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Magnetic resonance imaging of amyloid plaques using hollow manganese oxide nanoparticles conjugated with antibody aβ1–40 in a transgenic mouse model

Kim, Jae-Huna; Ha, Tae Linc; Im, Geun Hod; Yang, Jehoond; Seo, Sang Wonb; Lee, In Sue; Lee, Jung Heea,d

doi: 10.1097/WNR.0b013e32835ba850
BRAIN IMAGING
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In this study, we have shown the feasibility of hollow manganese oxide nanoparticles (HMON) conjugated with an antibody of Aβ1–40 peptide (abAβ40) (HMON-abAβ40) for MRI of amyloid plaques in APP/PS1 transgenic mice. MR brain images in APP/PS1 transgenic mice and their nontransgenic littermates were acquired using a 7.0 T MRI system before, and 24 and 72 h after an injection of HMON-abAβ40. After the injection of HMON-abAβ40, we found hyperenhanced spots in the frontal cortex area on T1-weighted MR images for transgenic mice, which corresponded qualitatively to amyloid plaques detected by thioflavin-S staining. For quantitative analysis, percent MR signal changes in six brain regions (olfactory cortex, frontal cortex, cerebral cortex, thalamus, hippocampus, and cerebellar cortex) were compared between transgenic and wild-type mice. We found significant increases in the percent MR signal changes in the olfactory cortex, frontal cortex, cerebral cortex, and hippocampus, but there were no significant differences in the thalamus and cerebellar cortex for transgenic mice compared with wild-type mice. This unique strategy allowed us to detect brain regions subjected to amyloid plaque deposition in Alzheimer’s disease transgenic mouse models and has a potential to be developed for human applications, which has a current utility in preclinical research, particularly in monitoring therapeutic response for drug development in Alzheimer’s disease.

Departments of aRadiology

bNeurology, Samsung Medical Center,Sungkyunkwan University School of Medicine

cDepartment of Applied Chemistry, Kyung Hee University

dCenter for Molecular and Cellular Imaging, Samsung Biomedical Research Institute, Seoul

eDepartment of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang, Korea

Correspondence to Jung Hee Lee, Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Korea Tel: +82 23 410 6459; fax: +82 23 410 0084; e-mail: hijunghee@skku.edu

Received September 23, 2012

Accepted October 17, 2012

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Introduction

Alzheimer’s disease is the most common cause of dementia, which is pathologically characterized by amyloid plaques and neurofibrillary tangles 1. There is a prevailing concept that an imbalance between the production and clearance of amyloid β (Aβ) is the initiating event in the pathogenesis of Alzheimer’s disease 2. According to the amyloid cascade hypothesis, it initially causes the aggregation of Aβ, leading to amyloid plaques that may progress to pathological events including the formation of neurofibrillary tangles, cortical atrophy, and dementia.

Along with rapid developments of technologies for molecular imaging, researchers have attempted to develop a method for imaging amyloid plaques in vivo over the last decade. PET technology using 11C-PIB (Pittsburgh compound B) has been used for the detection of amyloid plaques in patients with Alzheimer’s disease 3–5. However, PIB-PET imaging has several limitations in the detection of individual amyloid plaques: a low spatial resolution without an anatomical background, the short half-life of the radioactive ligand, and a general radiation problem. In addition, there is a deficiency for high-affinity binding of PIB to detect Aβ deposits, which limits the use in transgenic mice 6,7.

Owing to the favorable safety and easy accessibility of MRI techniques along with the high spatial resolution anatomical images it provides, researchers have continuously attempted to develop MRI techniques to detect amyloid plaques in vivo8–17. High-resolution T2-weighted or T2*-weighted MRI has been applied for the noninvasive detection of amyloid plaques on the basis of the speculation that amyloid plaques contain iron deposits 8–11. However, intrinsic T2 or T2* contrast arising from iron often fails to distinguish iron in the plaques from those in blood vessels or hemorrhages. Furthermore, these techniques only allow the detection of iron-containing plaques on the basis of the content of the plaque, not the size of the plaque 11.

