Global and local hard X-ray tomography of a centimeter-size tumor vessel tree.

The visualization of the vascular network in tumors down to the smallest vessels requires high spatial resolution and reasonable contrast. Stained corrosion casts of the microvasculature network guarantee superior X-ray absorption contrast and highest reproduction fidelity. Tomography of a centimeter-size tumor, however, is unfeasible at the spatial resolution needed to reveal the smallest vessels. Therefore, local tomography has been performed to visualize the smallest capillaries within the region of interest. These three-dimensional data show the detailed morphology, but the reconstructed absorption coefficients obtained in local tomography differ substantially from the absorption coefficients retrieved from the less detailed global tomography data. This paper deals with the adaptation of local tomography data using the global data and considers two-parameter histogram matching of the radiographs, sinogram extension, and multi-parameter cupping correction. It is demonstrated that two-parameter histogram matching of the radiographs already provides reasonable agreement. The change of the lens in front of the detector's camera, however, significantly affects the obtained local X-ray absorption coefficients in the tomograms predominantly owing to the dissimilar point-spread functions of the two configurations used, and much less to the fact that one of the data sets was acquired in a local geometry.

Vascular ß-amyloid and early astrocyte alterations impair cerebrovascular function and cerebral metabolism in transgenic arcAß mice.

Cerebrovascular lesions related to congophilic amyloid angiopathy (CAA) often accompany deposition of ß-amyloid (Aß) in Alzheimer's disease (AD), leading to disturbed cerebral blood flow and cognitive dysfunction, posing the question how cerebrovascular pathology contributes to the pathology of AD. To address this question, we characterised the morphology, biochemistry and functionality of brain blood vessels in transgenic arctic ß-amyloid (arcAß) mice expressing human amyloid precursor protein (APP) with both the familial AD-causing Swedish and Arctic mutations; these mice are characterised by strong CAA pathology. Mice were analysed at early, mid and late-stage pathology. Expression of the glucose transporter GLUT1 at the blood-brain barrier (BBB) was significantly decreased and paralleled by impaired in vivo blood-to-brain glucose transport and reduced cerebral lactate release during neuronal activation from mid-stage pathology onwards. Reductions in astrocytic GLUT1 and lactate transporters, as well as retraction of astrocyte endfeet and swelling consistent with neurovascular uncoupling, preceded wide-spread ß-amyloid plaque pathology. We show that CAA at later disease stages is accompanied by severe morphological alterations of brain blood vessels including stenoses, BBB leakages and the loss of vascular smooth muscle cells (SMCs). Together, our data establish that cerebrovascular and astrocytic pathology are paralleled by impaired cerebral metabolism in arcAß mice, and that astrocyte alterations occur already at premature stages of pathology, suggesting that astrocyte dysfunction can contribute to early behavioural and cognitive impairments seen in these mice.

Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease.

Substantial evidence from epidemiological, pathological, and clinical reports suggests that vascular factors are critical in the pathogenesis of Alzheimer's disease (AD), and changes in blood flow are currently the most reliable indicators of the disease. We previously reported that older APP23 transgenic (tg) mice have significant blood flow alterations correlated with structural modifications of blood vessels. For the present study, our objective was to analyze the age-dependent morphological and architectural changes of the cerebral vasculature of APP23 tg mice. To visualize the 3D arrangement of the entire brain vasculature, we used vascular corrosion casts. Already at young ages, when typically parenchymal amyloid plaques are not yet present, APP23 tg mice had significant alterations, particularly of the microvasculature, often accompanied by small deposits attached to the vessels. In older animals, vasculature abruptly ended at amyloid plaques, resulting in holes. Often, small deposits were sitting near or at the end of truncated vessels. Between such holes, the surrounding vascular array appeared more dense and showed features typical for angiogenesis. We propose that small amyloid aggregates associated with the microvasculature lead to morphological and architectural alterations of the vasculature, resulting in altered local blood flow. The characteristic early onset of vascular alterations suggests that imaging blood flow and/or vasculature architecture could be used as a tool for early diagnosis of the disease and to monitor therapies.

Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain.

