Glutamate Imaging of Mouse Models of Neurodegeneration

Malfunctions in the glutamatergic system of the central nervous system have been implicated in neurodegenerative diseases such as Alzheimer’s disease (AD), tauopathies, and Parkinson’s disease (PD). A non-invasive measurement of glutamate would enhance our understanding of neurodegenerative processes and potentially facilitate early diagnosis. The current method for measuring glutamate in vivo is proton magnetic resonance spectroscopy (1HMRS) although it has poor spatial resolution and weak sensitivity to glutamate changes. The primary objective of this thesis was to measure pathology induced changes in glutamate levels in mouse models of neurodegeneration using a novel magnetic resonance imaging technique, glutamate chemical exchange saturation transfer (GluCEST) imaging. Several studies were performed in three mouse models of neurodegeneration: the APP-PS1 transgenic model of amyloid-beta pathology of AD, the PS19 transgenic model of tau pathology, and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Glutamate levels derived from GluCEST imaging were correlated with results from 1HMRS and immunohistochemistry (IHC). The primary IHC antibodies that were investigated include markers of phosphorylated tau protein, synapse density, neuron density, glial cell reactivity, a glutamate transporter, and an NMDA receptor. GluCEST contrast correlated with 1HMRS-derived glutamate levels in the striatum of APP-PS1 mice (R2=0.91) and the thalamus of PS19 mice (R2=0.64). However, GluCEST detected deficits in PS19 mice four months earlier than 1HMRS, highlighting the method’s enhanced sensitivity to glutamate. Demonstrating the advantage of high spatial resolution, GluCEST imaging measured sub-hippocampal dynamics in glutamate levels in the aging PS19 mouse. A gradient in glutamate levels along the mouse hippocampus was also measured in vivo using GluCEST. While hippocampal glutamate levels were significantly decreased in early stages of PS19 tauopathy, glutamate levels in the dentate gyrus (DG) and cornu ammonis (CA1) increased at 9-13 months. Decreased GluCEST was concurrent with synapse loss and occurred before structural volume loss. Elevated GluCEST was associated with glial fibrillary acidic protein (GFAP) immunostaining in late stages of the PS19 tauopathy model and in the striatum of the MPTP PD model. Results of this work demonstrate the use of GluCEST imaging to study regional and temporal variations in glutamate in different pathologies associated with neurodegeneration

Imaging mouse models of neurodegeneration using multi-parametric MRI

Alzheimer’s disease (AD) is a devastating condition characterised by significant cognitive impairment and memory loss. Transgenic mouse models are increasingly being used to further our knowledge of the cause and progression of AD, and identify new targets for therapeutic intervention. These mice permit the study of specific pathological hallmarks of the disease, including intracellular deposits of hyperphosphorylated tau protein and extracellular amyloid plaques. In order to characterise these transgenic mice, robust biomarkers are required to evaluate neurodegenerative changes and facilitate preclinical evaluation of emerging therapeutics. In this work, a platform for in vivo structural imaging of the rTg4510 mouse model of tauopathy was developed and optimised. This was combined with a range of other clinically relevant magnetic resonance imaging (MRI) biomarkers including: arterial spin labelling, diffusion tensor imaging and chemical exchange saturation transfer. These techniques were applied in a single time-point study of aged rTg4510 mice, as well as a longitudinal study to serially assess neurodegeneration in the same cohort of animals. Doxycycline was administered to a subset of rTg4510 mice to suppress the tau transgene; this novel intervention strategy permitted the evaluation of the sensitivity of MRI biomarkers to the accumulation and suppression of tau. Follow-up ex vivo scans were acquired in order to assess the sensitivity of in vivo structural MRI to the current preclinical gold standard. High resolution structural MRI, when used in conjunction with advanced computational analysis, yielded high sensitivity to pathological changes occurring in the rTg4510 mouse. Atrophy was reduced in animals treated with doxycycline. All other MRI biomarkers were able to discriminate between doxycycline-treated and untreated rTg4510 mice as well as wildtype controls, and provided insight into complimentary pathological mechanisms occurring within the disease process. In addition, this imaging protocol was applied to the J20 mouse model of familial AD. This mouse exhibits widespread plaque formation, enabling the study of amyloid-specific pathological changes. Atrophy and deficits in cerebral blood flow were observed; however, the changes occurring in this model were markedly less than those observed in the rTg4510 mouse. This study was expanded to investigate the early-onset AD observed in individuals with Down’s syndrome (DS) by breeding the J20 mouse with the Tc1 mouse model of DS, permitting the relationship between genetics and neurodegeneration to be dissected. This thesis demonstrates the application of in vivo multi-parametric MRI to mouse models of neurodegeneration. All techniques were sensitive to pathological changes occurring in the models, and may serve as important biomarkers in clinical studies of AD. In addition, in vivo multi-parametric MRI permits longitudinal studies of the same animal cohort. This experimental design produces more powerful results, whilst contributing to worldwide efforts to reduce animal usage with respect to the 3Rs principles

