Identification of Cellular Infiltrates during Early Stages of Brain Inflammation with Magnetic Resonance Microscopy

A comprehensive view of brain inflammation during the pathogenesis of autoimmune encephalomyelitis can be achieved with the aid of high resolution non-invasive imaging techniques such as microscopic magnetic resonance imaging (μMRI). In this study we demonstrate the benefits of cryogenically-cooled RF coils to produce μMRI in vivo, with sufficient detail to reveal brain pathology in the experimental autoimmune encephalomyelitis (EAE) model. We could visualize inflammatory infiltrates in detail within various regions of the brain, already at an early phase of EAE. Importantly, this pathology could be seen clearly even without the use of contrast agents, and showed excellent correspondence with conventional histology. The cryogenically-cooled coil enabled the acquisition of high resolution images within short scan times: an important practical consideration in conducting animal experiments. The detail of the cellular infiltrates visualized by in vivo μMRI allows the opportunity to follow neuroinflammatory processes even during the early stages of disease progression. Thus μMRI will not only complement conventional histological examination but will also enable longitudinal studies on the kinetics and dynamics of immune cell infiltration

High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

Progress in clinical development of magnetic resonance imaging (MRI) substrate-sensors of enzymatic activity has been slow partly due to the lack of human efficacy data. We report here a strategy that may serve as a shortcut from bench to bedside. We tested ultra high-resolution 7T MRI (microMRI) of human surgical histology sections in a 3-year IRB approved, HIPAA compliant study of surgically clipped brain aneurysms. microMRI was used for assessing the efficacy of MRI substrate-sensors that detect myeloperoxidase activity in inflammation. The efficacy of Gd-5HT-DOTAGA, a novel myeloperoxidase (MPO) imaging agent synthesized by using a highly stable gadolinium (III) chelate was tested both in tissue-like phantoms and in human samples. After treating histology sections with paramagnetic MPO substrate-sensors we observed relaxation time shortening and MPO activity-dependent MR signal enhancement. An increase of normalized MR signal generated by ultra-short echo time MR sequences was corroborated by MPO activity visualization by using a fluorescent MPO substrate. The results of microMRI of MPO activity associated with aneurysmal pathology and immunohistochemistry demonstrated active involvement of neutrophils and neutrophil NETs as a result of pro-inflammatory signalling in the vascular wall and in the perivascular space of brain aneurysms

Advanced neuroimaging methods and biomarkers applied to preclinical models of multiple sclerosis and amyothropic lateral sclerosis

New paradigms are developed in magnetic resonance imaging for the advanced diagnosis of neurodegenerative diseases. In particular, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) preclinical research poorly focuses on functional connectivity in brain. Available animal models of MS and ALS are extensively used for analysis such drug testing and discovery of underlying mechanisms of pathogenesis. These diseases present, since neuronal lesions formation and neuroinflammation, a multilevel heterogeneity in mechanism of neurodegeneration and brain connectivity still not well understood. Moreover, they play a key role in pharmacological research, from the identification of a therapy target to the in vivo validation of the efficacy. More recently alterations in synchronized brain activity at rest in MS patients have been reported. At the best of our knowledge, functional imaging has not been applied yet in the assessment of new therapies in the preclinical models for MS and ALS. In this study, we aim to develop an innovative platform based on functional MRI in the resting state (rsfMRI), for the pre-clinical evaluation of new markers in MS and ALS. Moreover, the advancing in MRI techniques could assess new criteria of sensitivity and specificity in diagnosis; an additional analysis on diffusion MRI outcomes in MS preclinical models is added to this study

The Development and Validation of a Molecular Imaging Probe Targeted to Cathepsin D for the In-vivo Detection of Alzheimer Disease

