Magnetic Resonance Imaging in Tauopathy Animal Models

The microtubule-associated protein tau plays an important role in tauopathic diseases such as Alzheimer's disease and primary tauopathies such as progressive supranuclear palsy and corticobasal degeneration. Tauopathy animal models, such as transgenic, knock-in mouse and rat models, recapitulating tauopathy have facilitated the understanding of disease mechanisms. Aberrant accumulation of hyperphosphorylated tau contributes to synaptic deficits, neuroinflammation, and neurodegeneration, leading to cognitive impairment in animal models. Recent advances in molecular imaging using positron emission tomography (PET) and magnetic resonance imaging (MRI) have provided valuable insights into the time course of disease pathophysiology in tauopathy animal models. High-field MRI has been applied for in vivo imaging in animal models of tauopathy, including diffusion tensor imaging for white matter integrity, arterial spin labeling for cerebral blood flow, resting-state functional MRI for functional connectivity, volumetric MRI for neurodegeneration, and MR spectroscopy. In addition, MR contrast agents for non-invasive imaging of tau have been developed recently. Many preclinical MRI indicators offer excellent translational value and provide a blueprint for clinical MRI in the brains of patients with tauopathies. In this review, we summarized the recent advances in using MRI to visualize the pathophysiology of tauopathy in small animals. We discussed the outstanding challenges in brain imaging using MRI in small animals and propose a future outlook for visualizing tau-related alterations in the brains of animal models

Study the Longitudinal in vivo and Cross-Sectional ex vivo Brain Volume Difference for Disease Progression and Treatment Effect on Mouse Model of Tauopathy Using Automated MRI Structural Parcellation

Brain volume measurements extracted from structural MRI data sets are a widely accepted neuroimaging biomarker to study mouse models of neurodegeneration. Whether to acquire and analyze data in vivo or ex vivo is a crucial decision during the phase of experimental designs, as well as data analysis. In this work, we extracted the brain structures for both longitudinal in vivo and single-time-point ex vivo MRI acquired from the same animals using accurate automatic multi-atlas structural parcellation, and compared the corresponding statistical and classification analysis. We found that most gray matter structures volumes decrease from in vivo to ex vivo, while most white matter structures volume increase. The level of structural volume change also varies between different genetic strains and treatment. In addition, we showed superior statistical and classification power of ex vivo data compared to the in vivo data, even after resampled to the same level of resolution. We further demonstrated that the classification power of the in vivo data can be improved by incorporating longitudinal information, which is not possible for ex vivo data. In conclusion, this paper demonstrates the tissue-specific changes, as well as the difference in statistical and classification power, between the volumetric analysis based on the in vivo and ex vivo structural MRI data. Our results emphasize the importance of longitudinal analysis for in vivo data analysis

Automated morphometric analysis and phenotyping of mouse brains from structural µMR images

In light of the utility and increasing ubiquity of mouse models of genetic and neurological disease, I describefully automated pipelines for the investigation of structural microscopic magnetic resonance images of mouse brains – for both high-throughput phenotyping, and monitoring disease. Mouse models offer unparalleled insight into genetic function and brain plasticity, in phenotyping studies; and neurodegenerative disease onset and progression, in therapeutic trials. I developed two cohesive, automatic software tools, for Voxel- and Tensor-Based Morphometry (V/TBM) and the Boundary Shift Integral (BSI), in the mouse brain. V/TBM are advantageous for their ability to highlight morphological differences between groups, without laboriously delineating regions of interest. The BSI is a powerful and sensitive imaging biomarker for the detection of atrophy. The resulting pipelines are described in detail. I show the translation and application of open-source software developed for clinical MRI analysis to mouse brain data: for tissue segmentation into high-quality, subject-specific maps, using contemporary multi-atlas techniques; and for symmetric, inverse-consistent registration. I describe atlases and parameters suitable for the preclinical paradigm, and illustrate and discuss image processing challenges encountered and overcome during development. As proof of principle and to illustrate robustness, I used both pipelines with in and ex vivo mouse brain datasets to identify differences between groups, representing the morphological influence of genes, and subtle, longitudinal changes over time, in particular relation to Down syndrome and Alzheimer’s disease. I also discuss the merits of transitioning preclinical analysis from predominately ex vivo MRI to in vivo, where morphometry is still viable and fewer mice are necessary. This thesis conveys the cross-disciplinary translation of up-to-date image analysis techniques to the preclinical paradigm; the development of novel methods and adaptations to robustly process large cohorts of data; and the sensitive detection of phenotypic differences and neurodegenerative changes in the mouse brai