Monitoring Alzheimer's disease in transgenic mice with ultra high field magnetic resonance imaging

While aging remains one of the most significant risk factors for development of Alzheimer disease (AD), increasing evidence strongly points to the potential roles of cerebrovascular and white matter abnormalities in the disease development. A better understanding of the manner in which these abnormalities contribute to disease progression can be achieved by in vivo characterization of AD related pathologies. To this end, MR based techniques serve as effective non-invasive tools to longitudinally monitor changes in AD brain. In this thesis, a variety of MR based techniques were optimized and employed to longitudinally monitor the AD progression in transgenic mouse models of the disease at 9.4T and 17.6T. In Chapter 2, age-dependent blood flow alterations were examined in a Tg2576 mouse model of Alzheimer's disease using MR angiography at 17.6T. AD is linked to abnormalities in the vascular system. In Chapter 3, in vivo T2 changes were longitudinally monitored in the corpus callosum, of the Tg2576 mice. In Chapter 4, age-dependent regional brain T1 and T2 changes in healty mice were established at 17.6T. In vivo imaging of these mouse models at ultra-high magnetic field strengths can permit a better understanding of the underlying cellular mechanism of AD.The Centre for Medical Systems Biology (CMSB), Internationale Stichting Alzheimer Onderzoek and Alzheimer NederlandSolid state NMR/Biophysical Organic Chemistr

T1, diffusion tensor, and quantitative magnetization transfer imaging of the hippocampus in an Alzheimer's disease mouse model

Alzheimer's disease (AD) pathology causes microstructural changes in the brain. These changes, if quantified with magnetic resonance imaging (MRI), could be studied for use as an early biomarker for AD. The aim of our study was to determine if T1 relaxation, diffusion tensor imaging (DTI), and quantitative magnetization transfer imaging (qMTI) metrics could reveal changes within the hippocampus and surrounding white matter structures in ex vivo transgenic mouse brains overexpressing human amyloid precursor protein with the Swedish mutation. Delineation of hippocampal cell layers using DTI color maps allows more detailed analysis of T1-weighted imaging, DTI, and qMTI metrics, compared with segmentation of gross anatomy based on relaxation images, and with analysis of DTI or qMTI metrics alone. These alterations are observed in the absence of robust intracellular Aβ accumulation or plaque deposition as revealed by histology. This work demonstrates that multiparametric quantitative MRI methods are useful for characterizing changes within the hippocampal substructures and surrounding white matter tracts of mouse models of AD

In vivo magnetic resonance imaging and spectroscopy of Alzheimer__s disease in transgenic mice

The thesis describes the application of several different magnetic resonance (MR) techniques to study the effects of the progression of disease in a transgenic mouse model of Alzheimer's. Using MR imaging, the amyloid plaque deposition was visualized and the plaque load quantified in the same mice as they aged. Concurrently the transverse relaxation time (T2) was measured in affected brain regions and shown to decrease over time as plaque-load increased. To study the neurochemical profile in the mouse brain brain both one- (1D) and two-dimensional (2D) MR spectroscopic techniques were employed. 1D MRS is widely used in similar research, but has limited spectral resolution. To overcome this limitation, a 2D MRS technique was implemented and optimized for use in mouse brain. This technique, L-COSY, allowed the detection of several metabolites which were not visible using standard 1D MRS techniques. This technique was subsequently used to study the effects of Alzheimer's on the neurochemical profile. Observed changes were correlated with plaque deposition.UBL - phd migration 201

Development of a Multimodal MRI Study to Characterize Morpho-Functional Features in Rodent Models of Alzheimer's Disease