Background: Currently there is no widely accepted test to diagnose AD. The involvement of the lysosomal system in Alzheimer’s disease (AD) progression provides an opportunity to develop associated biomarkers. The lysosomal enzyme Cathepsin D (CatD) has been shown to be over-expressed in the AD brain before clinical onset. We have developed a dual modality contrast agent (CA) to detect CatD activity which consists of an HIV-1 Tat Cell Penetrating Peptide (CPP) conjugated to a CatD cleavage sequence and two imaging moieties consisting of a fluorescently- tagged probe and a DOTA cage for chelating Gallium-68. The purpose of this work was to validate CatD as an AD biomarker across multiple AD disease models and to test our novel CA in-vivo by means of optical near infra-red (NIR) fluorescence imaging and positron emission tomography (PET). Methods: Three transgenic (Tg) mouse AD model strains were tested for CatD expression by Western blot and immunohistochemistry analysis. The chosen mouse line (5XFAD) and controls were imaged at 5 and 12 months of age using an eXplore Optix scanner (GE Healthcare, Milwaukee, WI, USA). Next, mice at 2, 6 and 9 months of age were tested using an Inveon microPET system (Siemens Medical Solutions, Knoxville TN, USA) using a 68Ga-labeled CatD targeted CA. Results: All 3 AD mice demonstrated an elevation of CatD expression in parallel with AD pathology. The 5XFAD had the highest levels of CatD, making it the best mouse model to study CatD upregulation. The rate of the NIR CatD Targeted CA washout was significantly slower in the 5XFAD mice (

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

Volumetric Manganese Enhanced Magnetic Resonance Imaging in mice (mus musculus)

The present doctoral thesis introduces a method for semi-automatic volumetric analysis of the hippocampus and other distinct brain regions in laboratory mice. The method of volumetric manganese enhanced magnetic resonance imaging (vMEMRI) makes use of the paramagnetic property of the manganese ion, Mn2+, which results in a positive contrast enhancement of specific brain areas on the MR image and enables a more detailed image of brain morphology. The chemical similarity of Mn2+ to Calcium leads to an accumulation of Mn2+ in excited cells and consequentially an enhanced signal in certain brain regions in an activity dependent manner. However, one major drawback for vMEMRI is the toxicity of Mn2+. Therefore, the aims of the thesis have been: (1) Establishment of a MEMRI protocol in mice (2) Optimization of a Mn2+ application procedure to reduce toxic side effects (3) Development of an automatized method to determine hippocampal volume (4) Validation of vMEMRI analysis (5) Application of volumetric analysis in mouse models of psychopathology This thesis splits into 3 studies. Study 1 deals with Mn2+ toxicity and introduces an application method that considerably reduces the toxic side effects of Mn2+. Study 2 validates vMEMRI as a method to reliably determine hippocampal volume and explores its utilization it in animals with genetically and chemically modified hippocampi. Study 3 displays the application vMEMRI in a mouse model of a psychiatric disorder. Study 1 shows that a single application of Mn2+ in dosages used in current MEMRI studies leads to considerable toxic side effects measurable with physiological, behavioral and endocrine markers. In contrast, a fractionated application of a low dose of Mn2+ is proposed as an alternative to a single injection of a high dose. Repeated application of low dosages of 30 mg/kg Mn2+ showed less toxic side effects compared to the application schemes with higher dosages of 60 mg/kg. Additionally, the best vMEMRI signal contrast was seen for an injection protocol of 30 mg/kg 8 times with an inter-injection interval of 24 h (8x30/24 protocol). The impact of the 8x30/24 application protocol on longitudinal studies was tested by determining whether learning processes are disturbed. Mice were injected with the 8x30/24 protocol 2 weeks prior to receiving a single footshock. Manganese injected mice showed less contextual freezing to the shock context and a shock context reminder one month after shock application. Furthermore, mice showed increased hyperarousal and no avoidance of shock context related odors. This impairment in fear conditioning indicates a disturbed associative learning of Mn2+ injected mice. Therefore, it was investigated whether Mn2+ application shows a specific disturbance of hippocampus dependent learning. Mice were subjected to habitual and spatial learning protocols 12 h after each injection in a water cross-maze. There was no impairment in learning protocols which allowed for hippocampus-independent habitual learning. However, Mn2+ injected mice were specifically impaired in the hippocampus-dependent spatial learning protocol. Furthermore, it was shown that only mice with higher Mn2+ accumulation showed this impairment. Altogether, the results of this chapter argue for a fractionated application scheme such as 30 mg/kg every 24 h for 8 days to provide sufficient MEMRI signal contrast while minimizing toxic side effects. However, the treatment procedure has to be further improved to allow for an analysis of hippocampus-dependent learning processes as well. Because of the potential side effects, the vMEMRI method was applied as a final experiment in study 2 and 3. Study 2 introduces the method of vMEMRI, which allows, for the first time, an in vivo semi-automatic detection of hippocampal volume. Hippocampal volume of mice with genetically altered adult neurogenesis and those with chemically lesioned hippocampi could be analyzed with vMEMRI. Even the highly variable differences in hippocampal volume of these animals could be detected with vMEMRI. vMEMRI data correlated with manually obtained volumes and are in agreement with previously reported histological findings, indicating the high reliability of this method. Study 3 investigates the ability of vMEMRI to detect even small differences in brain morphology by examining volumetric changes of the hippocampus and other brain structures in a mouse model of PTSD supplemented with enriched housing conditions. It was shown, that exposure to a brief inescapable foot shock led to a volume reduction in both the left hippocampus and right central amygdala two months later. Enriched housing decreased the intensity of trauma-associated contextual fear independently of whether it was provided before or after the shock. vMEMRI analysis revealed that enriched housing led to an increase in whole brain volume, including the lateral ventricles and the hippocampus. Furthermore, the enhancement of hippocampal volume through enriched housing was accompanied by the amelioration of trauma-associated PTSD-like symptoms. Hippocampal volume gain and loss was mirrored by ex vivo ultramicroscopic measurements of the hippocampus. Together, these data demonstrate that vMEMRI is able to detect small changes in hippocampal and central amygdalar volumes induced by a traumatic experience in mice. In conclusion, vMEMRI proves to be very reliable and able to detect small volumetric differences in various brain regions in living mice. vMEMRI opens up a great number possibilities for future research determining neuroanatomical structure, volumes and activity in vivo as well as the ability to repeatedly determine such characteristics within each subject, given an improvement of the Mn2+ treatment protocols to minimize potential toxic side effects

