Functionalization and Characterization of Magnetic Nanoparticles for the Detection of Ferritin Accumulation in Alzheimer's Disease

Early diagnosis in Alzheimer's disease (AD), prior to the appearance of marked clinical symptoms, is critical to prevent irreversible neuronal damage and neural malfunction that lead to dementia and death. Therefore, there is an urgent need to generate new contrast agents which reveal by a noninvasive method the presence of some of the pathological signs of AD. In the present study, we demonstrate for the first time a new nanoconjugate composed of magnetic nanoparticles bound to an antiferritin antibody, which has been developed based on the existence of iron deposits and high levels of the ferritin protein present in areas with a high accumulation of amyloid plaques (particularly the subiculum in the hippocampal area) in the brain of a transgenic mouse model with five familial AD mutations. Both in vitro and after intravenous injection, functionalized magnetic nanoparticles were able to recognize and bind specifically to the ferritin protein accumulated in the subiculum area of the AD transgenic mice.Fil: Fernández Cabada, Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Politécnica de Madrid; EspañaFil: Martínez Serrano, Alberto. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; EspañaFil: Cussó, Lorena. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Universidad Carlos III de Madrid; España. Centro de Investigación Biomédica en Red de Salud Mental; EspañaFil: Desco, Manuel. Instituto de Investigacion Sanitaria Gregorio Marañón; España. Centro de Investigación Biomédica en Red de Salud Mental; España. Universidad Carlos III de Madrid; EspañaFil: Ramos Gómez, Milagros. Universidad Politécnica de Madrid; Españ

Computed microtomography visualization and quantification of mouse ischemic brain lesion by nonionic radio contrast agents.

AIM: To explore the possibility of brain imaging by microcomputed tomography (microCT) using x-ray contrasting methods to visualize mouse brain ischemic lesions after middle cerebral artery occlusion (MCAO). ----- METHODS: Isolated brains were immersed in ionic or nonionic radio contrast agent (RCA) for 5 days and subsequently scanned using microCT scanner. To verify whether ex-vivo microCT brain images can be used to characterize ischemic lesions, they were compared to Nissl stained serial histological sections of the same brains. To verify if brains immersed in RCA may be used afterwards for other methods, subsequent immunofluorescent labeling with anti-NeuN was performed. ----- RESULTS: Nonionic RCA showed better gray to white matter contrast in the brain, and therefore was selected for further studies. MicroCT measurement of ischemic lesion size and cerebral edema significantly correlated with the values determined by Nissl staining (ischemic lesion size: P=0.0005; cerebral edema: P=0.0002). Brain immersion in nonionic RCA did not affect subsequent immunofluorescent analysis and NeuN immunoreactivity. ----- CONCLUSION: MicroCT method was proven to be suitable for delineation of the ischemic lesion from the non-infarcted tissue, and quantification of lesion volume and cerebral edema

Voxel-wise comparisons of cellular microstructure and diffusion-MRI in mouse hippocampus using 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND)

A key challenge in medical imaging is determining a precise correspondence between image properties and tissue microstructure. This comparison is hindered by disparate scales and resolutions between medical imaging and histology. We present a new technique, 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND), for registering medical images with 3D histology to overcome these limitations. Ex vivo 120 × 120 × 200 μm resolution diffusion-MRI (dMRI) data was acquired at 7 T from adult C57Bl/6 mouse hippocampus. Tissue was then optically cleared using CLARITY and stained with cellular markers and confocal microscopy used to produce high-resolution images of the 3D-tissue microstructure. For each sample, a dense array of hippocampal landmarks was used to drive registration between upsampled dMRI data and the corresponding confocal images. The cell population in each MRI voxel was determined within hippocampal subregions and compared to MRI-derived metrics. 3D-BOND provided robust voxel-wise, cellular correlates of dMRI data. CA1 pyramidal and dentate gyrus granular layers had significantly different mean diffusivity (p > 0.001), which was related to microstructural features. Overall, mean and radial diffusivity correlated with cell and axon density and fractional anisotropy with astrocyte density, while apparent fibre density correlated negatively with axon density. Astrocytes, axons and blood vessels correlated to tensor orientation

