MR microimaging detects subfield-specific neuronal loss in the hippocampus

Poster Presentations P2

P2-351

FRAMIGHAM CARDIOVASCULAR RISK PROFILE CORRELATES WITH IMPAIRED HIPPOCAMPAL AND CORTICAL PERFUSION

Lidia Glodzik1, Henry Rusinek2, Miroslaw Brys3, Wai Tsui1, Elizabeth Pirraglia1, Lisa Mosconi1, Susan De Santi1,4, Yi Li1, Mony de Leon1,5, 1Center for Brain Health, Center of Excellence on Brain Aging, NYUSM, New York, NY, USA; 2Department of Radiology, NYUSM, New York, NY, USA; 3NYU Langone Medical Center, New York, NY, USA; 4 MedAvante Inc., Hamilton, NJ, USA; 5Nathan Kline Institute, Orangeburg, NY, USA. Contact e-mail: [email protected] Background: Contribution of vascular disease to AD pathology continues to be debated with the hope that modification of vascular risk would modify the course of AD. Vascular risk factors reduce cerebral blood flow (CBF) and cerebral vascular reactivity (VR) to hypercapnia, and thus contribute to tissue damage and cognitive decline. Hippocampal hemodynamic changes may constitute a mechanism linking atherosclerosis with cognitive impairment, given its considerable vulnerability to ischemia. Nonetheless, the information about the impact of vascular risk on hippocampal perfusion is minimal. Methods: Eighteen cognitively healthy elderly (NL, mean age 69.9 6 6.7) and 15 subjects with mild cognitive impairment (MCI, mean age 74.9 6 8.1), all without previous cerebro-vascular accident, were evaluated for a vascular disease burden with the Framingham Cardiovascular Risk Profile (FCRP). All underwent structural imaging and resting CBF assessment with arterial spin labeling at 3T MRI system. To reduce hippocampal susceptibility artifacts we used true fast imaging in steady precession sequence (TrueFISP). In twenty four subjects (NL ¼ 17, MCI ¼ 7), CBF was also measured after a carbon dioxide (CO2) re-breathing challenge Results: Across all subjects FCRP negatively correlated with hippocampal VR (rho ¼ -.41, p ¼ .049) and global cortical VR (rho ¼ -.46, p ¼ .02). The FCRP-VR relationships were most pronounced in the MCI group: hippocampus (rho ¼ -.77, p ¼ .04) and global cortex (rho ¼ -.83, p ¼ .02). No FCRP relationships were found for either volume or resting CBF measurements. The hippocampal VR was lower in MCI than in NL subjects (Z ¼ -2.1, p ¼ .04). This difference disappeared, however, after correction for FCRP. Conclusions: An elevated risk for vascular pathology is associated with a reduced dilatory response to CO2 in both hippocampal and cortical tissues. The VR is better related to an increased vascular risk than either resting CBF or brain volume and is compromised in the MCI stages of cognitive impairment. This last effect, however, could be at least in part attributed to the presence of vascular risk, emphasizing the contribution of vascular pathology to cognitive decline even in stroke-free subjects.

P2-352

HIGH RESOLUTION MRI REVEALS REDUCED HIPPOCAMPAL VOLUME IN APOE4 TARGETED REPLACEMENT MICE

Jesse M. Hunter1, Michael D. Cockman2, Allen D. Kline1, Scott Surridge3, Emily C. Rothstein1, Hong Liu2, Mingming Zhu2, Harlan Shannon2, Melissa Trowbridge2, Daniel A. Peterson3, Letia D. Peterson3, Patrick M. Sullivan4, Krish Krishnamurthy1, Micheal L. Hutton1, Steven M. Paul1, 1Eli Lilly and Company, Indianapolis, IN, USA; 2Covance Laboratories, Greenfield, IN, USA; 3Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Durham VA Medical Center, Duke University, Durham, NC, USA. Contact e-mail: hunterje@lilly. com Background: The epsilon 4 (E4) allele of apolipoprotein E (apoE) is the most significant genetic risk factor for developing late-onset Alzheimer’s disease (AD). Cognitively normal apoE4 carriers have altered glucose utilization in brain regions affected in AD as determined by PET. Functional MRI and EEG measurements during memory tasks suggest altered connectivity and broader utilization of brain regions even in young cognitively healthy E4 carriers. E4 is also associated with smaller hippocampal volume and faster rates of atrophy in AD patients. Hippocampal atrophy is also often found in epilepsy. We recently reported the emergence of a seizure