Several groups have developed molecular imaging techniques to visualize amyloid plaques in MRI using contrast agents that are conjugated with amyloid plaque-targeting ligands 14–17. Gadolinium-diethylenetriaminepentaacetic acid, monocrystalline iron oxide nanoparticles, or ultrasmall superparamagnetic iron oxide nanoparticles conjugated with a fragment of the Aβ peptide were applied to image amyloid plaques with the aid of mannitol or putrescine to open up the blood–brain barrier (BBB) 14–17. Their results showed that hyperenhanced T1 contrast of the amyloid plaques is more favorable for the specific detection of the plaques, excluding blood vessels or hemorrhages 14. However, nanoparticulated forms of iron oxide have been widely used in the field of molecular imaging for increasing sensitivity and as a facile method for conjugating the targeting ligands owing to the well-known properties of nanoparticulated forms.

Here, we show that hollow manganese oxide nanoparticles (HMON) developed in our laboratory can be used to detect amyloid plaques in Alzheimer’s disease transgenic mice. HMON is a T1-enhancing contrast agent that can be conjugated with targeting ligands on its surface and loaded with therapeutic agents in its central hollow area 18,19. In this report, we tested the feasibility of HMON conjugated with an antibody of Aβ1-40 (abAβ40) (HMON-abAβ40) for MRI of amyloid plaques in Alzheimer’s disease transgenic mice. For image analysis, we hypothesized that HMON-abAβ40 were accumulated and bound to plaques over time upon injection through the cisterna magna, whereas unbound HMON-abAβ40 washed out gradually. We used a template-based imaging processing technique for the quantification of amyloid plaques in the specific brain regions.

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Materials and methods

Preparation of HMON-abAβ40

HMON conjugated with abAβ40 to target amyloid plaque in Alzheimer’s disease transgenic mouse brain were prepared by conjugating the biotinylated antibody to the HMON-streptavidin hybrid (HMON-SA) nanoparticle, which has the readily connectable surface with biotinylated molecules. The HMON-SA, which carried five streptavidin proteins at the surface, was prepared using the previously reported procedure, which includes the successive steps of synthesis of HMON from an MnO–Mn3O4 core–shell nanoparticle, functionalizing the surface with biotins, and connecting the streptavidin proteins to the biotinylated surface 19.

For biotinylation of abAβ40, sulfo-NHS-biotin (0.5 ml, 4.4 mg/ml; EZ-Link Sulfo-NHS-biotin; Thermo Scientific, Rockford, Illinois, USA) was added to 3 ml of an aqueous solution containing CET (2 mg/ml) and incubated for 2 h with gentle shaking at 4°C. The biotinylated antibody was purified by eluting the resulting solution through a PD-10 column. To conjugate to HMON/SA, an aqueous suspension of HMON/SAs (1 mg) was added to 3 ml of an aqueous solution of biotinylated abAβ40. After the incubation for 2 h, nanoparticles were isolated from the reaction suspension by the centrifugation. The resulting HMON-abAβ40 was purified by repeating the redispersion in water and the centrifugation.

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Animal preparation

All experiments involving animals were performed according to a protocol approved by the Institutional Animal Care and Use Committee in the Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility and abided by the Institute of Laboratory Animal Resources guide. We used double transgenic mice expressing both a chimeric amyloid β-precursor protein (APP) and a mutant human presenilin 1 (PS1). Eight-week-old APP/PS1 transgenic mice were purchased from Jackson Laboratory (stock # 004462; Bar Harbor, Maine, USA) because this strain begins to develop amyloid plaques at 6–7 months of age and lead to abundant appearance in the hippocampus and cortex by 9 months of age 20. Mice were maintained as double hemizygotes by crossing with wild-type individuals on a C57BL/6 J F1 background strain (Jackson Laboratory). Offsprings were genotyped for the presence of the transgene by PCR amplification of genomic DNA extracted from tail biopsies. The transgenic mice (female, n=7) and their nontransgenic littermates (female, n=7) were randomly selected for this experiment. Because mice were required for in-vivo amyloid plaque imaging using MRI around 1 year of age, they were housed in the specific pathogen-free animal facility with free access to food and water (for 13 months).