Reward circuitry is perturbed in the absence of the serotonin transporter

The serotonin transporter (SERT) modulates the entire serotonergic system in the brain and influences both the dopaminergic and norepinephrinergic systems. These three systems are intimately involved in normal physiological functioning of the brain and implicated in numerous pathological conditions. Here we use high-resolution magnetic resonance imaging (MRI) and spectroscopy to elucidate the effects of disruption of the serotonin transporter in an animal model system: the SERT knock-out mouse. Employing manganese-enhanced MRI, we injected Mn^(2+) into the prefrontal cortex and obtained 3D MR images at specific time points in cohorts of SERT and normal mice. Statistical analysis of co-registered datasets demonstrated that active circuitry originating in the prefrontal cortex in the SERT knock-out is dramatically altered, with a bias towards more posterior areas (substantia nigra, ventral tegmental area, and Raphé nuclei) directly involved in the reward circuit. Injection site and tracing were confirmed with traditional track tracers by optical microscopy. In contrast, metabolite levels were essentially normal in the SERT knock-out by in vivo magnetic resonance spectroscopy and little or no anatomical differences between SERT knock-out and normal mice were detected by MRI. These findings point to modulation of the limbic cortical–ventral striatopallidal by disruption of SERT function. Thus, molecular disruptions of SERT that produce behavioral changes also alter the functional anatomy of the reward circuitry in which all the monoamine systems are involved

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn^(2+) into the prefrontal cortex indicated that DAT KO mice have a truncated Mn^(2+) distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn^(2+) transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here

Delineating neuroinflammation, parasite CNS invasion, and blood-brain barrier dysfunction in an experimental murine model of human African trypanosomiasis

Although Trypanosoma brucei spp. was first detected by Aldo Castellani in CSF samples taken from sleeping sickness patients over a century ago there is still a great deal of debate surrounding the timing, route and effects of transmigration of the parasite from the blood to the CNS. In this investigation, we have applied contrast-enhance magnetic resonance imaging (MRI) to study the effects of trypanosome infection on the blood-brain barrier (BBB) in the well-established GVR35 mouse model of sleeping sickness. In addition, we have measured the trypanosome load present in the brain using quantitative Taqman PCR and assessed the severity of the neuroinflammatory reaction at specific time points over the course of the infection. Contrast enhanced – MRI detected a significant degree of BBB impairment in mice at 14 days following trypanosome infection, which increased in a step-wise fashion as the disease progressed. Parasite DNA was present in the brain tissue on day 7 after infection. This increased significantly in quantity by day 14 post-infection and continued to rise as the infection advanced. A progressive increase in neuroinflammation was detected following trypanosome infection, reaching a significant level of severity on day 14 post-infection and rising further at later time-points. In this model stage-2 disease presents at 21 days post-infection. The combination of the three methodologies indicates that changes in the CNS become apparent prior to the onset of established stage-2 disease. This could in part account for the difficulties associated with defining specific criteria to distinguish stage-1 and stage-2 infections and highlights the need for improved staging diagnostics

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the R6/2 Mouse Model of HD

Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6/2 mouse model of HD expresses a mutant version of exon 1 HTT and develops motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Despite the vast number of studies that have been performed on this model, the association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood. In an attempt to link these factors, we have performed longitudinal assessments of behavior (rotarod, open field, passive avoidance) and of regional brain abnormalities determined through magnetic resonance imaging (MRI) (whole brain, striatum, cortex, hippocampus, corpus callosum), as well as an end-stage histological assessment. Detailed correlative analyses of these three measures were then performed. We found a gender-dependent emergence of motor impairments that was associated with an age-related loss of regional brain volumes. MRI measurements further indicated that there was no striatal atrophy, but rather a lack of striatal growth beyond 8 weeks of age. T2 relaxivity further indicated tissue-level changes within brain regions. Despite these dramatic motor and neuroanatomical abnormalities, R6/2 mice did not exhibit neuronal loss in the striatum or motor cortex, although there was a significant increase in neuronal density due to tissue atrophy. The deposition of the mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the brain. End-stage histopathological assessments were not found to be as robustly correlated with the longitudinal measures of brain atrophy or motor impairments. In conclusion, modeling pre-manifest and early progression of the disease in more slowly progressing animal models will be key to establishing which changes are causally related. © 2013 Rattray et al

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4.

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)(+) neuroblasts that migrate and integrate into olfactory bulb circuitry. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4), a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4(hi) astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creER(tm)4 lineage tracing demonstrated that it is these SVZ-generated Thbs4(hi) astrocytes, and not Dcx(+) neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production. Consequently, Thbs4 homozygous knockout mice (Thbs4(KO/KO)) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4(KO/KO) mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles