Faculty Bibliography

Transgenic mice are used increasingly to model brain amyloidosis, mimicking the pathogenic processes involved in Alzheimer's disease (AD). In this chapter, an in vivo strategy is described that has been successfully used to map amyloid-beta deposits in transgenic mouse models of AD with magnetic resonance imaging (MRI), utilizing both the endogenous contrast induced by the plaques attributed to their iron content and by selectively enhancing the signal from amyloid-beta plaques using molecular-targeting vectors labeled with MRI contrast agents. To obtain sufficient spatial resolution for effective and sensitive mouse brain imaging, magnetic fields of 7-Tesla (T) or more are required. These are higher than the 1.5-T field strength routinely used for human brain imaging. The higher magnetic fields affect contrast agent efficiency and dictate the choice of pulse sequence parameters for in vivo MRI, all addressed in this chapter. Two-dimensional (2D) multi-slice and three-dimensional (3D) MRI acquisitions are described and their advantages and limitations are discussed. The experimental setup required for mouse brain imaging is explained in detail, including anesthesia, immobilization of the mouse's head to reduce motion artifacts, and anatomical landmarks to use for the slice alignment procedure to improve image co-registration during longitudinal studies and for subsequent matching of MRI with histology.

Background: Immunotherapy targeting hyperphosphorylated tau is a promising prospect to mitigate the neurodegenerative effects of tauopathies. Assessing the effectiveness of such immunotherapies often involves sacrifice of the animal. However, Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) permits the longitudinal study of neuronal function with minimal risk to the animal. We hypothesize that tract-tracing MEMRI in a mouse model of tau pathology should enable non-invasive monitoring of various tau targeting therapies aimed at improving neuronal integrity. Methods: Twenty-five homozygous JNPL3 tangle transgenic mice underwent MEMRI at 6 months of age. Thirteen of the mice received tau immunotherapy with Tau379-408[P-Ser396,404] in alum adjuvant from 3 months of age, and twelve controls received an adjuvant alone. Imaging studies were performed on a 7-T micro-MRI. Mice were imaged pre-injection, then injected in one nostril with a solution of 2.5 M MnCl 2, under isoflurane anesthesia. Image sets were acquired at 1, 4, 8, 12, 24, 36 and 48 hours, and finally at 7 days (Fig 1). The datasets were processed using ImageJ. Normalized measurements for each mouse were plotted and fitted to a tract tracing bolus model using MATLAB. Fitting enabled the estimation of the timing (Pt) and intensity (Pv) of the bolus peak of Mn, and maximal slope of uptake (Sv). Results: A significant increase in maximal slope of manganese uptake, Sv, was observed in the mitral cell layer (35%, P <.005) and glomerular layer (36%, P <0.02) in treated JNPL3 mice compared to identical controls. There was also a significant increase in bolus peak value, Pv, in the mitral layer in the treated group (7%, P = 0.02). Furthermore, in the immunized mice, there was a strong trend for a decrease in the time to peak value, Pt (-9%P = 0.10), in the mitral cell layer, compared to the controls. Conclusions: Utilizing MEMRI's non-invasive, longitudinal measurements from 1 hour to 7 days, allowed us to detect substantial improvements in neuronal transport following tau immunotherapy. We are analyzing tau pathology in olfactory sections from these mice to assess the correlation of these benefits with clearance of tau lesions, which we have shown previously to occur with this treatment

