Alexandra Petiet

Gadolinium-staining reveals amyloid plaques in the brain of Alzheimer's transgenic mice

Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimers disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by μMRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.

Micro-MRI Study of Cerebral Aging: Ex Vivo Detection of Hippocampal Subfield Reorganization, Microhemorrhages and Amyloid Plaques in Mouse Lemur Primates

Mouse lemurs are non-human primate models of cerebral aging and neurodegeneration. Much smaller than other primates, they recapitulate numerous features of human brain aging, including progressive cerebral atrophy and correlation between regional atrophy and cognitive impairments. Characterization of brain atrophy in mouse lemurs has been done by MRI measures of regional CSF volume and by MRI measures of regional atrophy. Here, we further characterize mouse lemur brain aging using ex vivo MR microscopy (31 µm in-plane resolution). First, we performed a non-biased, direct volumetric quantification of dentate gyrus and extended Ammons horn. We show that both dentate gyrus and Ammons horn undergo an age-related reorganization leading to a growth of the dentate gyrus and an atrophy of the Ammons horn, even in the absence of global hippocampal atrophy. Second, on these first MR microscopic images of the mouse lemur brain, we depicted cortical and hippocampal hypointense spots. We demonstrated that their incidence increases with aging and that they correspond either to amyloid deposits or to cerebral microhemorrhages.

In Vivo Imaging Biomarkers in Mouse Models of Alzheimer's Disease: Are We Lost in Translation or Breaking Through?

Identification of biomarkers of Alzheimers Disease (AD) is a critical priority to efficiently diagnose the patients, to stage the progression of neurodegeneration in living subjects, and to assess the effects of disease-modifier treatments. This paper addresses the development and usefulness of preclinical neuroimaging biomarkers of AD. It is today possible to image in vivo the brain of small rodents at high resolution and to detect the occurrence of macroscopic/microscopic lesions in these species, as well as of functional alterations reminiscent of AD pathology. We will outline three different types of imaging biomarkers that can be used in AD mouse models: biomarkers with clear translational potential, biomarkers that can serve as in vivo readouts (in particular in the context of drug discovery) exclusively for preclinical research, and finally biomarkers that constitute new tools for fundamental research on AD physiopathogeny.

Models of neurodegenerative disease – Alzheimer’s Anatomical and amyloid plaque imaging

Alzheimers disease (AD) is an important social and economic issue for our societies. The development of therapeutics against this severe dementia requires assessing the effects of new drugs in animal models thanks to dedicated biomarkers. According to the amyloid cascade hypothesis, β-amyloid deposits are at the origin of most of the lesions associated with AD. These extracellular deposits are therefore one of the main targets in therapeutical strategies. Aβ peptides can be revealed histologically with specific dyes or antibodies, or by magnetic resonance microscopy (μMRI) that uses their association with iron as a source of signal. The microscopic size of the lesions necessitates the development of specific imaging protocols. Most protocols use T (2)-weighted sequences that reveal the aggregates as hypointense spots. This chapter describes histological methods that reveal amyloid plaques with specific stains and MR-imaging protocols for in vivo and ex vivo MR imaging of AD mice.

Multi-Spherical Diffusion MRI: Exploring Diffusion Time Using Signal Sparsity

Effective representation of the diffusion signals dependence on diffusion time is a sought-after, yet still unsolved, challenge in diffusion MRI (dMRI). We propose a functional basis approach that is specifically designed to represent the dMRI signal in this four-dimensional space – varying over gradient strength, direction and diffusion time. In particular , we provide regularization tools imposing signal sparsity and signal smoothness to drastically reduce the number of measurements we need to probe the properties of this multi-spherical space. We illustrate a novel application of our approach, which is the estimation of time-dependent q-space indices, on both synthetic data generated using Monte-Carlo simulations and in vivo data acquired from a C57Bl6 wild-type mouse. In both cases, we find that our regularization approach stabilizes the signal fit and index estimation as we remove samples, which may bring multi-spherical diffusion MRI within the reach of clinical application.

In vivo amyloid plaque detection using contrast-enhanced magnetic resonance microscopy in transgenic mice

regions of interest in dorsal hippocampus (Fig. 1). Images were acquired using rapid acquisition with relaxation enhancement (RARE) and echo-train multi-slice-multi-echo (MSME) sequences, from which T1 and T2 maps were generated, respectively; and a fast imaging with steady-state precession (FISP) protocol, from which both T1 and T2 maps were generated. Data from manually-selected regions of interest were analyzed using software scripts in IDL language (ITTVIS). Oneor two-way analyses of variance (ANOVA) with Fischer’s post-test were used to statistically assess the data. Tissue from the animals was examined for markers of astrogliosis and microglial activation. Results: T1 in hippocampus and cortex (p<0.01, Fig. 2) were significantly increased with age whilst T2 in hippocampus (p<0.05) and cortex (p<0.01, Fig. 3) were significantly decreased with age. Treatment with rosiglitazone significantly attenuated the age-related T1 increase in both regions (p<0.05, Fig. 2) and it increased T2 relaxation time in hippocampus of aged, but not young rats (p < 0.05, Fig. 3). Conclusions: The data demonstrate an age-related increase in T1 in hippocampus and cortex, which is attenuated by rosiglitazone; this correlates with astrocytosis (Fig. 4). We also show a decrease in T2 relaxation time in hippocampus and cortex, which correlates with an increase in microglial activation (Fig. 5), and report that rosiglitazone-treatment of aged rats increases hippocampal T2.

