Angiofil: a novel radio-contrast agent for post-mortem microangiography
Silke Grabherra, Marco Dominiettob, Lisa Yuc, Valentin Djonovd, Bert Müllere, Sebastian Friess*faInstitute of Forensic Medicine, University of Lausanne, 1005 Lausanne, Switzerland;bInstitute for Biomedical Engineering, ETH Zurich, 8093 Zurich, Switzerland;cMouse Imaging Centre, Toronto Centre for Phenogenomics, Toronto, Canada;dInstitute of Anatomy, University of Fribourg, 1700 Fribourg, Switzerland;eBiomaterials Science Center, University of Basel, 4031 Basel, Switzerland;fGloor Instruments AG, 8610 Uster, Switzerland
ABSTRACTThe radio-contrast agent Angiofil has recently been developed to be predominantely applied in forensic medicine.
Angiofil is a liquid radio-contrast agent based on iodine. Its viscosity is easy to adjust by the choice and theconcentration of the solvent. Therefore, it is well suited for penetrating vessels of different diameters. The liquid Angiofil
avoids the sedimentation of suspensions containing radio-opaque materials such as barium sulfate. The injection ofAngiofil into the vascular system of mice post-mortem results in remarkable data showing the vascular trees of tissues
and entire organs. Penetration into the surrounding tissue was not observed. Consequently, Angiofil has the potential toreach the performance of the established casting agent Microfil.
Keywords: Angiofil, radio-contrast agent, vascular tree, vascular phenotyping, micro computed tomography, microangiography

1. INTRODUCTION
The biomedical imaging market is currently seeking a steady growth. One large field comprises the development andmarketing of novel radio-contrast agents in order to enhance the imaging capabilities. The visualization of the vascularsystems of tissues, organs or entire small animals becomes more and more important. In particular, the detailed
morphological analysis of the vascular tree allows assessing the basic physiological conditions and metabolic functions.
Filling vessels with radio-opaque materials has been established. The application of several compounds have beenreported.1 Apart from the lyophilic salts, such as barium sulfate, iodine compounds were used because of its high x-ray
absorption. A powerful but time-consuming alternative is the filling of blood vessels with resins, often methyl
methacrylate. After polymerization the tissue can be dissolved and a fragile construct forms that can be easily imaged.
The present paper describes the performance of the lipophilic radio-contrast agent Angiofil in micro-angiography andmicro computed tomography. Based on applications in forensic medicine, the contrast agent has been patented.2 Experts
in bio-imaging have evaluated Angiofil within an international framework. The initial study3 included the injection into
ten mice for micro-angiography post-mortem. Whole body scans at isotropic 40 µm pixel size should permit to makevisible the vascular systems. It has to be uncovered if the dissection of individual organs really facilitates thesegmentation of the micro-vessels. Parameters such as vessel length and caliber should be quantified. Color-coding
should help to obtain detailed 3D representations of morphology of the vascular system.
Furthermore, Angiofil has to be comparatively evaluated with respect to established staining protocols. Such a
comparison should include barium sulfate that is known to be effective for lung vessel imaging in rodents.4 In addition,
the performance of Angiofil should be compared to casting procedures based on Microfil.
Another important issue to introduce Angiofil into the market is the handling. The procedure has to be relatively easy
and fast. The data quality has to be good enough that data processing such as intensity-based segmentation yields threedimensional images comparable to the casting. Finally, the waste management has to be considered.
*sebastian.friess@gloorinstruments.ch; phone 41 44 940-9955; fax 41 44 940-9914; www.angiofil.ch