The Translational Role of Diffusion Tensor Image Analysis in Animal Models of Developmental Pathologies

Diffusion Tensor Magnetic Resonance Imaging (DTI) has proven itself a powerful technique for clinical investigation of the neurobiological targets and mechanisms underlying developmental pathologies. The success of DTI in clinical studies has demonstrated its great potential for understanding translational animal models of clinical disorders, and preclinical animal researchers are beginning to embrace this new technology to study developmental pathologies. In animal models, genetics can be effectively controlled, drugs consistently administered, subject compliance ensured, and image acquisition times dramatically increased to reduce between-subject variability and improve image quality. When pairing these strengths with the many positive attributes of DTI, such as the ability to investigate microstructural brain organization and connectivity, it becomes possible to delve deeper into the study of both normal and abnormal development. The purpose of this review is to provide new preclinical investigators with an introductory source of information about the analysis of data resulting from small animal DTI studies to facilitate the translation of these studies to clinical data. In addition to an in depth review of translational analysis techniques, we present a number of relevant clinical and animal studies using DTI to investigate developmental insults in order to further illustrate techniques and to highlight where small animal DTI could potentially provide a wealth of translational data to inform clinical researchers

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40

Investigation of neuronal activity in a murine model of Alzheimer’s disease using in vivo two-photon calcium imaging

Alzheimer’s disease (AD) is one of the biggest challenges for biomedical research nowadays as with the growth of life span more and more people are affected by this disorder. Etiology of AD is unknown, yet growing evidence identifies alterations in neuronal activity as of the great importance for pathology. Although several significant studies of neuronal activity alteration in AD were done during the last decade, none of them addressed the question of the time course of these changes over the disease progression. Alzheimer’s disease (AD) is characterized by impairments of brain neurons that are responsible for the storage and processing of information. Studies have revealed decrease in the activity of neurons (Silverman et al., 2001; Prvulovic et al., 2005) and it was proposed that generalized hypoactivity and silencing of brain circuits takes place as formulated in the synaptic failure hypothesis (Selkoe, 2002). However, more recent studies also reported opposite effects – hyperexcitability and hyperactivity of neurons in the AD models (Busche et al., 2008; Sanchez et al., 2012; Liebscher et al., 2016). It still remains unclear if these are two sides of the same coin or if these are two stages, that follow each other. Moreover, it is not clear if observed neuronal activity alterations are caused by the dysfunction of individual neurons or if overall circuitry is disturbed because the crucial “activity controllers” (most probably - inhibitory neurons) alter their activity. This project aimed to examine spontaneous neuronal activity in the murine model of AD at the early stages of disease progression using chronic in vivo imaging to address the character and the stability of neuronal activity alterations as well relation of the activity alterations to amyloid plaque proximity. Compared to earlier studies the approach of in vivo awake calcium imaging used in the current study has many benefits for brain research. The main advantage is that brain activity can be measured without artifacts generated by anesthesia, which can exaggerate or mitigate experimental readouts. In this project, I used genetically encoded calcium indicator GCaMP6 that enables prolonged repetitive imaging of the same neurons in an intact environment. Recording of calcium transients in cell bodies of neurons was accompanied by in vivo imaging of Aβ plaques and followed by immunohistochemical staining of GCaMP6-expressing neurons to investigate how activity changes are correlated with proximity to the plaque. All the experiments were done in awake mice to ensure the absence of anesthesia-derived impact on spontaneous neuronal activity. My results support previously published reports of the increased proportion of hyperactive excitatory neurons in the AD mouse model. Importantly, my results also demonstrate that this increased activity is present in the awake state, is stable over a longer period of time (one month) and does not depend on the distance to the closest plaque. These findings support the hypothesis of permanent network alterations driving aberrant activity patterns that appear early in the disease progression, resulting in a chronic excitation/inhibition disbalance. Another important finding of my project is that individual neurons do not stay in the silent state and most of them remain functional demonstrating normal activity at the later time points. This finding requires further research as it has important implication for the development of the AD treatment, as in case many neurons remain functional and their normal neuronal activity can be recovered by addressing the cause of the circuit dysfunction with treatment. To summarize, the study presented in this PhD thesis is the first longitudinal study of neuronal activity changes in an AD mouse model, and while it provides important insight into pathology, it also emphasizes the importance of chronic in vivo studies to investigate neuronal activity and its role in the disease progression