For injection of the contrast agent through cisterna magna, the mice were anesthetized initially with 5% isoflurane in a mixture of O2 and N2 gases (3 : 7) delivered to a nosecone for spontaneous respiration and maintained with 1–2% isoflurane. A heating pad was used to maintain mice at physiological temperature throughout the procedure. The head of the mouse was carefully fixed using a bite/ear bar on a stereotaxic frame. HMON-abAβ40 was injected percutaneously into the cisterna magna using a 31 G needle at a rate of 1 µl/min (8 µl).

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In-vivo MRI

The MRI data were obtained using a 7.0 T MRI scanner (Bruker Biospin GmbH, Ettlingen, Germany) equipped with 400 mT/m actively shielded gradients with a 100 μs rise time. A birdcage coil (72 mm internal diameter) (Bruker Biospin, Fallanden, Switzerland) was used for excitation, and an actively decoupled phased array coil was used for receiving the signal. During MRI, the mice were under anesthesia with 1–2% isoflurane in a mixture of O2 and N2 gases (3 : 7) delivered to a nosecone for spontaneous respiration, and the body temperature was maintained at 36±1°C using a heating pad placed underneath while monitoring the mouse physiology. T1-weighted MR images were obtained using a 3D rapid acquisition with relaxation enhancement sequence (TR=200 ms; TE=7.5 ms), providing a 100 μm 3D isotropic resolution with an imaging time of 111 min. For each mouse, baseline image (D−1) was obtained before an injection of HMON-abAβ40. The HMON-abAβ40-enhanced MR images were obtained at 24 h (D+1) and 72 h (D+3) after injection. D+0 is the day of injection of a contrast agent.

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MRI data analysis

All MRI data were transformed into Analyze data format, and the brain skull was removed by manually drawing the brain region for all images. The spatial normalization process consists of two steps to ensure voxels’ correspondence between multiple images obtained at different scans for all mice. First, the skull-stripped mouse brain images were spatially normalized to the control C57B1/6 J mouse brain template using the SPM Mouse toolbox http://wbic.cam.ac.uk/~sjs80/spmmouse.html21, and then the normalized images were averaged to prepare the time-specific mean image across 14 mouse brain images for D−1, D+1, and D+3. Second, the skull-stripped mouse brain images were spatially normalized to the time-specific average images to avoid registration error induced by the enhancement of the contrast agent. Percent MR signal change was computed using the following formula:

where the background MR signal was measured from outside of the brain area. From the template of the mouse brain, six brain regions were chosen for analysis: olfactory cortex, frontal cortex, cerebral cortex, thalamus, hippocampus, and the cerebellar cortex.

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Histology

For staining amyloid plaques, the mouse was killed after MRI. The mouse was perfused and the brain was removed for fixation for 24 h. For thioflavin-S staining, the brain sections were mounted on slides and stained with a fresh filtered aqueous 1% thioflavin-S solution 12. The thioflavin-S-positive amyloid plaques were visualized under a fluorescence microscope.

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Statistical analysis

The differences in the mean change in the percent MR signal over time between wild type and Alzheimer’s disease transgenic mice were assessed by examining bar plots for each region of interest. For statistical analysis, we used the Mann–Whitney U-test to assess the difference in the percent MRI signal between the two groups. A P-value less than 0.05 was considered to be statistically significant only after Bonferroni’s correction for multiple comparisons [e.g. P<0.0167 (0.05/3)] for each brain region.

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Results

After injection of the contrast agent (HMON-abAβ40), we found hyperenhanced spots on T1-weighted MR images in Alzheimer’s disease transgenic mice. Figure 1 shows the HMON-abAβ40 enhanced T1-weighted MRI in an exemplar transgenic mouse. A few hyperenhanced spots were detected in the frontal cortex area, and these enhanced spots were corresponding to amyloid plaques detected by thioflavin-S staining. Figure 2 shows the imaging processing procedure for time-specific templates on D−1, D+1, and D+3. The mouse skull was removed manually and then mouse brain images were spatially normalized into the standard mouse brain. The distribution of intensity of mouse brain was different for each time point, because of different effects of image enhancement of the contrast agent over time. For time-specific mouse brain template, normalized mouse brains were averaged across 14 mouse brain images for each time point. The skull-stripped mouse brains were spatially normalized into time-specific templates for each time point. Figure 3 shows six specific brain areas and the statistical analysis of the mean changes in the percent MR signal between wild type and Alzheimer’s disease transgenic mice. A comparison of the mean percent MR signal of Alzheimer’s disease (n=7) and control mice (n=7) at D+3 showed that the Alzheimer’s disease group had a statistically higher mean percent MR signal than the control group in the olfactory cortex, frontal cortex, cerebral cortex, and hippocampus (P<0.05, corrected), but there were no significant differences in the thalamus and cerebellar cortex. Also, there were no significant differences of the percent MR signal between wild type and Alzheimer’s disease transgenic mice in any regions at D−1 and D+1.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