Developments in X-Ray Tomography VI, edited by Stuart R. Stock,
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2. MATERIALS & METHODS
Angiofil consists of two liquid components, namely the iodized oil (alkylesters of fatty acids) and the dedicated diluent.
The oil gives rise to high contrast of more than 2000 HU. Adding the solvent, one can adjust the viscosity of Angiofil. In
the present experiments, the iodized oil and the diluent were mixed with a ratio of 1:5. The related viscosity corresponds
to 3.4 mPas at the temperature of 20 °C.
For the initial study, ten wild-type mice were anesthetized applying the intra-peritoneal injection of ketamine andxylazine. Angiofil was manually injected at room temperature following two protocols.3 Upon fixing and complete
perfusion, the main afferent and efferent vessels of the organs were ligated, before the organs were removed and placedin a Petri dish to be immersed in paraformaldehyde. The specimens in the dishes were scanned by the micro CT system(ForBild scanner, Institute of Medical Physics, Erlangen, Germany) using an accelerating voltage of 40 kV and a voxel
size of 15 µm. The data were processed on a Leonardo workstation (Siemens). The merging of partial body scans and thebuilding of the 3D models were done by means of the Amira software package (Mercury Computer Systems). Colorcoding of vessel caliber and the subsequent remapping onto the surface of the 3D model were performed as previously
described.5
For the barium sulfate staining, two female nude mice (Balb/c, weight about 25 g) were anesthetized using isofluorane.
The contrast agent was injected into the tail vein of the mouse. Here, the suspension of 15 g barium sulfate in 200 ml
physiological sodium chloride solution was used. This suspension was filtered (pore size 40 µm, BD Falcon, USA) inorder to inject 500 µl that only contained particles with diameters of the micro-vessels. Just before injection, thesuspension was mixed for a period of 10 min in an ultrasound bath (Sonorex Digital 10P, Bandelin) at the temperature of
37 °C. Furthermore, the liquid mixture of 20 µl Angiofil and 60 µl solvent was applied in the same manner.
In the case of the barium sulfate suspension, the mouse survived for 5 min until it was sacrificed. In the case of Angiofil
staining, the mouse died within 2 min upon injection because of the non-physiological solvent used. Several organs,
namely lung, brain, liver, kidneys and spleen were isolated and fixed in paraformaldehyde for experiments at thesynchrotron radiation sources at HASYLAB/DESY, Hamburg, Germany, which have been operated by the GKSS
Research Center.
The barium sulfate stained lung, held in an Eppendorf container in liquid, was scanned at the beamline W 2 using thephoton energy of 38 keV, a value just above the K absorption edge of barium (37.44 keV). The pixel size corresponded
to 6.8 µm. The spatial resolution of 13.7 µm was determined by means of the modulated transfer function of a highly xray absorbing metal plate at the sample position. The tomograms were obtained by means of the standard filtered backprojection reconstruction algorithm out of 720 projections. The 3D dataset of the lung was generated combining fivetomograms at different height levels each with voxel precision.
The Angiofil stained lung was scanned in similar fashion at the beamline BW 2 using the photon energy of 17 keV andthe pixel size of 7.0 µm.
For Microfil experiments, female C57/BL6 mice were anesthetized with an intra-peritoneal injection mixture of
ketamine and xylazine, and a subcutaneous injection of 500 units of heparin was given. A midline incision was made andthe heart exposed. A catheter was inserted into the left ventricle and 1X PBS was allowed to flow in. The right atrium
was then cut for drainage and the flow of PBS continued at a constant pressure of 100 mmHg until the fluid exiting theatrium was clear and the organs were uniformly blanched. Then Angiofil (5 ml, 1:5 mixture contrast agent : solvent, v/v)
was infused at 150 mmHg constant pressure. Upon clamping the tubing line, the vessels were carefully ligated and theorgans were removed. For perfusion with Microfil silicone rubber injection compounds (MV-122, Flow-Tech Inc.), a
mixture of compound, diluent, and catalyst was infused at 160 mmHg pressure. Upon clamping the tubing line, Microfil
was allowed to polymerize during more than 90 min. Organs of interest were carefully removed. Upon dissection,
specimens were stored in neutral buffered formalin, and subsequently mounted in 1% agar for micro CT imaging on a
GE eXplore Locus SP specimen scanner. Kidneys were scanned at the accelerating voltage of 80 kV and a beam currentof 80 µA using the isotropic 20 µm pixel size.