Functional and structural substrates of increased dosage of Grik4 gene elucidated using multi-modal MRI

Grik4 is the gene responsible for encoding the high-affinity GluK4 subunit of the kainate receptors. Increased dosage of this subunit in the forebrain was linked to an increased level of anxiety, lack of social communication, and depression. On the synaptic level, abnormal synaptic transmission was also reported. The manifestations of this abnormal expression have not been investigated at the circuit level, nor the correlations between those circuits and the abnormal patterns of the behavior previously reported. In this line of work, we aspired to use different non-invasive magnetic resonance imaging (MRI) modalities to elucidate any disturbance that might stem from the increased dosage of Grik4 and how those changes might explain the abnormal behaviors. MRI offers a noninvasive way to look into the intact brain in vivo. Resting-state functional MRI casts light on how the brain function at rest on the network level and has the capability to detect any anomalies that might occur within or between those networks. On the microstructural level, the diffusion MRI is concerned with the underlying features of the tissues, using the diffusion of water molecules as a proxy for that end. Moving more macroscopically, using structural scans, voxel-based morphometry can detect subtle differences in the morphology of the different brain structures. We recorded videos of our animals performing two tasks that have long been linked to anxiety, the open field and the plus-maze tests before acquiring structural and functional scans. Lastly, we recorded blood-oxygenationlevel dependent (BOLD) signals in a different set of animals during electrical stimulation of specific white matter tracts in order to investigate how neuronal activity propagates. Our analysis showed a vast spectrum of changes in the transgenic group relative to the animals in the control group. On the resting-state networks level, we observed an increase in the within-network strength spanning different structures such as the hippocampus, some regions of the cortex, and the hypothalamus. The increased internal coherence or strength in the networks contrasted with a significant reduction in between-networks connectivity for some regions such as parts of the cortex and the hypothalamus, suggesting long-range network decorrelation. Supporting this idea, major white matter (WM) tracts, such as the corpus callosum and the hippocampal commissure, suffered from substantial changes compatible with an important reduction in myelination and/or a decrease in the mean axonal diameter. Macrostructurally speaking, the overexpression of GluK4 subunit had a bimodal effect, with expansion in some cortical areas in the transgenic animals accompanied by a shrinkage in the subcortical regions. Upon stimulating the brain with an electrical current, we noticed a difference in activity propagation between the two hemispheres. In transgenic animals, the evoked activity remained more confined to the stimulated hemisphere, again consistent with an impaired long-range connectivity. The structural changes both, at the micro and macro level, were in tight correlation with different aspects of the behavior including markers of anxiety such as the time spent in the open arms vs the closed arms in the plus-maze test and the time spent in the center vs the corners in the open field test. Our findings reveal how the disruption of kainate receptors, or more globally the glutamate receptors, and the abnormal synaptic transmission can translate into brain-wide changes in connectivity and alter the functional equilibrium between macro-and mesoscopic networks. The postsynaptic enhancement previously reported in the transgenic animals was here reflected in the BOLD signal and measured as an increase in the within-network strength. Importantly, the correlations between the structural changes and the behavior help to put the developmental changes and their behavioral ramifications into context. RESUMEN Grik4 es el gen responsable de codificar la subunidad GluK4 de alta afinidad de los receptores de kainato. El aumento de la dosis de esta subunidad