Can MRI measure myelin? Systematic review, qualitative assessment, and meta-analysis of studies validating microstructural imaging with myelin histology

Recent years have seen an increased understanding of the importance of myelination in healthy brain function and neuropsychiatric diseases. Non-invasive microstructural magnetic resonance imaging (MRI) holds the potential to expand and translate these insights to basic and clinical human research, but the sensitivity and specificity of different MR markers to myelination is a subject of debate. To consolidate current knowledge on the topic, we perform a systematic review and meta-analysis of studies that validate microstructural imaging by combining it with myelin histology. We find meta-analytic evidence for correlations between various myelin histology metrics and markers from different MRI modalities, including fractional anisotropy, radial diffusivity, macromolecular pool, magnetization transfer ratio, susceptibility and longitudinal relaxation rate, but not mean diffusivity. Meta-analytic correlation effect sizes range widely, between = 0.26 and = 0.82. However, formal comparisons between MRI-based myelin markers are limited by methodological variability, inconsistent reporting and potential for publication bias, thus preventing the establishment of a single most sensitive strategy to measure myelin with MRI. To facilitate further progress, we provide a detailed characterisation of the evaluated studies as an online resource. We also share a set of 12 recommendations for future studies validating putative MR-based myelin markers and deploying them in vivo in humans

Cortical circuit alterations precede motor impairments in Huntington's disease mice

Huntington's disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to longitudinally monitor the activity of identified single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in cortical network function, with an increase in activity that affects a large fraction of cells and occurs rather abruptly within one week, preceeding the onset of motor defects. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and human HD autopsy cases reveal a reduction in perisomatic inhibitory synaptic contacts on layer 2/3 pyramidal cells. Taken together, our study provides a time-resolved description of cortical network dysfunction in behaving HD mice and points to disturbed excitation/inhibition balance as an important pathomechanism in HD

Visualization of mouse barrel cortex using ex-vivo track density imaging

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo and T2-weighted imaging and gradient echo Ti/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3 mm)3 block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system. (C) 2013 Elsevier Inc. All rights reserved