Alzheimer’s Disease (AD) is the most common form of dementia, recognized by the World Health Organization as a global public health priority. It is a complex pathology characterized by the accumulation of the Amyloid-β (Aβ) peptide as extracellular plaques and of the intracellullar neuro fibrillary tangles (NFT) alongside with different events, such as chronic neuroinflammation and astrogliosis. None of the existing biomarkers is simultaneously specific for the pathology and sensitive to its progression. Metabolic and functional alterations are the earliest events described in the AD pathological cascade but shared with other form of dementia, while specific structural alterations occurs in a later stage of the disease. The use of transgenic models could simplify the development of new imaging biomarkers that would enable early diagnosis and making new treatments more effective. The objective of this work was to develop a multi-modal panel of magnetic resonance imaging (MRI) techniques and automated analysis pipelines, characterized by a high translational impact, in order to investigate the metabolic, functional and structural alterations in the brains of AD transgenic models. Results obtained in the APP23 transgenic mouse show that chemical exchange saturation transfer (CEST) imaging can be used to detect alterations in the brain uptake of the glucose analogue 2-deoxy-d-glucose (2DG) with a better resolution than PET and without the need of radioactive tracers. Moreover, a longitudinal study highlighted that significant structural and metabolic alterations can be found only in a late stage of the pathology. Furthermore, an advanced pipeline for the analysis of the rodent brain functional connectivity has been developed. This thesis demonstrates that the advantage to the experimental design adopted is simplifying longitudinal studies of the same animal cohort. The translation of the analysis pipelines adopted in human studies enables more powerful results and reduces the number of animals involved in research

CALIBRATED SHORT TR RECOVERY MRI FOR RAPID MEASUREMENT OF BRAIN-BLOOD PARTITION COEFFICIENT AND CORRECTION OF QUANTITATIVE CEREBRAL BLOOD FLOW

The high prevalence and mortality of cerebrovascular disease has led to the development of several methods to measure cerebral blood flow (CBF) in vivo. One of these, arterial spin labeling (ASL), is a quantitative magnetic resonance imaging (MRI) technique with the advantage that it is completely non-invasive. The quantification of CBF using ASL requires correction for a tissue specific parameter called the brain-blood partition coefficient (BBPC). Despite regional and inter-subject variability in BBPC, the current recommended implementation of ASL uses a constant assumed value of 0.9 mL/g for all regions of the brain, all subjects, and even all species. The purpose of this dissertation is 1) to apply ASL to a novel population to answer an important clinical question in the setting of Down syndrome, 2) to demonstrate proof of concept of a rapid technique to measure BBPC in mice to improve CBF quantification, and 3) to translate the correction method by applying it to a population of healthy canines using equipment and parameters suitable for use with humans. Chapter 2 reports the results of an ASL study of adults with Down syndrome (DS). This population is unique for their extremely high prevalence of Alzheimer’s disease (AD) and very low prevalence of systemic cardiovascular risk factors like atherosclerosis and hypertension. This prompted the hypothesis that AD pathology would lead to the development of perfusion deficits in people with DS despite their healthy cardiovascular profile. The results demonstrate that perfusion is not compromised in DS participants until the middle of the 6th decade of life after which measured global CBF was reduced by 31% (p=0.029). There was also significantly higher prevalence of residual arterial signal in older participants with DS (60%) than younger DS participants (7%, p = 0.005) or non-DS controls (0%, p \u3c 0.001). This delayed pattern of perfusion deficits in people with DS differs from observations in studies of sporadic AD suggesting that adults with DS benefit from an improved cardiovascular risk profile early in life. Chapter 3 introduces calibrated short TR recovery (CaSTRR) imaging as a rapid method to measure BBPC and its development in mice. This was prompted by the inability to account for potential changes in BBPC due to age, brain atrophy, or the accumulation of hydrophobic A-β plaques in the ASL study of people with DS in Chapter 2. The CaSTRR method reduces acquisition time of BBPC maps by 87% and measures a significantly higher BBPC in cortical gray matter (0.99±0.04 mL/g,) than white matter in the corpus callosum (0.93±0.05 mL/g, p=0.03). Furthermore, when CBF maps are corrected for BBPC, the contrast between gray and white matter regions of interest is improved by 14%. This demonstrates proof of concept for the CaSTRR technique. Chapter 4 describes the application of CaSTRR on healthy canines (age 5-8 years) using a 3T human MRI scanner. This represents a translation of the technique to a setting suitable for use with a human subject. Both CaSTRR and pCASL acquisitions were performed and further optimization brought the acquisition time of CaSTRR down to 4 minutes which is comparable to pCASL. Results again show higher BBPC in gray matter (0.83 ± 0.05 mL/g) than white matter (0.78 ± 0.04 mL/g, p = 0.007) with both values unaffected by age over the range studied. Also, gray matter CBF is negatively correlated with age (p = 0.003) and BBPC correction improved the contrast to noise ratio by 3.6% (95% confidence interval = 0.6 – 6.5%). In summary, the quantification of ASL can be improved using BBPC maps derived from the novel, rapid CaSTRR technique