Background: Amyloid plaques are a key pathological hallmark of Alzheimer's disease (AD). Their visualization is important for the diagnosis of AD, monitoring disease progression and evaluation of the efficacy of therapeutic interventions. We were the first group to use ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coupled to an amyloid targeting peptide to visualize amyloid plaques. USPIO's have been widely used in animal and human imaging, and have been found to be very safe. However, our approach, so far, has required intra-carotid or intra-femoral injection, with mannitol to break the blood brain barrier (BBB). In the current study we sought to develop and test non-toxic, bifunctional USPIO particles which could be introduced via the femoral vein, without mannitol. Methods: 13 to 18 month old APP/PS1 transgenic mice and age-matched wild-type (C57Bl/6J) control mice were used. The USPIO nanoparticles were linked to Ab1-42 (targeting to amyloid plaques) and polyethyleneglycol (PEG, to increase BBB permeability), using EDC/NHS coupling methods. The potential neurotoxicity of USPIO-PEG-Ab42 was assessed in N2a cells using a MTS Assay. MRI scans were performed on a 7T micro- MRI system consisting of a 7-Telsa 200-mm horizontal bore magnet. All mice were scanned 6-hrs after iv injection (0.2 mmol Fe/kg body weight) of the USPIO-PEG-Ab42. Ex vivo imaging of mouse brains was also performed. Serial coronal sections were subject to anti-Ab immunohistochemistry and Perl staining to identify the USPIO. Results: The USPIO particles were non-toxic. Figure 1A showsmMRI and matching histology in an APP/ PS1 Tg mouse and wild type mouse injected with USPIO-PEG-Ab. Numerous dark spots are evident in 1A. To the right of 1A is a higher magnification of an area of the MRI which is matched to double Perl and Ab stain in figure 1B. A coronal section matching to 1A is seen in 1C. In 1D the blue areas point to Perl stain positive dots corresponding to USPIO particles while the red arrow points to the amyloid plaque. 1E shows a wild-type mouse injected with USPIO-PEG-Ab. 1F documents the lack of plaques in the WT mouse. Conclusions: Our non-toxic, non-invasive m MRI method has great potential for the longitudinal assessment of amyloid burden

Background: Immunotherapy targeting hyperphosphorylated tau is a promising prospect to mitigate the neurodegenerative effects of tauopathies. Assessing the effectiveness of such immunotherapies often involves sacrifice of the animal. However, Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) permits the longitudinal study of neuronal function with minimal risk to the animal. We hypothesize that tract-tracing MEMRI in a mouse model of tau pathology should enable non-invasive monitoring of various tau targeting therapies aimed at improving neuronal integrity. Methods: Twenty-five homozygous JNPL3 tangle transgenic mice underwent MEMRI at 6 months of age. Thirteen of the mice received tau immunotherapy with Tau379-408[P-Ser396,404] in alum adjuvant from 3 months of age, and twelve controls received an adjuvant alone. Imaging studies were performed on a 7-T micro-MRI. Mice were imaged pre-injection, then injected in one nostril with a solution of 2.5 M MnCl 2, under isoflurane anesthesia. Image sets were acquired at 1, 4, 8, 12, 24, 36 and 48 hours, and finally at 7 days (Fig 1). The datasets were processed using ImageJ. Normalized measurements for each mouse were plotted and fitted to a tract tracing bolus model using MATLAB. Fitting enabled the estimation of the timing (Pt) and intensity (Pv) of the bolus peak of Mn, and maximal slope of uptake (Sv). Results: Asignificant increase in maximal slope of manganese uptake, Sv, was observed in the mitral cell layer (35%, P <.005) and glomerular layer (36%, P <0.02) in treated JNPL3 mice compared to identical controls. There was also a significant increase in bolus peak value, Pv, in the mitral layer in the treated group (7%, P = 0.02). Furthermore, in the immunized mice, there was a strong trend for a decrease in the time to peak value, Pt (-9%P = 0.10), in the mitral cell layer, compared to the controls. Conclusions: Utilizing MEMRI's non-invasive, longitudinal measurements from 1 hour to 7 days, allowed us to detect substantial improvements in neuronal transport following tau immunotherapy. We are analyzing tau pathology in olfactory sections from these mice to assess the correlation of these benefits with clearance of tau lesions, which we have shown previously to occur with this treatment