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Discussion

In this study, we have shown the ability of HMON-abAβ40 to detect amyloid plaques in the brain of Alzheimer’s disease transgenic mouse. They are visualized as hyperenhanced regions or spots in T1-weighted MR images, unlike hypoenhanced signals in T2-weighted or T2*-weighted MR images. These hyperenhanced spots correspond qualitatively to amyloid plaques detected by thioflavin-S stain.

For data analysis, we hypothesized that HMON-abAβ40 was accumulated and bound to the plaques over time upon injection through the cisterna magna, whereas the unbound HMON-abAβ40 washed out gradually. To ensure that these hyperenhanced MR signals are because of the accumulation of HMON-abAβ40 in amyloid plaques, we analyzed the mean percent signal change over time in the brain regions and compared them between wild type and Alzheimer’s disease transgenic mice. We chose the olfactory cortex, frontal cortex, cerebral cortex, and hippocampus for initial analysis, areas known to be sensitive to amyloid plaque deposition, and the thalamus and cerebellar cortex as control regions. Significant increases in the percent MR signals over time in the brain areas of olfactory cortex, frontal cortex, cerebral cortex, and hippocampus were observed for Alzheimer’s disease transgenic mice, contrasting with the results from wild-type mice. In agreement with previous studies 12,13,15,17, these brain areas are known to be particularly sensitive to amyloid plaque formation in Alzheimer’s disease transgenic mice. There were no differences in the percent MR signal changes in the thalamus and cerebellum between the Alzheimer’s disease transgenic and wild-type mice, as confirmed by our histology data as well as previous work that the thalamus and cerebellum are areas of the brain not subject to amyloid deposition in Alzheimer’s disease transgenic mouse models 22.

In other studies, an amyloid-labeling contrast agent was injected intravenously and delivered to the brain with the aid of mannitol or putrescine to open up the BBB 14–16. To utilize BBB breaking agents, the time to disrupt and the duration of BBB disruption need to be carefully determined, especially when coinjecting a contrast agent. To avoid these issues, we directly injected HMON-abAβ40 through the cisterna magna.

In our previous studies, we developed T1 contrast nanoparticles (HMON) as theragnostic MRI agents that have combined abilities as diagnostic imaging agents and vehicles to deliver therapeutic agents 18. We also showed that HMON conjugated with an antibody could be used for targeting specific tumor cells in vivo19. In this study, we show that HMON-abAβ40 can also be used for the detection of amyloid plaques in Alzheimer’s disease transgenic mice. The potential of HMON is that it can not only target amyloid plaques using amyloid targeting ligands attached to the surface of the HMON, but it can also be loaded with therapeutic agents in the interior void space. The current study is an initial step but shows a promising application of HMON for diagnosis and therapeutics in Alzheimer’s disease research.

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Conclusion

In this study, we developed a method to detect amyloid plaques in Alzheimer’s disease transgenic mouse using an HMON-abAβ40 contrast agent and a template-based imaging processing technique. This unique strategy could be useful for preclinical research related to Alzheimer’s disease, particularly in monitoring therapeutic response for drug development.

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Acknowledgements

This study was supported by the Korean Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A080855) (J.H.L.), the Basic Science Research Program through the National Research Foundation (NRF) grant funded by the Korean government (2010-0029410) (J.H.L.), and the National Research foundation of Korea (KRF) grant funded by the Korea government (MEST) (211–0017377 and 2010–0003950) (I.S.L.).

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Conflicts of interest

There are no conflicts of interest.

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Keywords:

Alzheimer’s disease; amyloid plaque; hollow manganese oxide nanoparticles; magnetic resonance imaging

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