPG for their ability to detect TSPO and xc- biomarkers, respectively, in the 5xFAD mouse model for AD. METHODS: Expression of TSPO and xc- system was assessed longitudinally (2-12 months of age) on 5xFAD mice and their respective controls by positron emission tomography (PET) imaging using radioligands [18F]DPA-714 and [18F]FSPG. In parallel, in the same mice, amyloid-ß plaque deposition was assessed with the amyloid PET radiotracer [18F]florbetaben. In vivo findings were correlated to ex vivo immunofluorescence staining of TSPO and xc- in microglia/macrophages and astrocytes on brain slices. Physiological changes of the brain tissue were assessed by magnetic resonance imaging (MRI) in 12-month-old mice. RESULTS: PET studies showed a significant increase in the uptake of [18F]DPA-714 and [18F]FSPG in the cortex, hippocampus, and thalamus in 5xFAD but not in WT mice over time. The results correlate with Aß plaque deposition. Ex vivo staining confirmed higher TSPO overexpression in both, microglia/macrophages and astrocytes, and overexpression of xc- in non-glial cells of 5xFAD mice. Additionally, the results show that Aß plaques were surrounded by microglia/macrophages overexpressing TSPO. MRI studies showed significant tissue shrinkage and microstructural alterations in 5xFAD mice compared to controls. CONCLUSIONS: TSPO and xc- overexpression can be assessed by [18F]DPA-714 and [18F]FSPG, respectively, and correlate with the level of Aß plaque deposition obtained with a PET amyloid tracer. These results position the two tracers as promising imaging tools for the evaluation of disease progression. Longitudinal in vivo study in the 5xFAD mouse model shows that TSPO and oxidative stress assessment through [18F]DPA-714 and [18F]FSPG-PET imaging, respectively, could serve as a potential tool for the evaluation of Alzheimer disease progression.

Assessing the Potential of Molecular Imaging for Myelin Quantification in Organotypic Cultures.

Ex vivo models for the noninvasive study of myelin-related diseases represent an essential tool to understand the mechanisms of diseases and develop therapies against them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes to quantify myelin in organotypic cultures. Methods: MRI testing was used to image mouse cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white (WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelin-targeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions: With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable for this goal, due to unspecific binding to tissues.

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease.

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aß) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aß plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aß at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aß plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aß accumulation, suggesting that BChE is a promising early biomarker for incipient AD.

Gut Microbiota Changes in Experimental Autoimmune Encephalomyelitis and Cuprizone Mice Models.

Multiple sclerosis (MS) is a chronic and neurodegenerative disease of the central nervous system (CNS) characterized by the immune mediated attack on axons and the subsequent demyelination. There is growing evidence that the gut microbiota of MS patients is altered; however, the connection between demyelination events and changes in the gut microbiota has not been determined. The objective of the current work was to characterize the microbial dysbiosis in two murine demyelinating models and to study the correlation between them. Concurrently, their suitability as predictors of microbial changes in MS patients was assessed. To this purpose, experimental autoimmune encephalomyelitis (EAE) and cuprizone (CPZ) models were induced in C57BL/6 mice that were monitored for 4 and 9 weeks, respectively. Fecal samples were collected during disease progression. Motor skill performance was evaluated by EAE scale measurement in EAE mice and demyelination by magnetic resonance imaging (MRI) in CPZ ones. EAE and CPZ mice revealed drastic microbial changes according to disease progression, adding a new layer of complexity to the understanding of demyelination and remyelination processes. Besides, the reported microbial changes replicate most of the characteristics that define the potential dysbiosis in MS patients. The controlled environment and stable diet that animals have in research centers offer an exceptional scenario to modify animal's microbiota and provide opportunities to study host microbiota interplay with restrained conditions not achievable in human studies. Nevertheless the slight differences from murine model's and patient's microbiota should be considered in the design of studies aiming to modulate the microbiota.

Functional rewiring across spinal injuries via biomimetic nanofiber scaffolds.

The regrowth of severed axons is fundamental to reestablish motor control after spinal-cord injury (SCI). Ongoing efforts to promote axonal regeneration after SCI have involved multiple strategies that have been only partially successful. Our study introduces an artificial carbon-nanotube based scaffold that, once implanted in SCI rats, improves motor function recovery. Confocal microscopy analysis plus fiber tracking by magnetic resonance imaging and neurotracer labeling of long-distance corticospinal axons suggest that recovery might be partly attributable to successful crossing of the lesion site by regenerating fibers. Since manipulating SCI microenvironment properties, such as mechanical and electrical ones, may promote biological responses, we propose this artificial scaffold as a prototype to exploit the physics governing spinal regenerative plasticity.

Aging Reduces the Functional Brain Networks Strength-a Resting State fMRI Study of Healthy Mouse Brain.

Resting-state functional magnetic resonance imaging (rsfMRI) is increasingly used to unravel the functional neuronal networks in health and disease. In particular, this technique of simultaneously probing the whole brain has found high interest in monitoring brain wide effects of cerebral disease and in evaluating therapeutic strategies. Such studies, applied in preclinical experimental mouse models, often require long-term observations. In particular during regeneration studies, easily several months of continuous monitoring are required to detect functional improvements. These long periods of following the functional deficits during disease evolution as well as the functional recoveries during therapeutic interventions represent a substantial fraction of the life span of the experimental animals. We have therefore aimed to decipher the role of healthy aging alone for changes in functional neuronal networks in mice, from developmental adolescence via adulthood to progressing aging. For this purpose, four different groups of C57Bl6 mice of varying age between 2 and 13 months were studied twice with 4 weeks separation using resting state fMRI at 9.4T. Dedicated data analysis including both Independent Component Analysis (ICA) followed by seed-based connectivity matrix compilation resulted in an inverse U-shape curve of functional connectivity (FC) strength in both the sensorimotor and default mode network (DMN). This inverse U-shape pattern presented a distinct maximum of FC strength at 8-9 months of age, followed by a continuous decrease during later aging phases. At progressed aging at 12-13 months, the reduction of connectivity strength varied between 25% and 70% with most connectivities showing a reduction in strength by approximately 50%. We recommend that these substantial age-dependent changes in FC strength must be considered in future longitudinal studies to discriminate focused disease-based functional deficits and therapy-related functional improvements from underlying independent age effects.