The formation of new blood vessels and the establishment of vascular networks are crucial during brain development, in the adult healthy brain, as well as in various diseases of the central nervous system. Here, we describe a step-by-step protocol for our recently developed method that enables hierarchical imaging and computational analysis of vascular networks in postnatal and adult mouse brains. The different stages of the procedure include resin-based vascular corrosion casting, scanning electron microscopy, synchrotron radiation and desktop microcomputed tomography imaging, and computational network analysis. Combining these methods enables detailed visualization and quantification of the 3D brain vasculature. Network features such as vascular volume fraction, branch point density, vessel diameter, length, tortuosity and directionality as well as extravascular distance can be obtained at any developmental stage from the early postnatal to the adult brain. This approach can be used to provide a detailed morphological atlas of the entire mouse brain vasculature at both the postnatal and the adult stage of development. Our protocol allows the characterization of brain vascular networks separately for capillaries and noncapillaries. The entire protocol, from mouse perfusion to vessel network analysis, takes ~10 d.

Nogo-A regulates vascular network architecture in the postnatal brain.

Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation µCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.

Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer's disease.

Neuropathological changes associated with Alzheimer's disease (AD) such as amyloidplaques, cerebral amyloid angiopathy, and related pathologies are reproduced in APP23 transgenic mice overexpressing amyloid precursor protein (APP) with the Swedish mutation. Magnetic resonance angiography (MRA) was applied to probe, in vivo, the cerebral arterial hemodynamics of these mice. Flow voids were detected at the internal carotid artery of 11-month-old APP23 mice. At the age of 20 months, additional flow disturbances were observed in large arteries at the circle of Willis. Vascular corrosion casts obtained from the same mice revealed that vessel elimination, deformation, or both had taken place at the sites where flow voids were detected by MRA. The detailed three-dimensional architecture of the vasculature visible in the casts assisted the identification of smaller vessels most likely formed as substitution or anastomosis within the circle of Willis. Angiograms and corrosion casts from nontransgenic, age-matched mice manifested no major abnormalities in the cerebrovascular arterial flow pattern. Because no transgene overexpression has been found in the cerebrovasculature of APP23 mice and no deposits of amyloid-beta (Abeta) were observed in large arteries in the region of the circle of Willis, the present results suggest that soluble Abeta may exert deleterious effects on the vasculature. Our findings support the idea that cerebral circulatory abnormalities evolving progressively could contribute to AD pathogenesis. The study also shows the power of MRA to identify changes of vascular function in genetically engineered mice. MRA as a noninvasive technique could be applied to test new therapeutic concepts in animal models of AD and in humans.

Integrin beta-3 genetic variants and risk of venous thromboembolism in colorectal cancer patients.

BACKGROUND: Integrin ß3 is involved in tumor and endothelial cell biology as well as in platelet aggregation. Herein, we evaluated the predictive potential of three germline single nucleotide polymorphisms (SNPs) in the integrin ß3 gene (rs3809865, rs5918 and rs4642) to predict the risk of venous thromboembolism (VTE) in colorectal cancer (CRC) patients, which is one of the leading causes of death among cancer patients. METHODS: 112 patients diagnosed with CRC enrolled in the prospective Vienna Cancer and Thrombosis Study (CATS) were assessed with a median follow-up of 46 months. DNA was isolated from venous blood samples and SNPs were analyzed by the PCR-RFLP method. RESULTS: VTE occurred in 12% (n=13) of all patients. The SNPs rs5918 and rs4642 were not associated with VTE risk. For rs3809565, 23% (n=11) of patients had the A/A genotype, 4% (n=2) had the A/T genotype, but none (0%) had the T/T genotype. In the univariate analysis, patients with the A/A genotype had a significantly higher risk to develop VTE compared to the other polymorphisms (P=0.0005 after Fine and Gray). In the multivariable analysis, the predictive value remained significant. CONCLUSIONS: This study identified the rs3809865 A/A genotype as an independent risk factor for VTE in CRC patients. Our findings would help identify high risk patients and would be essential for tailored anticoagulant prophylaxis.

Magnetic resonance angiography and vascular corrosion casting as tools in biomedical research: application to transgenic mice modeling Alzheimer's disease.

In vivo imaging technologies are presently receiving considerable attention in the biomedical and pharmaceutical research areas. One of the principal imaging modalities is magnetic resonance imaging (MRI). The multiparametric nature of MRI enables anatomical, functional and even molecular information to be obtained non-invasively from intact organisms at high spatial resolution. Here we describe the use of one MRI modality, namely angiography (MRA), to non-invasively study the arterial vascular architecture of APP23 transgenic mice modeling Alzheimer's disease. Because the spatial resolution of the technique is limited, the in vivo studies are complemented by a powerful analysis of the vasculature using vascular corrosion casting. Both techniques revealed age-dependent blood flow alterations and cerebrovascular abnormalities in these mice. Our experience suggests that MRA complemented by cast analysis are important tools to describe vascular alterations and test new therapy concepts in animal models of AD. Furthermore, being non-invasive, MRA can also be applied to studies in patients suffering from this disease.