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phenotype in aged apoE4 targeted replacement (TRE4) mice. Methods: To investigate structural brain changes in TRE4 mice, we performed high resolution in vivo MRI of young and aged TR mice with a 7 Tesla Varian spectrometer. Thirty-three slice images (250mm thickness, 98x98mm inplane resolution, no gap) were acquired for each mouse. Image analysis was performed using custom software or hand drawn regions of interest to determine volumes. All imaging and analysis was done blinded. Results: We report that hippocampal volume is reduced in aged TRE4 mice compared to TRE2 and TRE3 mice. Although young mice do not display a seizure phenotype, young female mice have reduced hippocampal volume and increased ventricular volume suggesting that apoE4 may result in reduced hippocampal development and predisposition to seizures. Seizure phenotype in TRE4 male mice is not completely penetrant, and aged TRE4 male mice with a severe seizure phenotype have reduced hippocampal volume in comparison to aged TRE4 male mice with very mild or no seizure phenotype suggesting a relationship between seizures and hippocampal volume in TRE4 mice. Stereological analysis of the hippocampus of TR mice is in progress and may reveal the cellular basis of the reduced hippocampal volume measured by MRI. Diffusion tensor imaging is also being employed to determine if TRE4 mice have further structural changes detectible by imaging. Conclusions: Our results provide evidence that old and young TRE4 mice have structural brain changes (reduced hippocampal volume) compared to TRE3 or TRE2 mice, suggesting a role for apoE4 in hippocampal integrity and possibly development.

P2-353

MR MICROIMAGING DETECTS SUBFIELDSPECIFIC NEURONAL LOSS IN THE HIPPOCAMPUS

Palamadai N. Venkatasubramanian1,2, Benjamin Banks3, George Iordanescu1,2, Jason C. Pych1,2, Alice M. Wyrwicz1,2, 1NorthShore University HealthSystem Research Institute, Evanston, IL, USA; 2Pritzker School of Medicine, University of Chicago, Chicago, IL, USA; 3Northwestern University, Evanston, IL, USA. Contact e-mail: pnvenkat2004@yahoo. com Background: Alzheimer’s disease is characterized by extensive neuronal loss in brain areas critical for learning and memory. Most APP transgenic mouse models of AD, however, do not demonstrate neuronal loss. Using MR microscopy we have detected neuronal loss specifically in the CA1 subfield of the hippocampus at the age of onset of impairment in hippocampally dependent behavioral tasks in a transgenic mouse that coexpresses five FAD mutations. Methods: Female 5xFAD transgenic mice coexpressing five FAD mutations [3 APP + 2 PS1] were used. Littermate wildtype mice were used as controls. At 2, 4 and 10 months of age, their performance in water maze and spontaneous alternation tasks was measured. Subsequently, brains fixed in paraformaldehyde were used for MR microimaging at 14.1T. T2-weighted spin-echo 3D images were acquired using the following imaging parameters: TR/TE 2500ms/40ms and isotropic voxel size 35mm. Results: MR microimaging provided a detailed view of neuroanatomy, particularly the subfields of the hippocampus. Images revealed differences between the genotypes in the MR contrast of the pyramidal cell layer. In 2M transgenic mice, the CA1 pyramidal cell layer appears as a solid, hypointense layer. In 4M transgenic mice, contrast diminished and in 10M transgenic mice, the contrast of the CA1 pyramidal cells was lost entirely. CA1 cell layer of wildtype mice, on the other hand, demonstrated no such age-dependent change in MR appearance. The observed loss of MR contrast suggests loss of CA1 pyramidal neurons as 5xFAD mice aged. Manual segmentation of CA1 pyramidal cell pixels revealed >60% decrease in volume between 2 and 4 months, as well as between 4 and 10 months in 5xFAD mice. CA2/ 3 volume did not change. Performance of 5xFAD mice in water maze and spontaneous alternation, hippocampally dependent tasks, began to decline at 4 months and worsened at 10 months, which coincides with the temporal pattern of neuronal loss in the CA1 subfield. Conclusions: Loss of CA1 neurons may be a critical step in the onset of AD neuropathology. MR microscopy could be a valuable tool to follow disease progression and assess