Alterations of the nigrostriatal pathway in a 6-OHDA rat model of Parkinson’s disease evaluated with multimodal MRI

Parkinson’s disease is characterized by neurodegeneration of the dopaminergic neurons in the substantia nigra pars compacta. The 6-hydroxydopamine (6-OHDA) rat model has been used to study neurodegeneration in the nigro-striatal dopaminergic system. The goal of this study was to evaluate the reliability of diffusion MRI and resting-state functional MRI biomarkers in monitoring neurodegeneration in the 6-OHDA rat model assessed by quantitative histology. We performed a unilateral injection of 6-OHDA in the striatum of Sprague Dawley rats to produce retrograde degeneration of the dopamine neurons in the substantia nigra pars compacta. We carried out a longitudinal study with a multi-modal approach combining structural and functional MRI together with quantitative histological validation to follow the effects of the lesion. Functional and structural connectivity were assessed in the brain of 6-OHDA rats and sham rats (NaCl injection) at 3 and 6 weeks post-lesioning using resting-state functional MRI and diffusion-weighted. Our results showed (i) increased functional connectivity in ipsi- and contra-lesioned regions of the cortico-basal ganglia network pathway including the motor cortex, the globus pallidus, and the striatum regions at 3 weeks; (ii) increased fractional anisotropy (FA) in the ipsi- and contralateral striatum of the 6-OHDA group at 3 weeks, and increased axial diffusivity (AD) and mean diffusivity in the ipsilateral striatum at 6 weeks; (iii) a trend for increased FA in both substantia nigra of the 6-OHDA group at 3 weeks. Optical density measurements of tyrosine-hydroxylase (TH) staining of the striatum showed good correlations with the FA and AD measurements in the striatum. No correlations were found between the number of TH-stained dopaminergic neurons and MRI measurements in the substantia nigra. This study suggested that (i) FA and AD were reliable biomarkers to evaluate neurodegeneration in the cortico-basal ganglia network of the 6-OHDA model, (ii) diffusion MRI and resting-state functional MRI (rsfMRI) were not sensitive enough to detect changes in the substantia nigra in this model.

Non-Parametric GraphNet-Regularized Representation of dMRI in Space and Time

Highlightsq&tgr;‐dMRI signal representation without making biophysical assumptions.Non‐parametric, time‐dependent reconstruction of the diffusion signal and EAP.Effective GraphNet regularization to reduce q&tgr;‐DWI sampling requirements.Analytic estimation of time‐dependent q‐space index functions (q&tgr;‐indices).Good test‐retest reproducibility of q&tgr;‐index trends in two wild‐type mice. Graphical abstract Figure. No Caption available. Abstract Effective representation of the four‐dimensional diffusion MRI signal – varying over three‐dimensional q‐space and diffusion time &tgr; – is a sought‐after and still unsolved challenge in diffusion MRI (dMRI). We propose a functional basis approach that is specifically designed to represent the dMRI signal in this q&tgr;‐space. Following recent terminology, we refer to our q&tgr;‐functional basis as “q&tgr;‐dMRI”. q&tgr;‐dMRI can be seen as a time‐dependent realization of q‐space imaging by Paul Callaghan and colleagues. We use GraphNet regularization – imposing both signal smoothness and sparsity – to drastically reduce the number of diffusion‐weighted images (DWIs) that is needed to represent the dMRI signal in the q&tgr;‐space. As the main contribution, q&tgr;‐dMRI provides the framework to – without making biophysical assumptions – represent the q&tgr;‐space signal and estimate time‐dependent q‐space indices (q&tgr;‐indices), providing a new means for studying diffusion in nervous tissue. We validate our method on both in‐silico generated data using Monte–Carlo simulations and an in‐vivo test‐retest study of two C57Bl6 wild‐type mice, where we found good reproducibility of estimated q&tgr;‐index values and trends. In the hopes of opening up new &tgr;‐dependent venues of studying nervous tissues, q&tgr;‐dMRI is the first of its kind in being specifically designed to provide open interpretation of the q&tgr;‐diffusion signal.