Simultaneous 3D visualization and quantification of murine bone and bone vasculature using micro-computed tomography and vascular replica.

Recent evidence suggests a close functional relationship between osteogenesis and angiogenesis as well as between bone remodeling and bone vascularization. Consequently, there is a need for visual inspection and quantitative analysis of the bone vasculature. We therefore adapted and implemented two different vascular corrosion casting (VCC) protocols using a polyurethane-based casting resin in mice for a true three-dimensional (3D), direct, and simultaneous measurement of bone tissue and vascular morphology by micro-computed tomography (microCT). For assessment of vascular replicas at the level of capillaries, a vascular contrast perfusion (VCP) protocol was devised using a contrast modality based on a barium sulfate suspension in conjunction with synchrotron radiation (SR) microCT. The vascular morphology quantified using the VCP protocol was compared quantitatively with the results of a previously established method, where the vascular network of cortical bone was derived indirectly from cortical porosity. The presented VCC and VCP protocols have the potential of serving as a valuable method for concomitant 3D quantitative morphometry of the bone tissue and its vasculature.

Novel three-dimensional analysis tool for vascular trees indicates complete micro-networks, not single capillaries, as the angiogenic endpoint in mice overexpressing human VEGF(165) in the brain.

To adequately supply tissues with oxygen and nutrients, the formation of functional vascular networks requires generation of normal, healthy vessels and their arrangement into an effective network architecture. While our knowledge about the development of single vessels significantly increased during the last years, mechanisms responsible for network formation are still poorly understood. This is probably due to the lack of suitable methods for quantification of structural properties of microvascular networks. Previously we showed that cerebral blood flow is not increased in mice exhibiting a 2- to 3-fold higher density of normal and perfused capillaries as a result of transgenic overexpression of the human vascular endothelial growth factor (VEGF(165)). Here we used vascular corrosion casting and hierarchical micro-computed tomography combined with a new network analysis tool to characterize the vascular architecture in gray and white matter of these mice. Our results indicate that VEGF overexpression leads to formation of additional micro-networks connected to higher order vessels rather than insertion of individual capillaries into the existing vessel structure. This implies that the smallest "angiogenic quantum", i.e. the final, stable result of angiogenesis and subsequent remodeling, is not a single microvessel, but a complete micro-network. In conclusion, high-resolution 3D imaging combined with network analysis can substantially improve our understanding of vascular architecture, beneficial for the development of therapeutic angiogenesis as a clinical tool for applications such as wound healing or treatment of ischemic diseases.

Hierarchical microimaging for multiscale analysis of large vascular networks.

There is a wide range of diseases and normal physiological processes that are associated with alterations of the vascular system in organs. Ex vivo imaging of large vascular networks became feasible with recent developments in microcomputed tomography (microCT). Current methods permit to visualize only limited numbers of physically excised regions of interests (ROIs) from larger samples. We developed a method based on modified vascular corrosion casting (VCC), scanning electron microscopy (SEM), and desktop and synchrotron radiation microCT (SRmicroCT) technologies to image vasculature at increasing levels of resolution, also referred to as hierarchical imaging. This novel approach allows nondestructive 3D visualization and quantification of large microvascular networks, while retaining a precise anatomical context for ROIs scanned at very high resolution. Scans of entire mouse brain VCCs were performed at 16-microm resolution with a desktop microCT system. Custom-made navigation software with a ROI selection tool enabled the identification of anatomical brain structures and precise placement of multiple ROIs. These were then scanned at 1.4-microm voxel size using SRmicroCT and a local tomography setup. A framework was developed for fast sample positioning, precise selection of ROIs, and sequential high-throughput scanning of a large numbers of brain VCCs. Despite the use of local tomography, exceptional image quality was achieved with SRmicroCT. This method enables qualitative and quantitative assessment of vasculature at unprecedented resolution and volume with relatively high throughput, opening new possibilities to study vessel architecture and vascular alterations in models of disease.