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Poster Presentations P2

therapy in preclinical studies that employ 5xFAD mice. (Supported by R01AG027424 and S10RR13880) P2-354

DETECTION OF AMYLOID PLAQUES IN ALZHEIMER’S TRANSGENIC MICE USING CONTRAST-ENHANCED MAGENTIC RESONANCE MICROIMAGING

Jing Yang, Minh Dung Hoang, Youssef Zaim Wadghiri, Thomas Wisniewski, NYU School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Amyloid plaques are a pathological hallmark of Alzheimer’s disease (AD). The visualization of amyloid plaquesis important to monitor AD progression and to evaluate efficacy of therapeutic interventions. Our group has developed several contrast-enhancing agents to detect amyloid plaques in vivo in AD transgenic mice using magnetic resonance microimaging (mMRI). However, each of our past studies involved intra-carotid injection of ligands in order to gain sufficient blood-brain-barrier penetration. This is highly invasive and not feasible for human studies. Therefore, a safer and less invasive method of ligand administration is required. In present study, we use ultrsamll superparamagnetic iron oxide (USPIO) nanoparticles to detect amyloid deposition for in vivo mMRI by femoral injection. Methods: mMRI was performed on APPK670N/M671L/PS1M146L transgenic AD mice and wild-type mice prior to and following intrafemoral injection with USPIO coupled to Ab1-42 in 15% mannitol solution. A 3D gradient echo T2*weighted sequence was used for imaging (TE ¼ 4 ms; TR ¼ 80 ms; flip angle ¼ 20 ), providing a 100 mm isotropic resolution with imaging times of 2h 20min. After in vivo mMRI, mice were anesthetized with sodium pentobarbital and perfused transaortically with PBS followed by 4% paraformaldehyde, and their brains were extracted for ex vivo MRI and histological analysis. Results: Amyloid plaques were detected in AD transgenic mice after intrafemoral injection of USPIO-Ab1-42 along with mannitol. The distribution of plaques detected by MRI was in excellent agreement with those seen in matched histological sections. In addition, the quantitative measurement of T2* values showed contrast injected APP/PS1mice have significantly reduced T2* values compared to wild-type mice. This reduction correlated with the amyloid burden determined histologically in APP/PS1 mice. Conclusions: Our results demonstrate that it is feasible to perform intrafemoral injection for amyloid plaque detection in AD transgenic mice using USPIO-Ab1-42 contrast ligands. USPIO has already been used in humans for CNS imaging in conditions such as multiple sclerosis and stroke. Hence if we are able to better develop this technology, this has potential to be used diagnostically in patients.

P2-355

FOLATE DEFICIENCY IMPAIRS BRAIN OXYGEN DELIVERY IN RAT: DETECTION BY NONINVASIVE NEAR INFRARED SPECTROSCOPY

Aron M. Troen, Bertan Hallacoglu, Angelo Sassaroli, Irwin H. Rosenberg, Sergio Fantini, Tufts University, Boston, MA, USA. Contact e-mail: aron. [email protected] Background: Dietary vascular risk factors, including poor folate status are powerful and potentially modifiable predictors of cognitive impairment among older adults. The prevalence of folate deficiency has been estimated at 5-10% of middle aged and elderly adults exposed to food folate fortification, and as much as 30% in unfortified populations. In mouse, we have shown consuming a diet deficient in vitamins folate, B12 and B6 to cause a 30% decrease in brain capillary density in association with significantly impaired performance on the Morris water maze. Similarly, folate deficiency in rat impairs cognition and causes cerebral microvascular damage, without concomitant neurodegeneration. Based on these observations we hypothesized that folate deficiency should result in functional decrements in cerebral blood volume, oxygen delivery and vascular reactivity, all of which should be detectable non-invasively by near-infrared spectroscopy (NIRS). Methods: We developed a NIRS device for non-invasive evaluation of these parameters in rat. We used this device to measure absolute tissue concentra-

