Proceedings – Advances in Vascular Casting
Imaging and Quantification of 3D Brain Vasculature in a Mouse Model for Alzheimer’s Disease Meyer, EP.1, Heinzer, S.2, Stampanoni, M.3, Ulmann-Schuler, A.1, Müller, R.2 and Krucker, T.4 1
Dept. of Neurobiology, University of Zurich, Zurich, Switzerland; Institute for Biomedical Engineering, ETH and University, Zurich, Switzerland 3 Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland; 4 Novartis Institutes for BioMedical Research Inc. Cambridge, USA Vascular factors associated with Alzheimer’s disease (AD) have recently gained increased attention. To investigate changes in brain vasculature, particularly of the microvascular architecture, we developed a hierarchical imaging framework to obtain large-volume, high-resolution 3D images from corrosion casted brains of transgenic mice modeling Alzheimer’s disease. 2
Keywords: Alzheimer’s disease, vasculature corrosion casting, morphology, high-resolution microCT, hierarchical imaging
Introduction The typical clinical picture of AD includes a progressive decline of memory function, often accompanied by other clinical signs such as agitation, aggression, sleep disturbances, and social withdrawal. Nonetheless, brain autopsy is needed to positively confirm the diagnosis. A high density of neuritic plaques, neurofibrillary tangles and vascular amyloid are characteristic neuropathological markers of AD [01, 02]. Plaques and tangles in the neuropil may affect neuronal function and also contribute to the neuronal damage. However, it is unclear whether their incidence correlates with the clinical signs and symptoms of cognitive impairment characteristic of the disease. Reduced blood flow has been reported as one of the most consistent physiological deficits in AD, and overt vascular irregularities have been found in biopsy material from pathologically confirmed AD cases [05]. However, it remains unclear whether the reduced cerebral blood flow and possible presence of regional microvascular abnormalities are a response to neuronal damage or factors initiating the characteristic neuropathology. The combination of advanced vascular corrosion cast and various imaging techniques (EM, micro-computed tomography (microCT) and synchrotron radiation microCT (SRmicroCT)) allows a thorough characterization and quantification of vasculature, especially microvasculature, through three-dimensional visualization. Material and Methods Animals: APP23 mice contain the human amyloid precursor protein APP751 cDNA with the Swedish double mutation at position 670/671 under the control of the neuron-specific Thy-1 promoter [03]. These animals express APP in seven-fold excess as compared to the endogenous murine APP and develop most of the histological hallmarks of AD. Vascular corrosion casting: Standard methods [04] were used for vascular corrosion casting. Briefly, animals are deeply anesthetized with pentobarbital (i.p.) and perfused via the left ventrical, first with ACSF/Heparin followed by 4% PFH. Immediately following the polymer PU4ii (vasQtec, Switzerland) was infused. Soft tissue is macerated and followed by decalcification with formic acid. Data acquisition: After the specimens were scanned at medium resolution (16 µm nominal voxel size) with a conventional µCT system (µCT 40, Scanco Medical, Switzerland), the image date was imported to custom-made ROI-picking software [04]. Regions of interest (ROIs) of 1 mm3 were selected in the frontal cortex and the hippocampus using custom made ROI selection software [Fig. _______________ * Corresponding author: e-mail:
[email protected]
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Proceedings – Advances in Vascular Casting
1]. High resolution scans (1.4 µm nominal voxel size) of these regions were then realized fully nondestructively using an additional motorized sample stage and a local tomography setup at the X-ray tomographic microscopy (XTM) station of the materials science (MS) beamline at the Swiss Light Source (SLS, PSI, Villigen, Switzerland) [07]. Image analysis: To quantify microvascular changes we use the imaging software Image-Pro Plus (Media Cybernetics, Inc., Des Moines, IA) to evaluate two-dimensional morphometry. In addition to visual assessment of structural changes, morphometrical and architectural indices are determined from the micro-tomographic examinations (both µCT and SRµCT) using software developed in house [06].
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Fig. 1: ROI selection software allows the exact localization of regions of interest in a brain cast scanned using conventional µCT. ROIs are then rescanned at the SLS with 1.4 µm resolution.
Results To assess vascular alterations in the APP23 mouse model, we focussed on hippocampal and cortical areas, because plaque and tangles, the histological hallmarks of Alzheimer’s disease, are primarily found in these two brain regions. They are the brain centers primarily responsible for cognition, particularly learning and memory. Through the combination of conventional µCT and local SRµCT, we scanned selected regions of cerebral vasculature in APP23 tg and wild-type (wt) animals, two scans located in the frontal cortex and two located in the hippocampus [Fig. 1]. Pre-scanning of full brain samples with µCT at medium resolution allowed identification of physiological regions such as hippocampus and cortex [Fig. 2]. ROIs could be marked and rescanned with SRµCT using our dedicated ROI selection and scan control software [Fig. 1]. To evaluate the vascular density, we determined the intervessel distance in selected sub-areas. Spheres were fitted mathematically between vessels using in-IPL software (Scanco Medical,
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Proceedings – Advances in Vascular Casting
Switzerland) and plotted as distribution histograms [Fig. 3]. In comparison with age-matched wt mice, young APP23 tg mice (