en el prosencéfalo se relacionó con un mayor nivel de ansiedad, falta de comunicación social y depresión. A nivel sináptico, también se informó una transmisión sináptica anormal. Las manifestaciones de esta expresión anormal no se han investigado a nivel de circuito, ni las correlaciones entre esos circuitos y los patrones anormales de la conducta previamente informada. En esta línea de trabajo, aspiramos a utilizar diferentes modalidades de imágenes por resonancia magnética (MRI) no invasivas para dilucidar cualquier alteración que pudiera derivarse del aumento de la dosis de Grik4 y cómo esos cambios podrían explicar los comportamientos anormales. La resonancia magnética ofrece una forma no invasiva de observar el cerebro intacto in vivo. La resonancia magnética funcional en estado de reposo arroja luz sobre cómo funciona el cerebro en reposo en el nivel de la red y tiene la capacidad de detectar cualquier anomalía que pueda ocurrir dentro o entre esas redes. En el nivel microestructural, la resonancia magnética de difusión se ocupa de las características subyacentes de los tejidos utilizando la difusión de moléculas de agua como un proxy para ese fin. Moviéndose más macroscópicamente, utilizando escaneos estructurales, la morfometría basada en vóxeles puede detectar diferencias sutiles en la morfología de las diferentes estructuras cerebrales. Grabamos videos de nuestros animales realizando dos tareas que durante mucho tiempo se han relacionado con la ansiedad, el campo abierto y las pruebas de laberinto positivo antes de adquirir escaneos estructurales y funcionales. Por último, registramos señales dependientes del nivel de oxigenación de la sangre (BOLD) en un grupo diferente de animales durante la estimulación eléctrica de tractos específicos de materia blanca para investigar cómo se propaga la actividad neuronal. Nuestro análisis mostró un amplio espectro de cambios en el grupo transgénico en relación con los animales en el grupo de control. En el nivel de las redes de estado de reposo, observamos un aumento en la fuerza dentro de la red que abarca diferentes estructuras como el hipocampo, algunas regiones de la corteza y el hipotálamo. La mayor coherencia interna o fuerza en las redes contrastó con una reducción significativa en la conectividad entre redes para algunas regiones como partes de la corteza y el hipotálamo, lo que sugiere una descorrelación de redes de largo alcance. Apoyando esta idea, los grandes tractos de materia blanca (WM), como el cuerpo calloso y la comisura del hipocampo, sufrieron cambios sustanciales compatibles con una importante reducción de la mielinización y / o una disminución del diámetro axonal medio. Macroestructuralmente hablando, la sobreexpresión de la subunidad GluK4 tuvo un efecto bimodal, con expansión en algunas áreas corticales en los animales transgénicos acompañada de una contracción en las regiones subcorticales. Al estimular el cerebro con una corriente eléctrica, notamos una diferencia en la propagación de la actividad entre las dos hemiesferas. En los animales transgénicos, la actividad evocada permaneció más confinada al hemisferio estimulado, de nuevo consistente con una conectividad de largo alcance deteriorada. Los cambios estructurales, tanto a nivel micro como macro, estaban en estrecha correlación con diferentes aspectos de la conducta, incluidos marcadores de ansiedad como el tiempo pasado con los brazos abiertos frente a los brazos cerrados en la prueba del laberinto positivo y el tiempo pasado en el centro vs las esquinas en la prueba de campo abierto. Nuestros hallazgos revelan cómo la interrupción de los receptores de kainato, o más globalmente los receptores de glutamato, y la transmisión sináptica anormal pueden traducirse en cambios de conectividad en todo el cerebro y alterar el equilibrio funcional entre las redes macro y mesoscópicas. La mejora postsináptica informada anteriormente en los animales transgénicos se reflejó aquí en la señal BOLD y se midió como un aumento en la fuerza dentro de la red. Es importante destacar que las correlaciones entre los cambios estructurales y elcomportamiento ayudan a contextualizar los cambios en el desarrollo y sus ramificaciones conductuales

