Jan Klohs

Detection of Cerebral Microbleeds with Quantitative Susceptibility Mapping in the Arcabeta Mouse Model of Cerebral Amyloidosis

Cerebral microbleeds (CMBs) are findings in patients with neurological disorders such as cerebral amyloid angiopathy and Alzheimers disease, and are indicative of an underlying vascular pathology. A diagnosis of CMBs requires an imaging method that is capable of detecting iron-containing lesions with high sensitivity and spatial accuracy in the presence of potentially confounding tissue abnormalities. In this study, we investigated the feasibility of quantitative magnetic susceptibility mapping (QSM), a novel technique based on gradient-recalled echo (GRE) phase data, for the detection of CMBs in the arcAβ mouse, a mouse model of cerebral amyloidosis. Quantitative susceptibility maps were generated from phase data acquired with a high-resolution T2∗-weighted GRE sequence at 9.4 T. We examined the influence of different regularization parameters on susceptibility computation; a proper adjustment of the regularization parameter minimizes streaking artifacts and preserves fine structures. In the present study, it is shown that QSM provides increased detection sensitivity of CMBs and improved contrast when compared with GRE magnitude imaging. Furthermore, QSM corrects for the blooming effect observed in magnitude and phase images and depicts both the localization and spatial extent of CMBs with high accuracy. Therefore, QSM may become an important tool for diagnosing CMBs in neurological diseases.

Contrast-Enhanced Magnetic Resonance Microangiography Reveals Remodeling of the Cerebral Microvasculature in Transgenic ArcAβ Mice

Amyloid-β (Aβ) deposition in the cerebral vasculature is accompanied by remodeling which has a profound influence on vascular integrity and function. In the current study we have quantitatively assessed the age-dependent changes of the cortical vasculature in the arcAβ model of cerebral amyloidosis. To estimate the density of the cortical microvasculature in vivo, we used contrast-enhanced magnetic resonance microangiography (CE-μMRA). Three-dimensional gradient echo datasets with 60 μm isotropic resolution were acquired in 4- and 24-month-old arcAβ mice and compared with wild-type (wt) control mice of the same age before and after administration of superparamagnetic iron oxide nanoparticles. After segmentation of the cortical vasculature from difference images, an automated algorithm was applied for assessing the number and size distribution of intracortical vessels. With CE-μMRA, cerebral arteries and veins with a diameter of less than the nominal pixel resolution (60 μm) can be visualized. A significant age-dependent reduction in the number of functional intracortical microvessels (radii of 20–80 μm) has been observed in 24-month-old arcAβ mice compared with age-matched wt mice, whereas there was no difference between transgenic and wt mice of 4 months of age. Immunohistochemistry demonstrated strong fibrinogen and Aβ deposition in small- and medium-sized vessels, but not in large cerebral arteries, of 24-month-old arcAβ mice. The reduced density of transcortical vessels may thus be attributed to impaired perfusion and vascular occlusion caused by deposition of Aβ and fibrin. The study demonstrated that remodeling of the cerebrovasculature can be monitored noninvasively with CE-μMRA in mice.

In Vivo Imaging of the Inflammatory Receptor CD40 After Cerebral Ischemia Using a Fluorescent Antibody

Background and Purpose— Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Methods— Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Results— Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. Conclusions— The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Imaging of cerebrovascular pathology in animal models of Alzheimer's disease.

In Alzheimers disease (AD), vascular pathology may interact with neurodegeneration and thus aggravate cognitive decline. As the relationship between these two processes is poorly understood, research has been increasingly focused on understanding the link between cerebrovascular alterations and AD. This has at last been spurred by the engineering of transgenic animals, which display pathological features of AD and develop cerebral amyloid angiopathy to various degrees. Transgenic models are versatile for investigating the role of amyloid deposition and vascular dysfunction, and for evaluating novel therapeutic concepts. In addition, research has benefited from the development of novel imaging techniques, which are capable of characterizing vascular pathology in vivo. They provide vascular structural read-outs and have the ability to assess the functional consequences of vascular dysfunction as well as to visualize and monitor the molecular processes underlying these pathological alterations. This article focusses on recent in vivo small animal imaging studies addressing vascular aspects related to AD. With the technical advances of imaging modalities such as magnetic resonance, nuclear and microscopic imaging, molecular, functional and structural information related to vascular pathology can now be visualized in vivo in small rodents. Imaging vascular and parenchymal amyloid-β (Aβ) deposition as well as Aβ transport pathways have been shown to be useful to characterize their dynamics and to elucidate their role in the development of cerebral amyloid angiopathy and AD. Structural and functional imaging read-outs have been employed to describe the deleterious affects of Aβ on vessel morphology, hemodynamics and vascular integrity. More recent imaging studies have also addressed how inflammatory processes partake in the pathogenesis of the disease. Moreover, imaging can be pivotal in the search for novel therapies targeting the vasculature.