Amyloid plaques are one of the pathological hallmarks of Alzheimer's disease (AD). The visualization of amyloid plaques in the brain is important to monitor AD progression and to evaluate the efficacy of therapeutic interventions. Our group has developed several contrast agents to detect amyloid plaques in vivo using magnetic resonance microimaging (muMRI) in AD transgenic mice, where we used intra-carotid mannitol to enhance blood-brain barrier (BBB) permeability. In the present study, we used ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, chemically coupled with Abeta1-42 peptide to detect amyloid deposition along with mannitol for in vivo muMRI by femoral intravenous injection. A 3D gradient multi-echo sequence was used for imaging with a 100mum isotropic resolution. The amyloid plaques detected by T2*-weighted muMRI were confirmed with matched histological sections. Furthermore, two different quantitative analyses were used. The region of interest-based quantitative measurement of T2* values showed contrast-injected APP/PS1 mice had significantly reduced T2* values compared to wild-type mice. In addition, the scans were examined with voxel-based morphometry (VBM) using statistical parametric mapping (SPM) for comparison of contrast-injected AD transgenic and wild-type mice. The regional differences seen in VBM comparing USPIO-Abeta1-42 injected APP/PS1 and wild-type mice correlated with the amyloid plaque distribution histologically, contrasting with no differences between the two groups of mice without contrast agent injection in regions of the brain with amyloid deposition. Our results demonstrated that both approaches were able to identify the differences between AD transgenic mice and wild-type mice, after injected with USPIO-Abeta1-42. The feasibility of using less invasive intravenous femoral injections for amyloid plaque detection in AD transgenic mice facilitates using this method for longitudinal studies in the pathogenesis of AD

Axonal transport perturbations are known to play a critical role in the pathological progression of Alzheimer's disease (AD); and Manganese-Enhanced MRI (MEMRI) provides a unique, non-invasive tool allowing for the in vivo evaluation of transport deficits in preclinical studies. In this paper, we provide a brief history of MEMRI, and review the current literature describing its biological basis. We propose a model of how manganese transport reflects both axonal and dendritic transport (termed 'neuronal transport'), and potentially, mitochondrial trafficking in neurons. A framework for the analysis of MEMRI data is provided. It summarizes the significance of the various parameters describing manganese transport and the pathophysiological events that can alter their relevance, such as neuronal loss, gliosis and excitotoxicity. Lastly, we review publications describing different animal models of AD pathology that suggest the expression of either mutated human tau or mutated human amyloid beta alters neuronal transport, as measured by MEMRI. In this way, MEMRI correlates the in vitro observation of impaired axonal transport and mitochondrial mislocalization related to AD lesions, with direct in vivo data. Therefore, MEMRI has the potential to become a unique tool for assessing the effect of new AD treatments aimed at restoring neuronal transport and mitochondrial trafficking

Vascular system development involves a complex, three-dimensional branching process that is critical for normal embryogenesis. In the brain, the arterial systems appear to develop in a stereotyped fashion, but no detailed quantitative analyses of the mouse embryonic cerebral arteries have been described. In this study, a gadolinium-based contrast perfusion method was developed to selectively enhance the cerebral arteries in fixed mouse embryos. Three-dimensional magnetic resonance micro-imaging (micro-MRI) data were acquired simultaneously from multiple embryos staged between 10 and 17 days of gestation, and a variety of image analysis methods was used to extract and analyze the cerebral arterial patterns. The results show that the primary arterial branches in the mouse brain are very similar between individuals, with the patterns established early and growth occurring by extension of the segments, while maintaining the underlying vascular geometry. To investigate the utility of this method for mutant mouse phenotype analysis, contrast-enhanced micro-MRI data were acquired from Gli2(-/-) mutant embryos and their wild-type littermates, showing several previously unreported vascular phenotypes in Gli2(-/-) embryos, including the complete absence of the basilar artery. These results demonstrate that contrast-enhanced micro-MRI provides a powerful tool for analyzing vascular phenotypes in a variety of genetically engineered mice

This paper describes colloidal particles that are designed to induce hyper-intensity contrast (T(1) relaxation) in MRI. The contrast agents consist of discrete gadolinium complexes tethered to 10 nm diameter silver nanoparticles. The gadolinium complexes (1) [Gd(DTPA-bisamido cysteine)](2-) and (2) [Gd(cystine-NTA)(2)](3-), undergo chemisorption to particle surfaces through thiol or disulfide groups, respectively, to form two new contrast agents. The resulting nanoparticulate constructs are characterized on the basis of their syntheses, composition, spectra and contrast enhancing power. The average r(1) relaxivities of the of the surface bound complexes (obtained at 9.4 T and 25 degrees C) are 10.7 and 9.7 s(-1) mM(-1), respectively, as compared to 4.7 s(-1) mM(-1) for the clinical agent Magnevist. Correspondingly, the respective whole particle relaxivities are 27927 and 13153 s(-1) mM(-1)