tions of oxygen free and bound hemoglobin, and tissue oxygen saturation, and related hemodynamic parameters at rest and during transient mild hypoxia and hypercapnia, in Sprague Dawley rats folate deficient diets for up to 20 weeks. Results: Total hemoglobin concentration [tHb] was 23% lower in folate deficient than in control rats at baseline, hypoxia and recovery (73 6 10 vs. 95 6 14 mM tHb (Mean 6 SD); p < 0.02). Brain tissue oxygen saturation (StO2%) was significantly lower in folate deficient than in control rats with baseline and recovery values of 55% 6 7% vs. 66% 6 4% (p < 0.03), and 43% 6 8% vs. 55% 6 6% (p < 0.01) during hypoxia for folate deficient vs. control rats respectively. However the change in brain StO2 during hypoxia was similar in both groups with a -13% 6 2% for control rats and -12% 6 4% for folate deficient rats. In contrast, arterial oxygen saturation did not differ by diet with baseline values of 96% 6 2% for both groups, indicating that the decrements detected by NIRS in brain were independent of blood oxygen carrying capacity. Conclusions: Our results point to microvascular rarefaction and diminished oxygen delivery as a basis of folate-deficiency associated cognitive impairment.

P2-356

GUIDING ANTI-RAGE MONOCLONAL ANTIBODY DEVELOPMENT FOR THE TREATMENT OF ALZHEIMER’S DISEASE VIA IMAGING

Feng Luo1, Gerard B. Fox1, Terese Seifert1, Prasant Chandran1, Zhen Wu2, Eve Barlow2, Jijie Gu2, Mary Leddy2, Chung-Ming Hsieh2, John Harlan3, Enrico DiGianmarrino2, Karl Walter3, Shaun McLoughlin3, Mark Day1, Hans Schoemaker4, Ralf Loebbert4, Alfred Hahn4, Volker Nimmrich4, Martin Schmidt4, 1Translational Imaging and Biomarkers, Abbott Laboratories, Abbott Park, IL, USA; 2Abbott Bioresearch Center, Abbott Laboratories, Worcester, MA, USA; 3Structural biology, Abbott Laboratories, Abbott Park, IL, USA; 4Neuroscience Research, Abbott Laboratories, Ludwigshafen, Germany. Contact e-mail: [email protected] Background: In contrast to the advancements seen in the employment of imaging in Alzheimer’s disease (AD) clinical trials, few studies have been reported that utilize imaging in animals for the development and translation of potential disease modifying agents. Receptor-for Advanced Glycation Endproducts, RAGE, is a receptor for amyloid-b (Ab) and other patholigands potentially contributing to the pathology in AD. Results obtained with Ab-overexpressing transgenic mice have shown RAGE-expression dependent cognitive decline and plaque formation. Previously, polyclonal antibodies to RAGE were used to show neutralization of Ab-induced decrease in CBF (Deane et al., 2003). Here we report the application of functional magnetic resonance imaging-cerebral blood volume (fMRI-CBV) methods for guiding and developing an anti-RAGE monoclonal antibody (mAb) for the treatment of AD. Methods: Two different fMRI-CBV imaging protocols were used. First, A-992401.0, an anti-RAGE mAb, and corresponding chimeric and humanized versions were used to attempt neutralization of Ab140 induced CBV decrease in C57/BL6 anesthetized young naı¨ve mice (Luo et al., 2008). CBV changes during acute treatment with A-992401.0 in aged APP-overexpressing mice (Tg2576) were also assessed. fMRI experiments were performed on a Bruker 7T/21cm scanner. Using AFNI DiffExp nonlinear modeling voxels with an F value  10, corresponding to p < 0.05 after Bonferroni correction, were considered significantly changed from baseline. Acetozolamide was used as a stimulus to validate fMRI CBV findings. Results: A-992401.0, and corresponding chimeric and humanized mAbs had comparable binding affinities in vitro and completely neutralized Ab1-40 induced decreases in CBV. In contrast, PBS and isotype control IgG1 molecule were ineffective. As a positive control, fMRI-CBV as used demonstrated the potent cerebral vascular dilatation effects of acetozolamide. Furthermore, we observed a significant increase in CBV in aged Tg2576 during the acute treatment with A-992401.0. Conclusions: Using in vivo fMRICBV methods, we demonstrated that A-992401.0 neutralized the Ab1-40 induced CBV-decrease and improved brain perfusion in aged Tg2576. Chimeric and humanized versions of A-992401.0 retain this property aligning this function to the epitope of this mAb. Changes in CBV/CBF may represent a biomarker for treatment of AD with A-992401.0.