Early Alterations in Functional Connectivity and White Matter Structure in a Transgenic Mouse Model of Cerebral Amyloidosis

Impairment of brain functional connectivity (FC) is thought to be an early event occurring in diseases with cerebral amyloidosis, such as Alzheimers disease. Regions sustaining altered functional networks have been shown to colocalize with regions marked with amyloid plaques burden suggesting a strong link between FC and amyloidosis. Whether the decline in FC precedes amyloid plaque deposition or is a consequence thereof is currently unknown. The sequence of events during early stages of the disease is difficult to capture in humans due to the difficulties in providing an early diagnosis and also in view of the heterogeneity among patients. Transgenic mouse lines overexpressing amyloid precursor proteins develop cerebral amyloidosis and constitute an attractive model system for studying the relationship between plaque and functional changes. In this study, ArcAβ transgenic and wild-type mice were imaged using resting-state fMRI methods across their life-span in a cross-sectional design to analyze changes in FC in relation to the pathology. Transgenic mice show compromised development of FC during the first months of postnatal life compared with wild-type animals, resulting in functional impairments that affect in particular the sensory-motor cortex already in preplaque stage. These functional alterations were accompanied by structural changes as reflected by reduced fractional anisotropy values, as derived from diffusion tensor imaging. Our results suggest cerebral amyloidosis in mice is preceded by impairment of neuronal networks and white matter structures. FC analysis in mice is an attractive tool for studying the implications of impaired neuronal networks in models of cerebral amyloid pathology.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke

AIM Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Fluorescence tomography technique optimized for noninvasive imaging of the mouse brain

In vivo molecular fluorescence tomography of brain disease mouse models has two very specific demands on the optical setup: the use of pigmented furry mice does not allow for a purely noncontact setup, and a high spatial accuracy is required on the dorsal side of the animal due to the location of the brain. We present an optimized setup and tomographic scheme that meet these criteria through a combined CW reflectance-transmittance fiber illumination approach and a charge-coupled device contactless detection scheme. To consider the anatomy of the mouse head and take short source detector separations into account, the forward problem was evaluated by a Monte Carlo simulation input with a magnetic resonance image of the animal. We present an evaluation of reconstruction performance of the setup under three different condition. (i) Using a simulated dataset, with well-defined optical properties and low noise, the reconstructed position accuracy is below 0.5 mm. (ii) Using experimental data on a cylindrical tissue-simulating phantom with well-defined optical properties, a spatial accuracy of about 1 mm was found. (iii) Finally, on an animal model with a fluorescent inclusion in the brain, the target position was reconstructed with an accuracy of 1.6 mm.

Longitudinal Assessment of Amyloid Pathology in Transgenic ArcAβ Mice Using Multi-Parametric Magnetic Resonance Imaging.

Magnetic resonance imaging (MRI) can be used to monitor pathological changes in Alzheimers disease (AD). The objective of this longitudinal study was to assess the effects of progressive amyloid-related pathology on multiple MRI parameters in transgenic arcAβ mice, a mouse model of cerebral amyloidosis. Diffusion-weighted imaging (DWI), T1-mapping and quantitative susceptibility mapping (QSM), a novel MRI based technique, were applied to monitor structural alterations and changes in tissue composition imposed by the pathology over time. Vascular function and integrity was studied by assessing blood-brain barrier integrity with dynamic contrast-enhanced MRI and cerebral microbleed (CMB) load with susceptibility weighted imaging and QSM. A linear mixed effects model was built for each MRI parameter to incorporate effects within and between groups (i.e. genotype) and to account for changes unrelated to the disease pathology. Linear mixed effects modelling revealed a strong association of all investigated MRI parameters with age. DWI and QSM in addition revealed differences between arcAβ and wt mice over time. CMBs became apparent in arcAβ mice with 9 month of age; and the CMB load reflected disease stage. This study demonstrates the benefits of linear mixed effects modelling of longitudinal imaging data. Moreover, the diagnostic utility of QSM and assessment of CMB load should be exploited further in studies of AD.

Reconstructing Cerebrovascular Networks under Local Physiological Constraints by Integer Programming

We introduce a probabilistic approach to vessel network extraction that enforces physiological constraints on the vessel structure. The method accounts for both image evidence and geometric relationships between vessels by solving an integer program, which is shown to yield the maximum a posteriori (MAP) estimate to a probabilistic model. Starting from an overconnected network, it is pruning vessel stumps and spurious connections by evaluating the local geometry and the global connectivity of the graph. We utilize a high-resolution micro computed tomography (μCT) dataset of a cerebrovascular corrosion cast to obtain a reference network and learn the prior distributions of our probabilistic model and we perform experiments on in-vivo magnetic resonance microangiography (μMRA) images of mouse brains. We finally discuss properties of the networks obtained under different tracking and pruning approaches.

Unveiling Molecular Events in the Brain by Noninvasive Imaging

Neuroimaging allows researchers and clinicians to noninvasively assess structure and function of the brain. With the advances of imaging modalities such as magnetic resonance, nuclear, and optical imaging; the design of target-specific probes; and/or the introduction of reporter gene assays, these technologies are now capable of visualizing cellular and molecular processes in vivo. Undoubtedly, the system biological character of molecular neuroimaging, which allows for the study of molecular events in the intact organism, will enhance our understanding of physiology and pathophysiology of the brain and improve our ability to diagnose and treat diseases more specifically. Technical/scientific challenges to be faced are the development of highly sensitive imaging modalities, the design of specific imaging probe molecules capable of penetrating the CNS and reporting on endogenous cellular and molecular processes, and the development of tools for extracting quantitative, biologically relevant information from imaging data. Today, molecular neuroimaging is still an experimental approach with limited clinical impact; this is expected to change within the next decade. This article provides an overview of molecular neuroimaging approaches with a focus on rodent studies documenting the exploratory state of the field. Concepts are illustrated by discussing applications related to the pathophysiology of Alzheimer’s disease.