Tissue Damage Quantification in Alzheimer\u27s Disease Brain via Magnetic Resonance Gradient Echo Plural Contrast Imaging (GEPCI)

Alzheimer’s disease (AD) affected approximately 48 million people worldwide in 2015. Its devastating consequences have stimulated an intense search for AD prevention and treatment. Clinically, AD is characterized by memory deficits and progressive cognitive impairment, leading to dementia. Over the past two to three decades, researchers have found that amyloidbeta (Aβ) plaques and neurofibrillary tau tangles occur during a long pre-symptomatic period (preclinical stage) before the onset of clinical symptoms. As a result, identification of the preclinical stage is essential for the initiation of prevention trials in asymptomatic individuals. Currently, Positron Emission Tomography (PET) imaging with injected 11C or 18F containing radiotracers (e.g., Pittsburgh compound B, PiB or florbetapir-fluorine-18, 18F-AV-45) is widely used to detect amyloid deposition in vivo and to identify this preclinical stage. However, PET scans are time consuming (about 1 hour), require injection of a radiotracer, thus, exposing the patient to ionizing radiation. After the preclinical stage, AD patients begin to show clinical symptoms, referred as a very mild or mild AD group. Post-mortem studies show that neuronal damage is the most proximate pathological substrate of cognitive impairment in AD compared with amyloid and tau deposition. Thus, a diagnostic tool is needed for detection of neuronal loss in vivo. As a faster, non-invasive, and radiation free imaging technique, Magnetic Resonance Imaging (MRI) plays an important role in the diagnosis of cognitive diseases. Conventional MRI yields superb definition of brain anatomy and structure and provide important volumetric information (e.g., brain atrophy). However, conventional MRI cannot provide microstructural and functional insight into the pathology of AD. The approach developed in Yablonskiy’s lab is based on the Gradient Echo Plural Contrast Imaging (GEPCI) protocol, which provides quantitative in vivo measurements of transverse relaxation properties of the tissue water 1H spins as determined from the gradient echo MRI signal. The measurements are corrected for macroscopic magnetic field inhomogeneity effects and physiologic-motion-driven fluctuations in magnetic field as these are the major artifacts present with the gradient echo technique. The principal relaxation property used in this dissertation is the tissue-specific transverse relaxation rate constant, R2*. The R2* value reflects the microscopic and mesoscopic magnetic field inhomogeneities rising from the complex tissuewater-environment within the human brain. In turn, changes in R2* reflect changes in the tissue’s microscopic and mesoscopic tissue structure. However, because of the presence of the cerebral blood vessel network, the magneticsusceptibility-driven blood-oxygen-level dependent (BOLD) effect also makes a significant contribution to R2*. A previously developed approach, quantitative BOLD (qBOLD), allows the separation of R2* into a tissue specific R2t* without blood vessel effects and the BOLD component. Quantifying the BOLD component allows the calculation of cerebral hemodynamics parameters, such as oxygen extraction fraction (OEF) and deoxygenated cerebral blood volume (dCBV). These parameters (R2*, R2t*, OEF, dCBV) describe structural and functional properties of tissue at the microstructural level in the human brain. In the study of normal aging, quantitative GEPCI measurements showed that R2t* increases with age while hemodynamic parameters, i.e., relative OEF and dCBV remain constant in most cerebral cortical regions. The comparison between quantitative GEPCI measurements and literature information suggest that (a) age-related increases in the cortical R2t* mostly reflect the age-related increases in the cellular packing density (or neuronal density); (b) regions in a brain characterized by higher R2t* contain a higher concentration of neurons with less developed cellular processes (dendrites, spines, etc.); and (c) brain regions characterized by lower R2t* represent regions with lower concentration of neurons but more developed cellular processes. In the Alzheimer study, R2* and R2t* together demonstrated significant differences among the normal, preclinical and mild AD groups. First, the results uncovered strong correlations between R2* and Aβ deposition measured by the PiB PET-tracer in several cortical regions (e.g., medial temporal lobe and precuneus). This finding indicates that R2* may be a potential surrogate marker for Aβ deposition. The strongest correlation was found in the medial temporal lobe (MTL), particularly in the parahippocampal cortex, which can be used to distinguish the normal and preclinical groups. Second, R2t* in the hippocampus, which characterized the hippocampal cellular integrity demonstrated much stronger correlations with psychometric tests than volume quantification of hippocampal atrophy. Importantly, decreased R2t* characterizing cellular damage was detected even in the hippocampal areas not affected by atrophy. In addition, R2t* significantly decreased in the mild AD group but was preserved in the preclinical group compared with the normal group. These results indicate a significant cellular density decrease in the mild group but not in the preclinical group, which is consistent with previous histological studies. In summary, GEPCI provides a new approach for evaluation of AD-related tissue pathology in vivo in the preclinical and early symptomatic stages of AD. Since MRI is widely available worldwide and does not require radiation exposure, it provides the opportunity to obtain new information on the pathogenesis of AD and for pre-screening cohorts (stratification) for clinical drug trials