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3. RESULTS & DISCUSSION
3.1 Imaging vasculature with the contrast agent Angiofil
The benefits, gained from the analysis of vascular systems by micro CT, have been demonstrated. This included theputative advantages and disadvantages of the contrast agents.6 Angiofil should fit well into the list of candidates forblood vessel imaging. Thus, it has the potential to become a valuable additive for vascular phenotyping. Imaging thevascular systems in whole body mice as well as in individually dissected organs by means of Angiofil gave rise toremarkably detailed overviews of the main vessel trees, as recently published.3 Within this study, the tomograms of thepartial body scans were merged to represent the entire vascular system in the 3D manner. By adjusting the region ofinterest, the main vessels of the vascular systems of different organs became visible and were color-coded according totheir caliber size. Vascular branches of the liver, vessels of spleen and kidneys were clearly detectable. The heart wascompletely filled with the contrast agent. So, cardiac and pulmonary vessels became partially visible. High-resolutionscans of individual organs revealed the micro-vessel morphology in detail. Within this study, the small arteries and veinsof the liver and the kidneys and even the capillaries of the lung were visualized down to the pixel size of 15 µm. Since
selected cross-sections were difficult to be evaluated, maximum intensity projections were generated for further analysis.
They gave a comprehensive overview of the vascular structure as demonstrated for the kidney in Fig. 1.

Fig. 1. (a) Representations of a high-resolution scan of a murine kidney visualizing the interlobular vessels in detail.
(b) After adjusting the region of interest, the smaller vessels of the cortex and of the glomeruli become included.
(c) Using the software Amira, the maximum intensity projections on the cube side create the iso-surface rendering of
the vascular surface.3

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A

Fig. 2. The high-resolution data of a part of the murine liver show the vascular tree with gray-value coded vessel calibers.3

Fig. 2 shows the high-resolution liver data. The vessels were quantified and remapped onto the surface of the vascularsystem according to their caliber, i.e. from light gray via dark gray to white. Light gray is used for thin vessels with mean
diameter between 50 and 100 µm, The dark gray corresponds to vessels with a mean diameter of 300 µm, whereas the
thickest vessels 400 µm wide in average are represented in white.
Angiofil stayed within the vessels for a long time. Thus, it is well suited for angiography. It does not penetrate throughthe vessel walls. Monitoring Angiofil in the chemically fixed tissue, it remained stable for days without detectableleakage.7 In the present study, we investigated organs more than one week after their perfusion with Angiofil, and did not
find any deterioration in image quality.
The vessels filled with Angiofil exhibit much higher x-ray absorption than the surrounding tissues. This high differencein absorption facilitates the automated segmentation of the vessel tree that is necessary to extract quantities such as
vessel length, caliber and bifurcation density. The intensity-based segmentation is also the first step to apply moresophisticated tools for erosion/dilatation, distance mapping, etc. In order to identify the morphological features thechemically fixed tissues is amenable to histological analysis.
3.2 Comparison of Angiofil and barium sulfate staining
The main difference between Angiofil and barium sulfate is the state of aggregation at the application temperatures. The
relatively heavy, insoluble barium salt is solid and gives rise to sedimentation phenomena, which finally lead to theinhomogeneous distribution within the vessels. Therefore, the vessels often appear disconnected.8 Angiofil is purely
liquid. Hence these sedimentation problems are avoided. The lung, however, can ‘filter’ the micrometer-sized barium
sulfate particles. This filtering effect results in high barium concentrations in the lung, which yields high-contrast images
as shown in Fig. 3. Angiofil staining of the lung leads to comparable images (cp. Fig. 3).

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Fig. 3. The 3D visualizations of mouse lungs perfused with Angiofil (top) and barium sulfate micro-particles (bottom) arebased on synchrotron-radiation based micro CT and generated by the software VGStudio Max 1.2 (Volume Graphics,
Heidelberg, Germany).4

The mouse lungs are well filled with the contrast agents. The barium sulfate particles 1 and 40 µm in diameter were often
larger than the arterial capillaries. Consequently, the lung acted as ‘physiological filter’ for particles larger than 5 µm.
Since Angiofil is not particle-based such phenomena are impossible. The liquid Angiofil, however, caused emboli in theheart chambers, which probably caused the death of the mouse and prevented significant flow via the arteries to otherorgans. External pumping (perfusion pump) may help to master this problem properly.9
It should be noted that Angiofil has further advantages. The viscosity triggers the embolization. It directly determines,
which calibers of the vessels are filled or not. Angiofil of high viscosity leads to micro-embolism and to the occlusion of
the micro-circulation.7 Lowering the viscosity by diluting the contrast agent, the oily liquid also penetrates into smaller
vessels. In general, one can recommend for micro-angiography ratios between 1:3 and 1:5 for the iodized oil vs. diluent,v/v. If only the main vessels should be visualized, the viscosity can be kept high. If the small vessels and capillaries areof interest, the viscosity should be decreased adding diluent.