Investigation of neuronal activity in a murine model of Alzheimer’s disease using in vivo two-photon calcium imaging

Alzheimer’s disease (AD) is one of the biggest challenges for biomedical research nowadays as with the growth of life span more and more people are affected by this disorder. Etiology of AD is unknown, yet growing evidence identifies alterations in neuronal activity as of the great importance for pathology. Although several significant studies of neuronal activity alteration in AD were done during the last decade, none of them addressed the question of the time course of these changes over the disease progression. Alzheimer’s disease (AD) is characterized by impairments of brain neurons that are responsible for the storage and processing of information. Studies have revealed decrease in the activity of neurons (Silverman et al., 2001; Prvulovic et al., 2005) and it was proposed that generalized hypoactivity and silencing of brain circuits takes place as formulated in the synaptic failure hypothesis (Selkoe, 2002). However, more recent studies also reported opposite effects – hyperexcitability and hyperactivity of neurons in the AD models (Busche et al., 2008; Sanchez et al., 2012; Liebscher et al., 2016). It still remains unclear if these are two sides of the same coin or if these are two stages, that follow each other. Moreover, it is not clear if observed neuronal activity alterations are caused by the dysfunction of individual neurons or if overall circuitry is disturbed because the crucial “activity controllers” (most probably - inhibitory neurons) alter their activity. This project aimed to examine spontaneous neuronal activity in the murine model of AD at the early stages of disease progression using chronic in vivo imaging to address the character and the stability of neuronal activity alterations as well relation of the activity alterations to amyloid plaque proximity. Compared to earlier studies the approach of in vivo awake calcium imaging used in the current study has many benefits for brain research. The main advantage is that brain activity can be measured without artifacts generated by anesthesia, which can exaggerate or mitigate experimental readouts. In this project, I used genetically encoded calcium indicator GCaMP6 that enables prolonged repetitive imaging of the same neurons in an intact environment. Recording of calcium transients in cell bodies of neurons was accompanied by in vivo imaging of Aβ plaques and followed by immunohistochemical staining of GCaMP6-expressing neurons to investigate how activity changes are correlated with proximity to the plaque. All the experiments were done in awake mice to ensure the absence of anesthesia-derived impact on spontaneous neuronal activity. My results support previously published reports of the increased proportion of hyperactive excitatory neurons in the AD mouse model. Importantly, my results also demonstrate that this increased activity is present in the awake state, is stable over a longer period of time (one month) and does not depend on the distance to the closest plaque. These findings support the hypothesis of permanent network alterations driving aberrant activity patterns that appear early in the disease progression, resulting in a chronic excitation/inhibition disbalance. Another important finding of my project is that individual neurons do not stay in the silent state and most of them remain functional demonstrating normal activity at the later time points. This finding requires further research as it has important implication for the development of the AD treatment, as in case many neurons remain functional and their normal neuronal activity can be recovered by addressing the cause of the circuit dysfunction with treatment. To summarize, the study presented in this PhD thesis is the first longitudinal study of neuronal activity changes in an AD mouse model, and while it provides important insight into pathology, it also emphasizes the importance of chronic in vivo studies to investigate neuronal activity and its role in the disease progression