O2-2 Détection par IRM haute résolution de plaques amyloïdes chez un primate : Le microcèbe

Introduction Le developpement de biomarqueurs pour diagnostiquer et suivre l’evolution de la maladie d’Alzheimer (MA) est un enjeu majeur pour la recherche. Chez les modeles murins de la MA, l’imagerie par RMN permet de detecter les plaques amyloides [1 ; 2]. Ces lesions apparaissent comme des spots hypointenses sur des images ponderees en T2*. Avant d’appliquer cette technique en clinique, il est necessaire de la developper chez un modele phylogenetiquement proche de l’homme. Nous avons donc cherche a mettre en evidence ces lesions par IRM chez un Primate : le microcebe murin [3]. Materiel et methodes Les cerveaux de 12 microcebes âges de 1 a 10 ans (n=6 jeunes et n=6 âges (âges moyens=2,5 et 7,5 ans respectivement)) ont ete incubes dans une solution de Gadolinium 2,5 mmol.l-1 (Dotarem® (Guerbet) dilue au 200eme) [4]. Des images ponderees en T2* ont ete enregistrees sur un spectrometre Clinique de 7T (Siemens, gradient 80mT/m, TR/TE=200/20 ms ; temps d’acquisition=5h ; resolution=(31×31×120)μm3). Les images ont ete analysees grâce au logiciel Anatomist. Une etude immunohistologique des cerveaux a ete realisee pour detecter l’amyloidose (coupes de 40μm, Ac 4G8(SIGNET)). Resultats Des lesions hypointenses ont ete detectees dans le cortex et l’hippocampe des microcebes. Le nombre de ces lesions etait plus important chez les animaux âges par rapport aux jeunes (Moyenne=4,83 et 0,17 chez les vieux et les jeunes ; U=0.5 ; p Conclusion L’IRM haute resolution permet de detecter des depots amyloides chez les microcebes. Les spots hypointenses ne correspondant pas aux plaques amyloides pourraient etre des alterations vasculaires. Une correlation entre le nombre de lesions hypointenses et l’âge a aussi ete mise en evidence. L’IRM permet donc de detecter des biomarqueurs du vieillissement cerebral chez les microcebes. Remerciements Association France-Alzheimer, National Institute on Aging (R01-AG020197).

P2b-16 Evaluation in-vivo par IRM des effets toxiques d’une immunothérapie anti-Alzheimer chez le primate microcèbe murin

Introduction L’immunotherapie anti-amyloide est une strategie therapeutique mise au point pour lutter contre la maladie d’Alzheimer (MA). Elle est basee sur l’induction d’une reponse immunitaire suite a l’injection de peptide amyloide β1-42 ou de ses derives. Ce traitement reduit la charge amyloide chez des modeles murins transgeniques (1). Cependant des cas d’encephalomyelites observes chez des patients humains vaccines avec le peptide Aβ1-42 montrent les limites de cette strategie (2). De nouvelles approches tentent de reduire les effets toxiques (inflammation et microhemorragies) lies a l’immunisation. La vaccination de souris transgeniques, modeles de la MA avec le peptide K6Aβ1-30 (un derive de l’Aβ1-42) entraIne une diminution des plaques amyloides et des deficits cognitifs sans augmenter les risques de microhemorragies (3). Notre etude evalue, grâce a un suivi IRM, les effets toxiques d’immunotherapie avec le K6Aβ1-30 et l’Aβ1-42 chez un modele primate, le microcebe murin. Materiel/methode Des microcebes âges de 5,5 a 6,5 ans ont ete vaccines avec les peptides K6Aβ1-30 (n=4) et Aβ1-42 (n=4). Des images RMN, 3D, ponderees en T2 (TR/TE= 2500/69 ; resolution= (234μm)3) et T2* (TR/TE= 40/8 ; resolution= (234μm)3) ont ete enregistrees a 7Tesla (Pharmascan-Bruker) avant immunisation puis 2 mois apres. Lors des sequences T2*, les animaux respiraient du carbogene (95% O2 - 5% CO2) afin de reduire le signal du sang circulant. Les zones hypointenses detectees sur ces images sont susceptibles d’etre des microhemorragies. Le volume global de ces zones a ete evalue grâce a un comptage manuel des voxels sur l’ensemble des coupes coronales du cerveau de l’animal. Resultats Apres deux mois d’immunisation aucun animal ne developpait de lesions inflammatoires detectees en IRM. Des foyers hypointenses etaient plus etendus apres immunisation (volumes globaux de 1.02±0.23mm3(avant) contre 1.25±0.23mm3(apres) F (1,6)=9.5 ; p<0.05). L’etendue des zones hypointenses n’etait pas significativement differentes pour les deux immunotherapies testees. Conclusion Cette etude preliminaire montre la possibilite d’evaluer des traitements anti-Alzheimer chez le primate microcebe murin âge. Elle demontre l’absence de lesions inflammatoires severes deux mois post-injection. Remerciements Region Martinique, Association France- Alzheimer, National Institute on Aging (R01-AG020197).