Myelin imaging and characterization by magnetic resonance imaging

280 p.Los axones neuronales están recubiertos de una membrana lipídica llamada mielina, que protege a los axones y posibilita una transmisión rápida y eficiente del impulso eléctrico. En ciertas patologías como la lesión cerebral traumática, la isquemia o principalmente, en la esclerosis múltiple, la pérdida de mielina o desmielinización da lugar a la muerte neuronal y por consiguiente a la pérdida de capacidades cognitivas. Este estado puede ser revertido por medio de la remielinización, en la que los oligodendrocitos mielinizantes del sistema nervioso central regeneran la vaina de mielina, evitando la degeneración de las neuronas. En los últimos años se ha realizado un esfuerzo considerable en el desarrollo de terapias remielinizantes. Para ello, es imprescindible el desarrollo de técnicas para la evaluación no-invasiva de estas terapias y una caracterización profunda de los procesos de desmielinización y remielinización. En este contexto, la imagen por resonancia magnética (IRM) juega un papel fundamental por su carácter no-invasivo, alta resolución y versatilidad.Los principales objetivos de esta tesis han sido el desarrollo de protocolos de IRM para la cuantificación de mielina y la caracterización de los procesos de remielinización y desmielinización a través de resonancia magnética funcional en reposo. Para ello se ha utilizado como base el modelo murinocuprizona, en la que la administración del tóxico da lugar a la desmielinización en el cerebro, seguido por la remielinización. Los datos y conclusiones obtenidas se han contrastado en otros modelos de ratón, como en modelos de Alzheimer o en ratones sanos envejecidos.A grandes rasgos, hemos podido concluir que la imagen ponderada en peso T2 es la más específica y sensible para la cuantificación de mielina en el modelo cuprizona. Por ello, en este trabajo se propone la utilización de la imagen ponderada en peso T2 para la evaluación de terapias remielinizantes en el modelo cuprizona. Sin embargo, el interés de realizar imagen multiparamétríca ha quedado al descubierto al realizar imagen de modelos de ratón de Alzheimer, pudiendo detectar patología no relacionada con pérdida de mielina en zonas de materia blanca.Así mismo, hemos podido comprobar como la desmielinización conlleva la pérdida de la conectividad y función cerebral y la remielinización posibilita la recuperación por medio de la resonancia magnética funcional en reposo. Además, el potencial agente remielinizante clemastina, ha demostrado su capacidad de promover la remielinización a nivel anatómico y funcional tras 2 semanas de tratamiento. Finalmente, se ha realizado un estudio para determinar el efecto del envejecimiento en la conectividad del cerebro. Hemos podido observar que en ratones sanos, se ha observado un incremento de la conectividad cerebral hasta el mes 8, seguido de un descenso hasta el mes 13, probablemente debido a la neurodegeneración.En este trabajo hemos contribuido al desarrollo de terapias remielinizantes, por un lado, desarrollando protocolos de imagen para la cuantificación de mielina en modelos animales y por otro lado, caracterizando la desmielinización y remielinización a nivel funcional y anatómico