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3.3 Comparison of Angiofil and Microfil
Alternatively to conventional radio-opaque liquids or particle-based suspensions, casting techniques have commonly
been used to stain the vessels post-mortem.10 Here, blood vessels have been filled with resins, such as methyl
methacrylate, that polymerize within a certain period of time. Upon removal of the biological tissue, a solid but fragile
3D network of the vessel morphology can be obtained. Such a construct can be easily imaged taking advantage of tools
such as (synchrotron radiation-based) micro CT or confocal microscopy.
Compared to Angiofil, the casting methods including Microfil casting are more difficult and time-consuming, since
mixing and polymerization are additional steps to be performed. Even more important, the tissues have to be post-treated
and are not available for histology.

e. 4"Fig. 4. The maximum intensity projection images were obtained from mouse kidneys stained with Angiofil (top) and casted
with Microfil (bottom).

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In order to demonstrate that Angiofil is as powerful as Microfil, Fig. 4 shows the direct comparison using mouse
kidneys. The two images are maximum projections of the vessel tree. They nicely show the typical morphology of thekidney vascularization.
The maximum intensity projection images show that both procedures can be readily used to visualize the vessel trees ofdedicated organs. Microfil, however, results in enhanced image clarity, because the radio-contrast of Microfil is higher
than that of Angiofil.

4. CONCLUSION & OUTLOOKThe radio-contrast agent Angiofil provides images of the vascular system in rodents with micrometer resolution that arecomparable to established staining and casting procedures but do have significant advantages especially in the handlingand time efficiency. The images obtained show that Angiofil provides contrast in the vascular system without the timeconsuming sample preparation steps.
Angiofil is a transparent orange-colored liquid, which is not ideal for blood vessel labeling. A rather dark and opaqueappearance would permit the user to directly see the perfusion near the organ’s surface. This could be achieved adding
Sudan’s Black or Cibacron Blue to the Angiofil liquid.
Although Angiofil is restricted to post-mortem applications such as forensic or small animal research, it is obvious that
Angiofil has the potential to replace casting agents or particle-based contrast agents. Casts, however, have certain
advantages, because they can be subjected to scanning electron microscopy analysis, which principally allows forimaging down to nanometer scale. Nevertheless, casting often fails to maintain the entire vascular network including
capillaries. The cast specimens are typically prone to damage during transport. Finally, casting is a time-consuming
process that involves the critical polymerization. Here, perfusion of individual organs with Angiofil or injecting thecontrast agent into the animal is straightforward. If the radio-contrast of Angiofil can be significantly improved, it will
replace castings in many applications.
In conclusion, Angiofil compares well to established compounds while offering clear benefits. There is no need of timeconsuming sample preparation. Upon micro CT scanning, the organs are readily available for further comparative
histological analysis. The user can tailor the viscosity of the contrast agent and thus trigger caliber-dependentvisualization of the vessels. Angiofil is a pure liquid, thus no sedimentation or filtering effects by body tissue isobserved. Reengineering Angiofil in order to achieve an even increased radio-contrast is desirable, since this property
currently prevents being the clear alternative to casting.

ACKNOWLEDGEMENTS
The authors kindly acknowledge the support of Richard Dirnhofer and Erich Gygax, who largely provided infrastructure
and personal commitment, Heinz Lüscher for constant support and a stimulating environment and Felix Beckmann andJulia Herzen for the support during the data acquisition at HASYLAB/DESY. Financial support is greatly acknowledged
from Forim-X, Fumedica, Virtopsy Foundation, Universities of Lausanne, Bern, Fribourg, and from the Swiss Federal
Institute of Technology. The HASYLAB kindly provided beamtime in the frame of an approved proposal.

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